Reprogramming of blood cells into induced pluripotent stem cells as a new cell source for cartilage repair.

PubMed

Li, Yueying; Liu, Tie; Van Halm-Lutterodt, Nicholas; Chen, JiaYu; Su, Qingjun; Hai, Yong

2016-02-17

An attempt was made to reprogram peripheral blood cells into human induced pluripotent stem cell (hiPSCs) as a new cell source for cartilage repair. We generated chondrogenic lineage from human peripheral blood via hiPSCs using an integration-free method. Peripheral blood cells were either obtained from a human blood bank or freshly collected from volunteers. After transforming peripheral blood cells into iPSCs, the newly derived iPSCs were further characterized through karyotype analysis, pluripotency gene expression and cell differentiation ability. iPSCs were differentiated through multiple steps, including embryoid body formation, hiPSC-mesenchymal stem cell (MSC)-like cell expansion, and chondrogenic induction for 21 days. Chondrocyte phenotype was then assessed by morphological, histological and biochemical analysis, as well as the chondrogenic expression. hiPSCs derived from peripheral blood cells were successfully generated, and were characterized by fluorescent immunostaining of pluripotent markers and teratoma formation in vivo. Flow cytometric analysis showed that MSC markers CD73 and CD105 were present in monolayer cultured hiPSC-MSC-like cells. Both alcian blue and toluidine blue staining of hiPSC-MSC-chondrogenic pellets showed as positive. Immunohistochemistry of collagen II and X staining of the pellets were also positive. The sulfated glycosaminoglycan content was significantly increased, and the expression levels of the chondrogenic markers COL2, COL10, COL9 and AGGRECAN were significantly higher in chondrogenic pellets than in undifferentiated cells. These results indicated that peripheral blood cells could be a potential source for differentiation into chondrogenic lineage in vitro via generation of mesenchymal progenitor cells. This study supports the potential applications of utilizing peripheral blood cells in generating seed cells for cartilage regenerative medicine in a patient-specific and cost-effective approach.

Amniotic-Fluid Stem Cells: Growth Dynamics and Differentiation Potential after a CD-117-Based Selection Procedure

PubMed Central

Arnhold, S.; Glüer, S.; Hartmann, K.; Raabe, O.; Addicks, K.; Wenisch, S.; Hoopmann, M.

2011-01-01

Amniotic fluid (AF) has become an interesting source of fetal stem cells. However, AF contains heterogeneous and multiple, partially differentiated cell types. After isolation from the amniotic fluid, cells were characterized regarding their morphology and growth dynamics. They were sorted by magnetic associated cell sorting using the surface marker CD 117. In order to show stem cell characteristics such as pluripotency and to evaluate a possible therapeutic application of these cells, AF fluid-derived stem cells were differentiated along the adipogenic, osteogenic, and chondrogenic as well as the neuronal lineage under hypoxic conditions. Our findings reveal that magnetic associated cell sorting (MACS) does not markedly influence growth characteristics as demonstrated by the generation doubling time. There was, however, an effect regarding an altered adipogenic, osteogenic, and chondrogenic differentiation capacity in the selected cell fraction. In contrast, in the unselected cell population neuronal differentiation is enhanced. PMID:21437196

Dental mesenchymal stem cells encapsulated in an alginate hydrogel co-delivery microencapsulation system for cartilage regeneration.

PubMed

Moshaverinia, Alireza; Xu, Xingtian; Chen, Chider; Akiyama, Kentaro; Snead, Malcolm L; Shi, Songtao

2013-12-01

Dental-derived mesenchymal stem cells (MSCs) are promising candidates for cartilage regeneration, with a high capacity for chondrogenic differentiation. This property helps make dental MSCs an advantageous therapeutic option compared to current treatment modalities. The MSC delivery vehicle is the principal determinant for the success of MSC-mediated cartilage regeneration therapies. The objectives of this study were to: (1) develop a novel co-delivery system based on TGF-β1 loaded RGD-coupled alginate microspheres encapsulating periodontal ligament stem cells (PDLSCs) or gingival mesenchymal stem cells (GMSCs); and (2) investigate dental MSC viability and chondrogenic differentiation in alginate microspheres. The results revealed the sustained release of TGF-β1 from the alginate microspheres. After 4 weeks of chondrogenic differentiation in vitro, PDLSCs and GMSCs as well as human bone marrow mesenchymal stem cells (hBMMSCs) (as positive control) revealed chondrogenic gene expression markers (Col II and Sox-9) via qPCR, as well as matrix positively stained by Toluidine Blue and Safranin-O. In animal studies, ectopic cartilage tissue regeneration was observed inside and around the transplanted microspheres, confirmed by histochemical and immunofluorescent staining. Interestingly, PDLSCs showed more chondrogenesis than GMSCs and hBMMSCs (p<0.05). Taken together, these results suggest that RGD-modified alginate microencapsulating dental MSCs make a promising candidate for cartilage regeneration. Our results highlight the vital role played by the microenvironment, as well as value of presenting inductive signals for viability and differentiation of MSCs. Copyright © 2013 Acta Materialia Inc. All rights reserved.

Development of a model system to analyze chondrogenic differentiation of mesenchymal stem cells

PubMed Central

Ruedel, Anke; Hofmeister, Simone; Bosserhoff, Anja-Katrin

2013-01-01

High-density cell culture is widely used for the analysis of cartilage development of human mesenchymal stem cells (HMSCs) in vitro. Several cell culture systems, as micromass, pellet culture and alginate culture, are applied by groups in the field to induce chondrogenic differentiation of HMSCs. A draw back of all model systems is the high amount of cells necessary for the experiments. Further, handling of large experimental approaches is difficult due to culturing e.g. in 15 ml tubes. Therefore, we aimed to develop a new model system based on “hanging drop” cultures using 10 to 100 fold less cells. Here, we demonstrate that differentiation of chondrogenic cells was induced as previously shown in other model systems. Real time RT-PCR analysis demonstrated that Collagen type II and MIA/CD-RAP were upregulated during culturing whereas for induction of hypertrophic markers like Collagen type X and AP-2 epsilon treatment with TGF beta was needed. To further test the system, siRNA against Sox9 was used and effects on chondrogenic gene expression were evaluated. In summary, the hanging drop culture system was determined to be a promising tool for in vitro chondrogenic studies. PMID:24294400

Dental mesenchymal stem cells encapsulated in alginate hydrogel co-delivery microencapsulation system for cartilage regeneration

PubMed Central

Moshaverinia, Alireza; Xu, Xingtian; Chen, Chider; Akiyama, Kentaro; Snead, Malcolm L; Shi, Songtao

2013-01-01

Dental-derived MSCs are promising candidates for cartilage regeneration, with high chondrogenic differentiation capacity. This property contributes to making dental MSCs an advantageous therapeutic option compared to current treatment modalities. The MSC delivery vehicle is the principal determinant for the success of MSC-mediated cartilage regeneration therapies. The objectives of this study were to: (1) develop a novel co-delivery system based on TGF-β1 loaded RGD-coupled alginate microspheres encapsulating Periodontal Ligament Stem Cells (PDLSCs) or Gingival Mesenchymal Stem Cells (GMSCs); and (2) investigate dental MSC viability and chondrogenic differentiation in alginate microspheres. The results revealed the sustained release of TGF-β1 from the alginate microspheres. After 4 weeks of chondrogenic differentiation in vitro, PDLSCs, GMSCs as well as human bone marrow mesenchymal stem cells (hBMMSC) (as positive control) revealed chondrogenic gene expression markers (Col II and Sox-9) via qPCR, as well as matrix positively stained by toluidine blue and safranin-O. In animal studies, ectopic cartilage tissue regeneration was observed inside and around the transplanted microspheres, confirmed by histochemical and immunofluorescent staining. Interestingly, PDLSCs showed more chondrogenesis than GMSCs and hBMMSCs (P<0.05). Taken together, these results suggest that RGD-modified alginate microencapsulating dental MSCs make a promising candidate for cartilage regeneration. Our results highlight the vital role played by the microenvironment, as well as value of presenting inductive signals for viability and differentiation of MSCs. PMID:23891740

Isolation, characterization, and differentiation of stem cells for cartilage regeneration.

PubMed

Beane, Olivia S; Darling, Eric M

2012-10-01

The goal of tissue engineering is to create a functional replacement for tissues damaged by injury or disease. In many cases, impaired tissues cannot provide viable cells, leading to the investigation of stem cells as a possible alternative. Cartilage, in particular, may benefit from the use of stem cells since the tissue has low cellularity and cannot effectively repair itself. To address this need, researchers are investigating the chondrogenic capabilities of several multipotent stem cell sources, including adult and extra-embryonic mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs). Comparative studies indicate that each cell type has advantages and disadvantages, and while direct comparisons are difficult to make, published data suggest some sources may be more promising for cartilage regeneration than others. In this review, we identify current approaches for isolating and chondrogenically differentiating MSCs from bone marrow, fat, synovium, muscle, and peripheral blood, as well as cells from extra-embryonic tissues, ESCs, and iPSCs. Additionally, we assess chondrogenic induction with growth factors, identifying standard cocktails used for each stem cell type. Cell-only (pellet) and scaffold-based studies are also included, as is a discussion of in vivo results.

Gene expression profile in human induced pluripotent stem cells: Chondrogenic differentiation in vitro, part A

PubMed Central

Suchorska, Wiktoria Maria; Augustyniak, Ewelina; Richter, Magdalena; Trzeciak, Tomasz

2017-01-01

Human induced pluripotent stem cells (hiPSCs) offer promise in regenerative medicine, however more data are required to improve understanding of key aspects of the cell differentiation process, including how specific chondrogenic processes affect the gene expression profile of chondrocyte-like cells and the relative value of cell differentiation markers. The main aims of the present study were as follows: To determine the gene expression profile of chondrogenic-like cells derived from hiPSCs cultured in mediums conditioned with HC-402-05a cells or supplemented with transforming growth factor β3 (TGF-β3), and to assess the relative utility of the most commonly used chondrogenic markers as indicators of cell differentiation. These issues are relevant with regard to the use of human fibroblasts in the reprogramming process to obtain hiPSCs. Human fibroblasts are derived from the mesoderm and thus share a wide range of properties with chondrocytes, which also originate from the mesenchyme. Thus, the exclusion of dedifferentiation instead of chondrogenic differentiation is crucial. The hiPSCs were obtained from human primary dermal fibroblasts during a reprogramming process. Two methods, both involving embryoid bodies (EB), were used to obtain chondrocytes from the hiPSCs: EBs formed in a chondrogenic medium supplemented with TGF-β3 (10 ng/ml) and EBs formed in a medium conditioned with growth factors from HC-402-05a cells. Based on immunofluorescence and reverse transcription-quantiative polymerase chain reaction analysis, the results indicated that hiPSCs have the capacity for effective chondrogenic differentiation, in particular cells differentiated in the HC-402-05a-conditioned medium, which present morphological features and markers that are characteristic of mature human chondrocytes. By contrast, cells differentiated in the presence of TGF-β3 may demonstrate hypertrophic characteristics. Several genes [paired box 9, sex determining region Y-box (SOX) 5, SOX6, SOX9 and cartilage oligomeric matrix protein] were demonstrated to be good markers of early hiPSC chondrogenic differentiation: Insulin-like growth factor 1, Tenascin-C, and β-catenin were less valuable. These observations provide valuable data on the use of hiPSCs in cartilage tissue regeneration. PMID:28447755

Human platelet lysate successfully promotes proliferation and subsequent chondrogenic differentiation of adipose-derived stem cells: a comparison with articular chondrocytes.

PubMed

Hildner, F; Eder, M J; Hofer, K; Aberl, J; Redl, H; van Griensven, M; Gabriel, C; Peterbauer-Scherb, A

2015-07-01

Fetal calf serum (FCS) bears a potential risk for carrying diseases and eliciting immune reactions. Nevertheless, it still represents the gold standard as medium supplement in cell culture. In the present study, human platelet lysate (PL) was tested as an alternative to FCS for the expansion and subsequent chondrogenic differentiation of human adipose-derived stem cells (ASCs). ASCs were expanded with 10% FCS (group F) or 5% PL (group P). Subsequently, three-dimensional (3D) micromass pellets were created and cultured for 5 weeks in chondrogenic differentiation medium. Additionally, the de- and redifferentiation potential of human articular chondrocytes (HACs) was evaluated and compared to ASCs. Both HACs and ASCs cultured with PL showed strongly enhanced proliferation rates. Redifferentiation of HACs was possible for cells expanded up to 3.3 population doublings (PD). At this stage, PL-expanded HACs demonstrated better redifferentiation potential than FCS-expanded cells. ASCs could also be differentiated following extended passaging. Glycosaminoglycan (GAG) quantification and qRT-PCR of 10 cartilage related markers demonstrated a tendency for increased chondrogenic differentiation of PL-expanded ASCs compared to cells expanded with FCS. Histologically, collagen type II but also collagen type X was mainly present in group P. The present study demonstrates that PL strongly induces proliferation of ASCs, while the chondrogenic differentiation potential is retained. HACs also showed enhanced proliferation and even better redifferentiation when previously expanded with PL. This suggests that PL is superior to FCS as a supplement for the expansion of ASCs and HACs, particularly with regard to chondrogenic (re)differentiation. Copyright © 2013 John Wiley & Sons, Ltd.

Dental pulp stem cell-derived chondrogenic cells demonstrate differential cell motility in type I and type II collagen hydrogels.

PubMed

Yao, Li; Flynn, Nikol

2018-06-01

Advances in the development of biomaterials and stem cell therapy provide a promising approach to regenerating degenerated discs. The normal nucleus pulposus (NP) cells exhibit similar phenotype to chondrocytes. Because dental pulp stem cells (DPSCs) can be differentiated into chondrogenic cells, the DPSCs and DPSCs-derived chondrogenic cells encapsulated in type I and type II collagen hydrogels can potentially be transplanted into degenerated NP to repair damaged tissue. The motility of transplanted cells is critical because the cells need to migrate away from the hydrogels containing the cells of high density and disperse through the NP tissue after implantation. The purpose of this study was to determine the motility of DPSC and DPSC-derived chondrogenic cells in type I and type II collagen hydrogels. The time lapse imaging that recorded cell migration was analyzed to quantify the cell migration velocity and distance. The cell viability of DPSCs in native or poly(ethylene glycol) ether tetrasuccinimidyl glutarate (4S-StarPEG)-crosslinked type I and type II collagen hydrogels was determined using LIVE/DEAD cell viability assay and AlamarBlue assay. DPSCs were differentiated into chondrogenic cells. The migration of DPSCs and DPSC-derived chondrogenic cells in these hydrogels was recorded using a time lapse imaging system. This study was funded by the Regional Institute on Aging and Wichita Medical Research and Education Foundation, and the authors declare no competing interest. DPSCs showed high cell viability in non-crosslinked and crosslinked collagen hydrogels. DPSCs migrated in collagen hydrogels, and the cell migration speed was not significantly different in either type I collagen or type II collagen hydrogels. The migration speed of DPSC-derived chondrogenic cells was higher in type I collagen hydrogel than in type II collagen hydrogel. Crosslinking of type I collagen with 4S-StarPEG significantly reduced the cell migration speed of DPSC-derived chondrogenic cells. After implantation of collagen hydrogels encapsulating DPSCs or DPSC-derived chondrogenic cells, the cells can potentially migrate from the hydrogels and migrate into the NP tissue. This study also explored the differential cell motility of DPSCs and DPSC-derived chondrogenic cells in these collagen hydrogels. Copyright © 2018 Elsevier Inc. All rights reserved.

Effect of in vitro chondrogenic differentiation of autologous mesenchymal stem cells on cartilage and subchondral cancellous bone repair in osteoarthritis of temporomandibular joint.

PubMed

Chen, K; Man, C; Zhang, B; Hu, J; Zhu, S S

2013-02-01

This study investigated the effects of in vitro chondrogenic differentiated mesenchymal stem cells (MSCs) on cartilage and subchondral cancellous bone in temporomandibular joint osteoarthritis (TMJOA). Four weeks after induction of osteoarthritis (OA), the joints received hylartin solution, non-chondrogenic MSCs or in vitro chondrogenic differentiated MSCs. The changes in cartilage and subchondral cancellous bone were evaluated by histology, reverse transcription polymerase chain reaction and micro-computed tomography (CT). Implanted cells were tracked using Adeno-LacZ labelling. The differentiated MSC-treated group had better histology than the MSC-treated group at 4 and 12 weeks, but no difference at 24 weeks. Increased mRNA expression of collegan II, aggeran, Sox9 and decreased matrix metalloproteinase 13 (MMP13) were observed in differentiated MSC-treated groups compared to the undifferentiated MSC-treated group at 4 weeks. The differentiated MSC-treated group had decreased bone volume fraction, trabecular thickness and bone surface density, and increased trabecular spacing in the subchondral cancellous bone than the undifferentiated MSC-treated group. Transplanted cells were observed at cartilage, subchondral bone, and the synovial membrane lining at 4 weeks. Intra-articular injection of MSCs could delay the progression of TMJOA, and in vitro chondrogenic induction of MSCs could enhance the therapeutic effects. This provides new insights into the role of MSCs in cell-based therapies for TMJOA. Copyright © 2012 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Scaffold composition affects cytoskeleton organization, cell-matrix interaction and the cellular fate of human mesenchymal stem cells upon chondrogenic differentiation.

PubMed

Li, Yuk Yin; Choy, Tze Hang; Ho, Fu Chak; Chan, Pui Barbara

2015-06-01

The stem cell niche, or microenvironment, consists of soluble, matrix, cell and mechanical factors that together determine the cellular fates and/or differentiation patterns of stem cells. Collagen and glycosaminoglycans (GAGs) are important scaffolding materials that can mimic the natural matrix niche. Here, we hypothesize that imposing changes in the scaffold composition or, more specifically, incorporating GAGs into the collagen meshwork, will affect the morphology, cytoskeletal organization and integrin expression profiles, and hence the fate of human mesenchymal stem cells (MSCs) upon the induction of differentiation. Using chondrogenesis as an example, we microencapsulated MSCs in three scaffold systems that had varying matrix compositions: collagen alone (C), aminated collagen (AC) and aminated collagen with GAGs (ACG). We then induced the MSCs to differentiate toward a chondrogenic lineage, after which, we characterized the cell viability and morphology, as well as the level of cytoskeletal organization and the integrin expression profile. We also studied the fate of the MSCs by evaluating the major chondrogenic markers at both the gene and protein level. In C, MSC chondrogenesis was successfully induced and MSCs that spread in the scaffolds had a clear actin cytoskeleton; they expressed integrin α2β1, α5 and αv; promoted sox9 nuclear localization transcription activation; and upregulated the expression of chondrogenic matrix markers. In AC, MSC chondrogenesis was completely inhibited but the scaffold still supported cell survival. The MSCs did not spread and they had no actin cytoskeleton; did not express integrin α2 or αv; they failed to differentiate into chondrogenic lineage cells even on chemical induction; and there was little colocalization or functional interaction between integrin α5 and fibronectin. In ACG, although the MSCs did not express integrin α2, they did express integrin αv and there was strong co-localization and hence functional binding between αv and fibronectin. In addition, vimentin was the dominant cytoskeletal protein in these cells, and the chondrogenic marker genes were expressed but at a much lower level than in the MSCs encapsulated in C alone. This work suggests the importance of controlling the matrix composition as a strategy to manipulate cell-matrix interactions (through changes in the integrin expression profile and cytoskeleton organization), and hence stem cell fates. Copyright © 2015 Elsevier Ltd. All rights reserved.

Intervertebral disc-derived stem cells: implications for regenerative medicine and neural repair.

PubMed

Erwin, W Mark; Islam, Diana; Eftekarpour, Eftekhar; Inman, Robert D; Karim, Muhammad Zia; Fehlings, Michael G

2013-02-01

An in vitro and in vivo evaluation of intervertebral disc (IVD)-derived stem/progenitor cells. To determine the chondrogenic, adipogenic, osteogenic, and neurogenic differentiation capacity of disc-derived stem/progenitor cells in vitro and neurogenic differentiation in vivo. Tissue repair strategies require a source of appropriate cells that could be used to replace dead or damaged cells and tissues such as stem cells. Here we examined the potential use of IVD-derived stem cells in regenerative medicine approaches and neural repair. Nonchondrodystrophic canine IVD nucleus pulposus (NP) cells were used to generate stem/progenitor cells (NP progenitor cells [NPPCs]) and the NPPCs were differentiated in vitro into chondrogenic, adipogenic, and neurogenic lineages and in vivo into the neurogenic lineage. NPPCs were compared with bone marrow-derived mesenchymal (stromal) stem cells in terms of the expression of stemness genes. The expression of the neural crest marker protein 0 and the Brachyury gene were evaluated in NP cells and NPPCs. NPPCs contain stem/progenitor cells and express "stemness" genes such as Sox2, Oct3/4, Nanog, CD133, Nestin, and neural cell adhesion molecule but differ from mesenchymal (stromal) stem cells in the higher expression of the Nanog gene by NPPCs. NPPCs do not express protein 0 or the Brachyury gene both of which are expressed by the totality of IVD NP cells. The percentage of NPPCs within the IVD is 1% of the total as derived by colony-forming assay. NPPCs are capable of differentiating along chondrogenic, adipogenic, and neurogenic lineages in vitro and into oligodendrocyte, neuron, and astroglial specific precursor cells in vivo within the compact myelin-deficient shiverer mouse. We propose that the IVD NP represents a regenerative niche suggesting that the IVD could represent a readily accessible source of precursor cells for neural repair and regeneration.

Formation of Cartilage and Synovial Tissue by Human Gingival Stem Cells

PubMed Central

Larjava, Hannu; Loison-Robert, Ludwig-Stanislas; Berbar, Tsouria; Owen, Gethin R.; Berdal, Ariane; Chérifi, Hafida; Gogly, Bruno; Häkkinen, Lari; Fournier, Benjamin P.J.

2014-01-01

Human gingival stem cells (HGSCs) can be easily isolated and manipulated in culture to investigate their multipotency. Osteogenic differentiation of bone-marrow-derived mesenchymal stem/stromal cells has been well documented. HGSCs derive from neural crests, however, and their differentiation capacity has not been fully established. The aim of the present report was to investigate whether HGSCs can be induced to differentiate to osteoblasts and chondrocytes. HGSCs were cultured either in a classical monolayer culture or in three-dimensional floating micromass pellet cultures in specific differentiation media. HGSC differentiation to osteogenic and chondrogenic lineages was determined by protein and gene expression analyses, and also by specific staining of cells and tissue pellets. HGSCs cultured in osteogenic differentiation medium showed induction of Runx2, alkaline phosphatase (ALPL), and osterix expression, and subsequently formed mineralized nodules consistent with osteogenic differentiation. Interestingly, HGSC micromass cultures maintained in chondrogenic differentiation medium showed SOX9-dependent differentiation to both chondrocyte and synoviocyte lineages. Chondrocytes at different stages of differentiation were identified by gene expression profiles and by histochemical and immunohistochemical staining. In 3-week-old cultures, peripheral cells in the micromass cultures organized in layers of cuboidal cells with villous structures facing the medium. These cells were strongly positive for cadherin-11, a marker of synoviocytes. In summary, the findings indicate that HGSCs have the capacity to differentiate to osteogenic, chondrogenic, and synoviocyte lineages. Therefore, HGSCs could serve as an alternative source for stem cell therapies in regenerative medicine for patients with cartilage and joint destructions, such as observed in rheumatoid arthritis. PMID:25003637

Fibroblast growth factor receptors in in vitro and in vivo chondrogenesis: relating tissue engineering using adult mesenchymal stem cells to embryonic development.

PubMed

Hellingman, Catharine A; Koevoet, Wendy; Kops, Nicole; Farrell, Eric; Jahr, Holger; Liu, Wei; Baatenburg de Jong, Robert J; Frenz, Dorothy A; van Osch, Gerjo J V M

2010-02-01

Adult mesenchymal stem cells (MSCs) are considered promising candidate cells for therapeutic cartilage and bone regeneration. Because tissue regeneration and embryonic development may involve similar pathways, understanding common pathways may lead to advances in regenerative medicine. In embryonic limb development, fibroblast growth factor receptors (FGFRs) play a role in chondrogenic differentiation. The aim of this study was to investigate and compare FGFR expression in in vivo embryonic limb development and in vitro chondrogenesis of MSCs. Our study showed that in in vitro chondrogenesis of MSCs three sequential stages can be found, as in embryonic limb development. A mesenchymal condensation (indicated by N-cadherin) is followed by chondrogenic differentiation (indicated by collagen II), and hypertrophy (indicated by collagen X). FGFR1-3 are expressed in a stage-dependent pattern during in vitro differentiation and in vivo embryonic limb development. In both models FGFR2 is clearly expressed by cells in the condensation phase. No FGFR expression was observed in differentiating and mature hyaline chondrocytes, whereas hypertrophic chondrocytes stained strongly for all FGFRs. To evaluate whether stage-specific modulation of chondrogenic differentiation in MSCs is possible with different subtypes of FGF, FGF2 and FGF9 were added to the chondrogenic medium during different stages in the culture process (early or late). FGF2 and FGF9 differentially affected the amount of cartilage formed by MSCs depending on the stage in which they were added. These results will help us understand the role of FGF signaling in chondrogenesis and find new tools to monitor and control chondrogenic differentiation.

In vivo cartilage formation using chondrogenic-differentiated human adipose-derived mesenchymal stem cells mixed with fibrin glue.

PubMed

Jung, Sung-No; Rhie, Jong Won; Kwon, Ho; Jun, Young Joon; Seo, Je-Won; Yoo, Gyeol; Oh, Deuk Young; Ahn, Sang Tae; Woo, Jihyoun; Oh, Jieun

2010-03-01

Human adipose-derived mesenchymal stem cells (MSCs) were differentiated into chondrogenic MSCs, and fibrin glue was used together to explore the feasibility of whether cartilages can be generated in vivo by injecting the differentiated cells. Mesenchymal stem cells extracted from human adipose were differentiated into chondrogenic MSCs, and such differentiated cells mixed with fibrin glue were injected subcutaneously into the back of the nude mouse. In addition to visual evaluation of the tissues formed after 4, 8, and 12 weeks, hematoxylin-eosin staining, Masson trichrome staining, measurement of glycosaminoglycan concentration using dimethylmethylene blue, agreecan through reverse transcriptase-polymerase chain reaction, type II collagen, and expression of SOX-9 were verified. Moreover, the results were compared with 2 groups of controls: 1 control group that received only injection of chondrogenic-differentiated MSC and the supporting control group that received only fibrin glue injection. For the experimental group, cartilage-like tissues were formed after 4, 8, and 12 weeks. Formation of cartilage tissues was not observed in any of 4, 8, and 12 weeks of the control group. The supporting control group had only a small structure formation after 4 weeks, but the formed structure was completely decomposed by the 8th and 12th weeks. The range of staining dramatically increased with time at 4, 8, and 12 weeks in Masson trichrome staining. The concentration of glycosaminoglycan also increased with time. The increased level was statistically significant with more than 3 times more after 8 weeks compared with 4 weeks and more than 2 times more after 12 weeks compared with 8 weeks. Also, in reverse transcriptase-polymerase chain reaction at 4, 8, and 12 weeks, all results expressed a cartilage-specific gene called aggrecan, type II collagen, and SOX-9. The study verified that the chondrogenic-differentiated MSCs derived from human adipose tissues with fibrin glue can proliferate and form new cartilage. Our findings suggest that formation of cartilages in vivo is possible.

Extracellular Protease Inhibition Alters the Phenotype of Chondrogenically Differentiating Human Mesenchymal Stem Cells (MSCs) in 3D Collagen Microspheres.

PubMed

Han, Sejin; Li, Yuk Yin; Chan, Barbara Pui

2016-01-01

Matrix remodeling of cells is highly regulated by proteases and their inhibitors. Nevertheless, how would the chondrogenesis of mesenchymal stem cells (MSCs) be affected, when the balance of the matrix remodeling is disturbed by inhibiting matrix proteases, is incompletely known. Using a previously developed collagen microencapsulation platform, we investigated whether exposing chondrogenically differentiating MSCs to intracellular and extracellular protease inhibitors will affect the extracellular matrix remodeling and hence the outcomes of chondrogenesis. Results showed that inhibition of matrix proteases particularly the extracellular ones favors the phenotype of fibrocartilage rather than hyaline cartilage in chondrogenically differentiating hMSCs by upregulating type I collagen protein deposition and type II collagen gene expression without significantly altering the hypertrophic markers at gene level. This study suggests the potential of manipulating extracellular proteases to alter the outcomes of hMSC chondrogenesis, contributing to future development of differentiation protocols for fibrocartilage tissues for intervertebral disc and meniscus tissue engineering.

Extracellular Protease Inhibition Alters the Phenotype of Chondrogenically Differentiating Human Mesenchymal Stem Cells (MSCs) in 3D Collagen Microspheres

PubMed Central

Han, Sejin; Li, Yuk Yin; Chan, Barbara Pui

2016-01-01

Matrix remodeling of cells is highly regulated by proteases and their inhibitors. Nevertheless, how would the chondrogenesis of mesenchymal stem cells (MSCs) be affected, when the balance of the matrix remodeling is disturbed by inhibiting matrix proteases, is incompletely known. Using a previously developed collagen microencapsulation platform, we investigated whether exposing chondrogenically differentiating MSCs to intracellular and extracellular protease inhibitors will affect the extracellular matrix remodeling and hence the outcomes of chondrogenesis. Results showed that inhibition of matrix proteases particularly the extracellular ones favors the phenotype of fibrocartilage rather than hyaline cartilage in chondrogenically differentiating hMSCs by upregulating type I collagen protein deposition and type II collagen gene expression without significantly altering the hypertrophic markers at gene level. This study suggests the potential of manipulating extracellular proteases to alter the outcomes of hMSC chondrogenesis, contributing to future development of differentiation protocols for fibrocartilage tissues for intervertebral disc and meniscus tissue engineering. PMID:26760956

Comparative Effect of Physicomechanical and Biomolecular Cues on Zone-Specific Chondrogenic Differentiation of Mesenchymal Stem Cells

PubMed Central

Moeinzadeh, Seyedsina; Shariati, Seyed Ramin Pajoum; Jabbari, Esmaiel

2016-01-01

Current tissue engineering approaches to regeneration of articular cartilage rarely restore the tissue to its normal state because the generated tissue lacks the intricate zonal organization of the native cartilage. Zonal regeneration of articular cartilage is hampered by the lack of knowledge for the relation between physical, mechanical, and biomolecular cues and zone-specific chondrogenic differentiation of progenitor cells. This work investigated in 3D the effect of TGF-β1, zone-specific growth factors, optimum matrix stiffness, and adding nanofibers on the expression of chondrogenic markers specific to the superficial, middle, and calcified zones of articular cartilage by the differentiating human mesenchymal stem cells (hMSCs). Growth factors included BMP-7, IGF-1, and hydroxyapatite (HA) for the superficial, middle, and calcified zones, respectively; optimum matrix stiffness was 80 kPa, 2.1 MPa, and 320 MPa; and nanofibers were aligned horizontal, random, and perpendicular to the gel surface. hMSCs with zone-specific cell densities were encapsulated in engineered hydrogels and cultured with or without TGF-β1, zone-specific growth factor, optimum matrix modulus, and fiber addition and cultured in basic chondrogenic medium. The expression of encapsulated cells was measured by mRNA, protein, and biochemical analysis. Results indicated that zone-specific matrix stiffness had a dominating effect on chondrogenic differentiation of hMSCs to the superficial and calcified zone phenotypes. Addition of aligned nanofibers parallel to the direction of gel surface significantly enhanced expression of Col II in the superficial zone chondrogenic differentiation of hMSCs. Conversely, biomolecular factor IGF-1 in combination with TGF-β1 had a dominating effect on the middle zone chondrogenic differentiation of hMSCs. Results of this work could potentially lead to the development of multilayer grafts mimicking the zonal organization of articular cartilage. PMID:27038568

A reliable protocol for the isolation of viable, chondrogenically differentiated human mesenchymal stem cells from high-density pellet cultures.

PubMed

Ullah, Mujib; Hamouda, Houda; Stich, Stefan; Sittinger, Michael; Ringe, Jochen

2012-12-01

Administration of chondrogenically differentiated mesenchymal stem cells (MSC) is discussed as a promising approach for the regenerative treatment of injured or diseased cartilage. The high-density pellet culture is the standard culture for chondrogenic differentiation, but cells in pellets secrete extracellular matrix (ECM) that they become entrapped in. Protocols for cell isolation from pellets often result in cell damage and dedifferentiation towards less differentiated MSC. Therefore, our aim was to develop a reliable protocol for the isolation of viable, chondrogenically differentiated MSC from high-density pellet cultures. Human bone marrow MSC were chondrogenically stimulated with transforming growth factor-β3, and the cartilaginous structure of the pellets was verified by alcian blue staining of cartilage proteoglycans, antibody staining of cartilage collagen type II, and quantitative real-time reverse-transcription polymerase chain reaction of the marker genes COL2A1 and SOX9. Trypsin and collagenases II and P were tested alone or in combination, and for different concentrations and times, to find a protocol for optimized pellet digestion. Whereas trypsin was not able to release viable cells, 90-min digestion with 300 U of collagenase II, 20 U of collagenase P, and 2 mM CaCl2 worked quite well and resulted in about 2.5×10(5) cells/pellet. The protocol was further optimized for the separation of released cells and ECM from each other. Cells were alcian blue and collagen type II positive and expressed COL2A1 and SOX9, verifying a chondrogenic character. However, they had different morphological shapes. The ECM was also uniformly alcian blue and collagen type II positive but showed different organizational and structural forms. To conclude, our protocol allows the reliable isolation of a defined number of viable, chondrogenically differentiated MSC from high-density pellet cultures. Such cells, as well as the ECM components, are of interest as research tools and for cartilage tissue engineering.

Effects of chitosan and bioactive glass modifications of knitted and rolled polylactide-based 96/4 L/D scaffolds on chondrogenic differentiation of adipose stem cells.

PubMed

Ahtiainen, Katja; Sippola, Laura; Nurminen, Manu; Mannerström, Bettina; Haimi, Suvi; Suuronen, Riitta; Hyttinen, Jari; Ylikomi, Timo; Kellomäki, Minna; Miettinen, Susanna

2015-01-01

The performance of biodegradable knitted and rolled 3-dimensional (3D) polylactide-based 96/4 scaffolds modified with bioactive glass (BaG) 13-93, chitosan and both was compared with regard to the viability, proliferation and chondrogenic differentiation of rabbit adipose stem cells (ASCs). Scaffold porosities were determined by micro-computed tomography (μCT). Water absorption and degradation of scaffolds were studied during 28-day hydrolysis in Tris-buffer. Viability, number and differentiation of ASCs in PLA96/4 scaffolds were examined in vitro. The dimensions of the scaffolds were maintained during hydrolysis and mass loss was detected only in the BaG13-93 containing scaffolds. ASCs adhered and proliferated on each scaffold type. Cell aggregation and expression of chondral matrix components improved in all scaffold types in chondrogenic medium. Signs of hypertrophy were detected in the modified scaffolds but not in the plain PLA96/4 scaffold. Chondrogenic differentiation was most enhanced in the presence of chitosan. These findings indicate that the plain P scaffold provided a good 3D-matrix for ASC proliferation whereas the addition of chitosan to the PLA96/4 scaffold induced chondrogenic differentiation independent of the medium. Accordingly, a PLA96/4 scaffold modified by chitosan could provide a functional and bioactive basis for tissue-engineered chondral implants. Copyright © 2012 John Wiley & Sons, Ltd.

Reconstruction of cartilage with clonal mesenchymal stem cell-acellular dermal matrix in cartilage defect model in nonhuman primates.

PubMed

Ma, Anlun; Jiang, Li; Song, Lijun; Hu, Yanxin; Dun, Hao; Daloze, Pierre; Yu, Yonglin; Jiang, Jianyuan; Zafarullah, Muhammad; Chen, Huifang

2013-07-01

Articular cartilage defects are commonly associated with trauma, inflammation and osteoarthritis. Mesenchymal stem cell (MSC)-based therapy is a promising novel approach for repairing articular cartilage. Direct intra-articular injection of uncommitted MSCs does not regenerate high-quality cartilage. This study explored utilization of a new three-dimensional, selected chondrogenic clonal MSC-loaded monkey acellular dermal matrix (MSC-ADM) scaffold to repair damaged cartilage in an experimental model of knee joint cartilage defect in Cynomolgus monkeys. MSCs were characterized for cell size, cell yield, phenotypes, proliferation and chondrogenic differentiation capacity. Chondrogenic differentiation assays were performed at different MSC passages by sulfated glycosaminoglycans (sGAG), collagen, and fluorescence activated cell sorter (FACS) analysis. Selected chondrogenic clonal MSCs were seeded onto ADM scaffold with the sandwich model and MSC-loaded ADM grafts were analyzed by confocal microscopy and scanning electron microscopy. Cartilage defects were treated with normal saline, clonal MSCs and clonal MSC-ADM grafts, respectively. The clinical parameters, and histological and immunohistochemical examinations were evaluated at weeks 8, 16, 24 post-treatment, respectively. Polyclonal and clonal MSCs could differentiate into the chondrogenic lineage after stimulation with suitable chondrogenic factors. They expressed mesenchymal markers and were negative for hematopoietic markers. Articular cartilage defects were considerably improved and repaired by selected chondrogenic clonal MSC-based treatment, particularly, in MSC-ADM-treated group. The histological scores in MSC-ADM-treated group were consistently higher than those of other groups. Our results suggest that selected chondrogenic clonal MSC-loaded ADM grafts could improve the cartilage lesions in Cynomolgus monkey model, which may be applicable for repairing similar human cartilage defects. Copyright © 2013 Elsevier B.V. All rights reserved.

Comparison of Four Protocols to Generate Chondrocyte-Like Cells from Human Induced Pluripotent Stem Cells (hiPSCs).

PubMed

Suchorska, Wiktoria Maria; Augustyniak, Ewelina; Richter, Magdalena; Trzeciak, Tomasz

2017-04-01

Stem cells (SCs) are a promising approach to regenerative medicine, with the potential to treat numerous orthopedic disorders, including osteo-degenerative diseases. The development of human-induced pluripotent stem cells (hiPSCs) has increased the potential of SCs for new treatments. However, current methods of differentiating hiPSCs into chondrocyte-like cells are suboptimal and better methods are needed. The aim of the present study was to assess four different chondrogenic differentiation protocols to identify the most efficient method of generating hiPSC-derived chondrocytes. For this study, hiPSCs were obtained from primary human dermal fibroblasts (PHDFs) and differentiated into chondrocyte-like cells using four different protocols: 1) monolayer culture with defined growth factors (GF); 2) embryoid bodies (EBs) in a chondrogenic medium with TGF-β3 cells; 3) EBs in chondrogenic medium conditioned with human chondrocytes (HC-402-05a cell line) and 4) EBs in chondrogenic medium conditioned with human chondrocytes and supplemented with TGF-β3. The cells obtained through these four protocols were evaluated and compared at the mRNA and protein levels. Although chondrogenic differentiation of hiPSCs was successfully achieved with all of these protocols, the two fastest and most cost-effective methods were the monolayer culture with GFs and the medium conditioned with human chondrocytes. Both of these methods are superior to other available techniques. The main advantage of the conditioned medium is that the technique is relatively simple and inexpensive while the directed method (i.e., monolayer culture with GFs) is faster than any protocol described to date because it is does not require additional steps such as EB formation.

Chondrogenic induction of mesenchymal stromal/stem cells from Wharton's jelly embedded in alginate hydrogel and without added growth factor: an alternative stem cell source for cartilage tissue engineering.

PubMed

Reppel, Loïc; Schiavi, Jessica; Charif, Naceur; Leger, Léonore; Yu, Hao; Pinzano, Astrid; Henrionnet, Christel; Stoltz, Jean-François; Bensoussan, Danièle; Huselstein, Céline

2015-12-30

Due to their intrinsic properties, stem cells are promising tools for new developments in tissue engineering and particularly for cartilage tissue regeneration. Although mesenchymal stromal/stem cells from bone marrow (BM-MSC) have long been the most used stem cell source in cartilage tissue engineering, they have certain limits. Thanks to their properties such as low immunogenicity and particularly chondrogenic differentiation potential, mesenchymal stromal/stem cells from Wharton's jelly (WJ-MSC) promise to be an interesting source of MSC for cartilage tissue engineering. In this study, we propose to evaluate chondrogenic potential of WJ-MSC embedded in alginate/hyaluronic acid hydrogel over 28 days. Hydrogels were constructed by the original spraying method. Our main objective was to evaluate chondrogenic differentiation of WJ-MSC on three-dimensional scaffolds, without adding growth factors, at transcript and protein levels. We compared the results to those obtained from standard BM-MSC. After 3 days of culture, WJ-MSC seemed to be adapted to their new three-dimensional environment without any detectable damage. From day 14 and up to 28 days, the proportion of WJ-MSC CD73(+), CD90(+), CD105(+) and CD166(+) decreased significantly compared to monolayer marker expression. Moreover, WJ-MSC and BM-MSC showed different phenotype profiles. After 28 days of scaffold culture, our results showed strong upregulation of cartilage-specific transcript expression. WJ-MSC exhibited greater type II collagen synthesis than BM-MSC at both transcript and protein levels. Furthermore, our work highlighted a relevant result showing that WJ-MSC expressed Runx2 and type X collagen at lower levels than BM-MSC. Once seeded in the hydrogel scaffold, WJ-MSC and BM-MSC have different profiles of chondrogenic differentiation at both the phenotypic level and matrix synthesis. After 4 weeks, WJ-MSC, embedded in a three-dimensional environment, were able to adapt to their environment and express specific cartilage-related genes and matrix proteins. Today, WJ-MSC represent a real alternative source of stem cells for cartilage tissue engineering.

Polyglycolic acid-hyaluronan scaffolds loaded with bone marrow-derived mesenchymal stem cells show chondrogenic differentiation in vitro and cartilage repair in the rabbit model.

PubMed

Patrascu, Jenel M; Krüger, Jan Philipp; Böss, Hademar G; Ketzmar, Anna-Katharina; Freymann, Undine; Sittinger, Michael; Notter, Michael; Endres, Michaela; Kaps, Christian

2013-10-01

In cartilage repair, scaffold-assisted one-step approaches are used to improve the microfracture (Mfx) technique. Since the number of progenitors in Mfx is low and may further decrease with age, aim of our study was to analyze the chondrogenic potential of freeze-dried polyglycolic acid-hyaluronan (PGA-HA) implants preloaded with mesenchymal stem cells (MSCs) in vitro and in a rabbit articular cartilage defect model. Human bone marrow-derived MSC from iliac crest were cultured in freeze-dried PGA-HA implants for chondrogenic differentiation. In a pilot study, implants were loaded with autologous rabbit MSC and used to cover 5 mm × 6 mm full-thickness femoral articular cartilage defects (n = 4). Untreated defects (n = 3) served as controls. Gene expression analysis and histology showed induction of typical chondrogenic marker genes like type II collagen and formation of hyaline-like cartilaginous tissue in MSC-laden PGA-HA implants. Histological evaluation of rabbit repair tissue formation after 30 and 45 days showed formation of repair tissue, rich in chondrocytic cells and of a hyaline-like appearance. Controls showed no articular resurfacing, tissue repair in the subchondral zone and fibrin formation. These results suggest that MSC-laden PGA-HA scaffolds have chondrogenic potential and are a promising option for stem cell-mediated cartilage regeneration. Copyright © 2013 Wiley Periodicals, Inc.

Chondrogenic differentiation potential of human mesenchymal stem cells photoencapsulated within poly(ethylene glycol)-arginine-glycine-aspartic acid-serine thiol-methacrylate mixed-mode networks.

PubMed

Salinas, Chelsea N; Cole, Brook B; Kasko, Andrea M; Anseth, Kristi S

2007-05-01

Chondrogenesis of human mesenchymal stem cells (hMSCs) encapsulated in poly(ethylene glycol) (PEG)-based hydrogels was studied in the presence and absence of 5 ng/mL transforming growth factor beta and chondrogenic medium to better understand the role of the gel environment on this process. The lack of any cell-polymer interactions led to decreasing cell viability, as measured using adenosine triphosphate, over a 14-day period. The extent of chondrogenic differentiation was evaluated by immunostaining, and although viability dramatically decreased, cells cultured in chondrogenic differentiation medium expressed higher levels of collagen type II. Cells cultured in hMSC control medium remained undifferentiated and continued to express CD105, a MSC marker. To increase cell survival, arginine-glycine-aspartic acid-serine (RGDS) was incorporated into gels using a novel mixed-mode thiol-ene reaction by synthesizing a cysteine-cysteine-arginine-glycine-aspartic acid-serine-cysteine-cysteine-glycine, N-terminus to C-terminus peptide sequence with pendant cysteine residues. A concentration of 5 mM RGDS incorporated into the network maintained 75% viability in control cultures. Further studies demonstrated that 5-mM RGDS chondrogenic cultures had greater gene expression for aggrecan and collagen II in conjunction with producing twice as much glycosaminoglycan as 0-mM chondrogenic cultures and 7 times that of control cultures. Incorporation of this peptide sequence not only allows for sustained viability, but also contributes to initiating chondrogenesis.

An electromagnetic compressive force by cell exciter stimulates chondrogenic differentiation of bone marrow-derived mesenchymal stem cells.

PubMed

Park, Sang-Hyug; Sim, Woo Young; Park, Sin Wook; Yang, Sang Sik; Choi, Byung Hyune; Park, So Ra; Park, Kwideok; Min, Byoung-Hyun

2006-11-01

In this study, we present a biological micro-electromechanical system and its application to the chondrogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells (MSCs). Actuated by an electromagnetic force, the micro cell exciter was designed to deliver a cyclic compressive load (CCL) with various magnitudes. Two major parts in the system are an actuator and a cartridge-type chamber. The former has a permanent magnet and coil, and the latter is equipped with 7 sample dishes and 7 metal caps. Mixed with a 2.4% alginate solution, the alginate/MSC layers were positioned in the sample dishes; the caps contained chondrogenic defined medium without transforming growth factor-beta (TGF-beta). Once powered, the actuator coil-derived electromagnetic force pulled the metal caps down, compressing the samples. The cyclic load was given at 1-Hz frequency for 10 min twice a day. Samples in the dishes without a cap served as a control. The samples were analyzed at 3, 5, and 7 days after stimulation for cell viability, biochemical assays, histologic features, immunohistochemistry, and gene expression of the chondrogenic markers. Applied to the alginate/MSC layer, the CCL system enhanced the synthesis of cartilage-specific matrix proteins and the chondrogenic markers, such as aggrecan, type II collagen, and Sox9. We found that the micromechanically exerted CCL by the cell exciter was very effective in enhancing the chondrogenic differentiation of MSCs, even without using exogenous TGF-beta.

Hyaline cartilage formation and tumorigenesis of implanted tissues derived from human induced pluripotent stem cells.

PubMed

Saito, Taku; Yano, Fumiko; Mori, Daisuke; Kawata, Manabu; Hoshi, Kazuto; Takato, Tsuyoshi; Masaki, Hideki; Otsu, Makoto; Eto, Koji; Nakauchi, Hiromitsu; Chung, Ung-il; Tanaka, Sakae

2015-01-01

Induced pluripotent stem cells (iPSCs) are a promising cell source for cartilage regenerative medicine. Meanwhile, the risk of tumorigenesis should be considered in the clinical application of human iPSCs (hiPSCs). Here, we report in vitro chondrogenic differentiation of hiPSCs and maturation of the differentiated hiPSCs through transplantation into mouse knee joints. Three hiPSC clones showed efficient chondrogenic differentiation using an established protocol for human embryonic stem cells. The differentiated hiPSCs formed hyaline cartilage tissues at 8 weeks after transplantation into the articular cartilage of NOD/SCID mouse knee joints. Although tumors were not observed during the 8 weeks after transplantation, an immature teratoma had developed in one mouse at 16 weeks. In conclusion, hiPSCs are a potent cell source for regeneration of hyaline articular cartilage. However, the risk of tumorigenesis should be managed for clinical application in the future.

Stable subcutaneous cartilage regeneration of bone marrow stromal cells directed by chondrocyte sheet.

PubMed

Li, Dan; Zhu, Lian; Liu, Yu; Yin, Zongqi; Liu, Yi; Liu, Fangjun; He, Aijuan; Feng, Shaoqing; Zhang, Yixin; Zhang, Zhiyong; Zhang, Wenjie; Liu, Wei; Cao, Yilin; Zhou, Guangdong

2017-05-01

In vivo niche plays an important role in regulating differentiation fate of stem cells. Due to lack of proper chondrogenic niche, stable cartilage regeneration of bone marrow stromal cells (BMSCs) in subcutaneous environments is always a great challenge. This study explored the feasibility that chondrocyte sheet created chondrogenic niche retained chondrogenic phenotype of BMSC engineered cartilage (BEC) in subcutaneous environments. Porcine BMSCs were seeded into biodegradable scaffolds followed by 4weeks of chondrogenic induction in vitro to form BEC, which were wrapped with chondrocyte sheets (Sheet group), acellular small intestinal submucosa (SIS, SIS group), or nothing (Blank group) respectively and then implanted subcutaneously into nude mice to trace the maintenance of chondrogenic phenotype. The results showed that all the constructs in Sheet group displayed typical cartilaginous features with abundant lacunae and cartilage specific matrices deposition. These samples became more mature with prolonged in vivo implantation, and few signs of ossification were observed at all time points except for one sample that had not been wrapped completely. Cell labeling results in Sheet group further revealed that the implanted BEC directly participated in cartilage formation. Samples in both SIS and Blank groups mainly showed ossified tissue at all time points with partial fibrogenesis in a few samples. These results suggested that chondrocyte sheet could create a chondrogenic niche for retaining chondrogenic phenotype of BEC in subcutaneous environment and thus provide a novel research model for stable ectopic cartilage regeneration based on stem cells. In vivo niche plays an important role in directing differentiation fate of stem cells. Due to lack of proper chondrogenic niche, stable cartilage regeneration of bone marrow stromal cells (BMSCs) in subcutaneous environments is always a great challenge. The current study demonstrated that chondrocyte sheet generated by high-density culture of chondrocytes in vitro could cearte a chondrogenic niche in subcutaneous environment and efficiently retain the chondrogenic phenotype of in vitro BMSC engineered cartilage (vitro-BEC). Furthermore, cell tracing results revealed that the regenerated cartilage mainly derived from the implanted vitro-BEC. The current study not only proposes a novel research model for microenvironment simulation but also provides a useful strategy for stable ectopic cartilage regeneration of stem cells. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Chondrogenic Differentiation of Defined Equine Mesenchymal Stem Cells Derived from Umbilical Cord Blood for Use in Cartilage Repair Therapy

PubMed Central

Desancé, Mélanie; Contentin, Romain; Bertoni, Lélia; Gomez-Leduc, Tangni; Branly, Thomas; Jacquet, Sandrine; Betsch, Jean-Marc; Batho, Agnès; Legendre, Florence; Audigié, Fabrice

2018-01-01

Cartilage engineering is a new strategy for the treatment of cartilage damage due to osteoarthritis or trauma in humans. Racehorses are exposed to the same type of cartilage damage and the anatomical, cellular, and biochemical properties of their cartilage are comparable to those of human cartilage, making the horse an excellent model for the development of cartilage engineering. Human mesenchymal stem cells (MSCs) differentiated into chondrocytes with chondrogenic factors in a biomaterial appears to be a promising therapeutic approach for direct implantation and cartilage repair. Here, we characterized equine umbilical cord blood-derived MSCs (eUCB-MSCs) and evaluated their potential for chondrocyte differentiation for use in cartilage repair therapy. Our results show that isolated eUCB-MSCs had high proliferative capacity and differentiated easily into osteoblasts and chondrocytes, but not into adipocytes. A three-dimensional (3D) culture approach with the chondrogenic factors BMP-2 and TGF-β1 potentiated chondrogenic differentiation with a significant increase in cartilage-specific markers at the mRNA level (Col2a1, Acan, Snorc) and the protein level (type II and IIB collagen) without an increase in hypertrophic chondrocyte markers (Col10a1 and Mmp13) in normoxia and in hypoxia. However, these chondrogenic factors caused an increase in type I collagen, which can be reduced using small interfering RNA targeting Col1a2. This study provides robust data on MSCs characterization and demonstrates that eUCB-MSCs have a great potential for cartilage tissue engineering. PMID:29439436

Depletion of histone demethylase KDM2A enhanced the adipogenic and chondrogenic differentiation potentials of stem cells from apical papilla

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dong, Rui; Yao, Rui; Du, Juan

Mesenchymal stem cells (MSCs) are a reliable resource for tissue regeneration, but the molecular mechanism underlying directed differentiation remains unclear; this has restricted potential MSC applications. The histone demethylase, lysine (K)-specific demethylase 2A (KDM2A), is evolutionarily conserved and ubiquitously expressed members of the JmjC-domain-containing histone demethylase family. A previous study determined that KDM2A can regulate the cell proliferation and osteo/dentinogenic differentiation of MSCs. It is not known whether KDM2A is involved in the other cell lineages differentiation of MSCs. Here, we show that depletion of KDM2A by short hairpin RNAs can enhance adipogenic and chondrogenic differentiation potentials in human stemmore » cells from apical papilla (SCAPs). We found that the stemness-related genes, SOX2, and the embryonic stem cell master transcription factor, NANOG were significantly increased after silence of KDM2A in SCAPs. Moreover, we found that knock-down of the KDM2A co-factor, BCOR also up-regulated the mRNA levels of SOX2 and NANOG. Furthermore, Chromatin immunoprecipitation assays demonstrate that silence of KDM2A increased the histone H3 Lysine 4 (H3K4) trimethylation in the SOX2 and NANOG locus and regulates its expression. In conclusion, our results suggested that depletion of KDM2A enhanced the adipogenic and chondrogenic differentiation potentials of SCAPs by up-regulated SOX2 and NANOG, BCOR also involved in this regulation as co-factor, and provided useful information to understand the molecular mechanism underlying directed differentiation in MSCs. - Highlights: • Depletion of KDM2A enhances adipogenic/chondrogenic differentiation in SCAPs. • Depletion of KDM2A enhances the differentiation of SCAPs by activate SOX2 and NANOG. • Silence of KDM2A increases histone H3 Lysine 4 trimethylation in SOX2 and NANOG. • BCOR is co-factor of KDM2A involved in the differentiation regulation.« less

The effect of magnetic stimulation on the osteogenic and chondrogenic differentiation of human stem cells derived from the adipose tissue (hASCs)

NASA Astrophysics Data System (ADS)

Lima, João; Gonçalves, Ana I.; Rodrigues, Márcia T.; Reis, Rui L.; Gomes, Manuela E.

2015-11-01

The use of magnetic nanoparticles (MNPs) towards the musculoskeletal tissues has been the focus of many studies, regarding MNPs ability to promote and direct cellular stimulation and orient tissue responses. This is thought to be mainly achieved by mechano-responsive pathways, which can induce changes in cell behavior, including the processes of proliferation and differentiation, in response to external mechanical stimuli. Thus, the application of MNP-based strategies in tissue engineering may hold potential to propose novel solutions for cell therapy on bone and cartilage strategies to accomplish tissue regeneration. The present work aims at studying the influence of MNPs on the osteogenic and chondrogenic differentiation of human adipose derived stem cells (hASCs). MNPs were incorporated in hASCs and cultured in medium supplemented for osteogenic and chondrogenic differentiation. Cultures were maintained up to 28 days with/without an external magnetic stimulus provided by a magnetic bioreactor, to determine if the MNPs alone could affect the osteogenic or chondrogenic phenotype of the hASCs. Results indicate that the incorporation of MNPs does not negatively affect the viability nor the proliferation of hASCs. Furthermore, Alizarin Red staining evidences an enhancement in extracellular (ECM) mineralization under the influence of an external magnetic field. Although not as evident as for osteogenic differentiation, Toluidine blue and Safranin-O stainings also suggest the presence of a cartilage-like ECM with glycosaminoglycans and proteoglycans under the magnetic stimulus provided. Thus, MNPs incorporated in hASCs under the influence of an external magnetic field have the potential to induce differentiation towards the osteogenic and chondrogenic lineages.

Noninvasive Monitoring of Three-Dimensional Chondrogenic Constructs Using Molecular Beacon Nanosensors.

PubMed

Tay, Li Min; Wiraja, Christian; Yeo, David C; Wu, Yingnan; Yang, Zheng; Chuah, Yon Jin; Lee, Eng Hin; Kang, Yuejun; Xu, Chenjie

2017-01-01

Chondrogenic differentiation of human mesenchymal stem cells (MSCs) in three-dimensional hydrogel holds promise as a method for repairing injured articular cartilage. Given MSC plasticity (its potential to mature into alternative lineages), nondestructive monitoring is critical for the optimization of chondrogenic differentiation conditions and the evaluation of the final product. However, conventional validation/assessments of the differentiation process (i.e., quantitative reverse transcription polymerase chain reaction [qRT-PCR] and histology) are end-point assays requiring disruption of the sample. This report introduces molecular beacon (MB)-based nanosensors to achieve noninvasive monitoring of chondrogenic differentiation. These nanosensors consist of biodegradable poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) encapsulating MBs to detect Type II Collagen (Col2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs that serve as lineage-specific and housekeeping biomarkers, respectively. The sustainable release of MBs from MB-NPs allows longitudinal monitoring of MSCs undergoing chondrogenic differentiation over a period of 28 days. Dual-colored MB loading ensures accurate assessment of Col2 mRNA expression level, where potential heterogeneity in nanosensor uptake and retention by MSCs are taken into account. When normalized nanosensor signal was compared against qRT-PCR result, a tight correlation was observed (R 2  = 0.9301). Finally, nanosensor usage was compatible with MSC potency with minimal influence on chondrogenic, adipogenic, and osteogenic differentiation.

Glycosaminoglycan-Mimetic Signals Direct the Osteo/Chondrogenic Differentiation of Mesenchymal Stem Cells in a Three-Dimensional Peptide Nanofiber Extracellular Matrix Mimetic Environment.

PubMed

Arslan, Elif; Guler, Mustafa O; Tekinay, Ayse B

2016-04-11

Recent efforts in bioactive scaffold development focus strongly on the elucidation of complex cellular responses through the use of synthetic systems. Designing synthetic extracellular matrix (ECM) materials must be based on understanding of cellular behaviors upon interaction with natural and artificial scaffolds. Hence, due to their ability to mimic both the biochemical and mechanical properties of the native tissue environment, supramolecular assemblies of bioactive peptide nanostructures are especially promising for development of bioactive ECM-mimetic scaffolds. In this study, we used glycosaminoglycan (GAG) mimetic peptide nanofiber gel as a three-dimensional (3D) platform to investigate how cell lineage commitment is altered by external factors. We observed that amount of fetal bovine serum (FBS) presented in the cell media had synergistic effects on the ability of GAG-mimetic nanofiber gel to mediate the differentiation of mesenchymal stem cells into osteogenic and chondrogenic lineages. In particular, lower FBS concentration in the culture medium was observed to enhance osteogenic differentiation while higher amount FBS promotes chondrogenic differentiation in tandem with the effects of the GAG-mimetic 3D peptide nanofiber network, even in the absence of externally administered growth factors. We therefore demonstrate that mesenchymal stem cell differentiation can be specifically controlled by the combined influence of growth medium components and a 3D peptide nanofiber environment.

Overexpression of TGF-β1 enhances chondrogenic differentiation and proliferation of human synovium-derived stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Yong Il; Ryu, Jae-Sung; Yeo, Jee Eun

2014-08-08

Highlights: • Continuous TGF-β1 overexpression in hSD-MSCs did not influence their phenotypes. • Retroviral-mediated transduction of TGFB1 in hSD-MSCs enhances cell proliferation. • TGF-β1 overexpression did not effect to adipo- or osteogenic potential of hSD-MSCs. • TGF-β1 overexpression in hSD-MSCs could stimulate and accelerate chondrogenesis. - Abstract: Transforming growth factor-beta (TGF-β) superfamily proteins play a critical role in proliferation, differentiation, and other functions of mesenchymal stem cells (MSCs). During chondrogenic differentiation of MSCs, TGF-β up-regulates chondrogenic gene expression by enhancing the expression of the transcription factor SRY (sex-determining region Y)-box9 (Sox9). In this study, we investigated the effect of continuousmore » TGF-β1 overexpression in human synovium-derived MSCs (hSD-MSCs) on immunophenotype, differentiation potential, and proliferation rate. hSD-MSCs were transduced with recombinant retroviruses (rRV) encoding TGF-β1. The results revealed that continuous overexpression of TGF-β1 did not affect their phenotype as evidenced by flow cytometry and reverse transcriptase PCR (RT-PCR). In addition, continuous TGF-β1 overexpression strongly enhanced cell proliferation of hSD-MSCs compared to the control groups. Also, induction of chondrogenesis was more effective in rRV-TGFB-transduced hSD-MSCs as shown by RT-PCR for chondrogenic markers, toluidine blue staining and glycosaminoglycan (GAG)/DNA ratio. Our data suggest that overexpression of TGF-β1 positively enhances the proliferation and chondrogenic potential of hSD-MSCs.« less

Monomeric, porous type II collagen scaffolds promote chondrogenic differentiation of human bone marrow mesenchymal stem cells in vitro

NASA Astrophysics Data System (ADS)

Tamaddon, M.; Burrows, M.; Ferreira, S. A.; Dazzi, F.; Apperley, J. F.; Bradshaw, A.; Brand, D. D.; Czernuszka, J.; Gentleman, E.

2017-03-01

Osteoarthritis (OA) is a common cause of pain and disability and is often associated with the degeneration of articular cartilage. Lesions to the articular surface, which are thought to progress to OA, have the potential to be repaired using tissue engineering strategies; however, it remains challenging to instruct cell differentiation within a scaffold to produce tissue with appropriate structural, chemical and mechanical properties. We aimed to address this by driving progenitor cells to adopt a chondrogenic phenotype through the tailoring of scaffold composition and physical properties. Monomeric type-I and type-II collagen scaffolds, which avoid potential immunogenicity associated with fibrillar collagens, were fabricated with and without chondroitin sulfate (CS) and their ability to stimulate the chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells was assessed. Immunohistochemical analyses showed that cells produced abundant collagen type-II on type-II scaffolds and collagen type-I on type-I scaffolds. Gene expression analyses indicated that the addition of CS - which was released from scaffolds quickly - significantly upregulated expression of type II collagen, compared to type-I and pure type-II scaffolds. We conclude that collagen type-II and CS can be used to promote a more chondrogenic phenotype in the absence of growth factors, potentially providing an eventual therapy to prevent OA.

The Distinct Effects of Estrogen and Hydrostatic Pressure on Mesenchymal Stem Cells Differentiation: Involvement of Estrogen Receptor Signaling.

PubMed

Zhao, Ying; Yi, Fei-Zhou; Zhao, Yin-Hua; Chen, Yong-Jin; Ma, Heng; Zhang, Min

2016-10-01

This study aimed to investigate the differential and synergistic effects of mechanical stimulation and estrogen on the proliferation and osteogenic or chondrogenic differentiation potential of bone marrow mesenchymal stem cells (BMSCs) and the roles of estrogen receptor (ER) in them. BMSCs were isolated and cultured using the whole bone marrow adherence method, and flow cytometry was used to identify the surface marker molecules of BMSCs. Cells were pre-treated with 1 nM 17β-estradiol or 1 nM of the estrogen receptor antagonist tamoxifen. Then, the cells were stimulated with hydrostatic pressure. Assessment included flow cytometry analysis of the cell cycle; immunofluorescent staining for F-actin; protein quantification for MAPK protein; and mRNA analysis for Col I, OCN, OPN and BSP after osteogenic induction and Sox-9, Aggrecan and Col-II after chondrogenic induction. Hydrostatic pressure (90 kPa/1 h) and 1 nM 17β-estradiol enhanced the cellular proliferation ability and the cytoskeleton activity but without synergistic biological effects. Estrogen activated ERKs and JNKs simultaneously and promoted the osteogenic differentiation, whereas the pressure just caused JNK-1/2 activation and promoted the chondrogenic differentiation of BMSCs. Estrogen had antagonism effect on chondrogenic promotion of hydrostatic pressure. Mechanobiological effects of hydrostatic pressure are closely associated with ERα activity. MAPK molecules and F-actin were likely to be important mediator molecules in the ER-mediated mechanotransduction of BMSCs.

Second-harmonic generation microscopy used to evaluate the effect of the dimethyl sulfoxide in the cryopreservation process in collagen fibers of differentiated chondrocytes

NASA Astrophysics Data System (ADS)

Andreoli-Risso, M. F.; Duarte, A. S. S.; Ribeiro, T. B.; Bordeaux-Rego, P.; Luzo, A.; Baratti, M. O.; Adur, J.; de Thomaz, A. A.; Pelegati, V. B.; Carvalho, H. F.; Cesar, C. L.; Kharmadayan, P.; Costa, F. F.; Olalla-Saad, S. T.

2012-03-01

Cartilaginous lesions are a significant public health problem and the use of adult stem cells represents a promising therapy for this condition. Cryopreservation confers many advantages for practitioners engaged in cell-based therapies. However, conventional slow freezing has always been associated with damage and mortality due to intracellular ice formation, cryoprotectant toxicity, and dehydration. The aim of this work is to observe the effect of the usual Dimethyl Sulfoxide (DMSO) cryopreservation process on the architecture of the collagen fiber network of chondrogenic cells from mesenchymal stem cells by Second Harmonic Generation (SHG) microscopy. To perform this study we used Mesenchymal Stem Cells (MSC) derived from adipose tissue which presents the capacity to differentiate into other lineages such as osteogenic, adipogenic and chondrogenic lineages. Mesenchymal stem cells obtained after liposuction were isolated digested by collagenase type I and characterization was carried out by differentiation of mesodermic lineages, and flow cytometry using specific markers. The isolated MSCs were cryopreserved by the DMSO technique and the chondrogenic differentiation was carried out using the micromass technique. We then compared the cryopreserved vs non-cryopreserved collagen fibers which are naturally formed during the differentiation process. We observed that noncryopreserved MSCs presented a directional trend in the collagen fibers formed which was absent in the cryopreserved MSCs. We confirmed this trend quantitatively by the aspect ratio obtained by Fast Fourier Transform which was 0.76 for cryopreserved and 0.52 for non-cryopreserved MSCs, a statistical significant difference.

The synergistic effects of shear stress and cyclic hydrostatic pressure modulate chondrogenic induction of human mesenchymal stem cells.

PubMed

Hosseini, Motahare-Sadat; Tafazzoli-Shadpour, Mohammad; Haghighipour, Nooshin; Aghdami, Naser; Goodarzi, Alireza

2015-10-01

In this study, we examined chondrogenic regulation of 2 types of mesenchymal stem cells seeded on the bioengineered substrate in monolayer cultures under mechanically defined conditions to mimic the in vivo microenvironment of chondrocytes within articular cartilage tissues. Human adipose-derived mesenchymal stem cells (ASCs) and bone marrow mesenchymal stem cells (BSCs) were exposed to 0.2 Pa shear stress, 3 MPa cyclic hydrostatic pressure, and combined loading with different sequences on chemically designed medical-grade silicone rubber, while no soluble growth factors were added to the culture medium. The expression levels of chondrogenic-specific genes of SOX9, aggrecan, and type II collagen (Col II) were measured. Results were compared to those of cells treated by biological growth factor. Gene expression patterns were dependent on the loading regime. Moreover, the source of mesenchymal stem cells (adipose or bone marrow) was influential in gene expression. Overall, enhanced expression of chondrogenic markers was found through application of mechanical stimuli. The response was generally found to be significantly promoted when the 2 loading regimes were superimposed. Differentiation of ASCs was shown by a modest increase in gene expression profiles. In general, BSCs expressed higher levels of chondrogenic gene expression than ASCs after 3 weeks. A greater effect on Col II and SOX9 mRNA expression was observed when combined loadings were applied. Results may be applied in determining the proper loading sequence for obtaining functional target cells in cartilage engineering applications.

Impact of low oxygen tension on stemness, proliferation and differentiation potential of human adipose-derived stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Jane Ru; Pingguan-Murphy, Belinda; Wan Abas, Wan Abu Bakar

2014-05-30

Highlights: • Hypoxia maintains the stemness of adipose-derived stem cells (ASCs). • ASCs show an increased proliferation rate under low oxygen tension. • Oxygen level as low as 2% enhances the chondrogenic differentiation potential of ASCs. • HIF-1α may regulate the proliferation and differentiation activities of ASCs under hypoxia. - Abstract: Adipose-derived stem cells (ASCs) have been found adapted to a specific niche with low oxygen tension (hypoxia) in the body. As an important component of this niche, oxygen tension has been known to play a critical role in the maintenance of stem cell characteristics. However, the effect of O{submore » 2} tension on their functional properties has not been well determined. In this study, we investigated the effects of O{sub 2} tension on ASCs stemness, differentiation and proliferation ability. Human ASCs were cultured under normoxia (21% O{sub 2}) and hypoxia (2% O{sub 2}). We found that hypoxia increased ASC stemness marker expression and proliferation rate without altering their morphology and surface markers. Low oxygen tension further enhances the chondrogenic differentiation ability, but reduces both adipogenic and osteogenic differentiation potential. These results might be correlated with the increased expression of HIF-1α under hypoxia. Taken together, we suggest that growing ASCs under 2% O{sub 2} tension may be important in expanding ASCs effectively while maintaining their functional properties for clinical therapy, particularly for the treatment of cartilage defects.« less

Hyaluronan influence on the onset of chondrogenic differentiation of mesenchymal stem cells.

PubMed

Dvorakova, Jana; Velebny, Vladimir; Kubala, Lukas

2008-10-01

Hyaluronan (HA) is an abundant component of chondrogenic tissue hence it is often used as a fundamental constituent in cartilage tissue substitutes. However, effects of different molecular weight HA on chondrogenic differentiation are not clear. The aim was to evaluate modulation of mesenchymal stem cell (MSC) early chondrogenesis by HA of molecular weights 100, 600 and 1 500 kDa. HA was applied on MSCs cultured in a pellet system for one, two and three weeks. Chondrogenesis was evaluated by determinations of gene expression of transcription factor Sox-9 and extracellular matrix proteins collagen type II and XI, aggrecan, and COMP by Real-Time PCR and completed with histological analysis. Upon chondrogenic induction, the respective pellets revealed active transcription of the chondrogenic genes together with proceeding accumulation of glycosaminoglycan (GAG) rich extracellular matrix. Sox-9 was also expressed in non-chondrogenic MSC controls. HA treated pellets were not significantly influenced on day 7 of culture. However, on day 14, lowered expression in some of the extracellular matrix proteins appeared together with a moderately smaller amount of GAG content in pellet sections. Nevertheless, the analysis on day 21 has demonstrated that HA did not affect the outcome of the differentiation by the end of the culture. Any difference regarding the molecular weight of the HA was not found. It could be speculated that HA induced a time shift in the phase of the dominant matrix protein onset which was in full compensated by the end of the evaluated time period. Thus, data suggest that HA of any tested molecular weight does not significantly modulate chondrogenesis of MSCs in pellet system.

Responses to altered oxygen tension are distinct between human stem cells of high and low chondrogenic capacity.

PubMed

Anderson, Devon E; Markway, Brandon D; Bond, Derek; McCarthy, Helen E; Johnstone, Brian

2016-10-20

Lowering oxygen from atmospheric level (hyperoxia) to the physiological level (physioxia) of articular cartilage promotes mesenchymal stem cell (MSC) chondrogenesis. However, the literature is equivocal regarding the benefits of physioxic culture on preventing hypertrophy of MSC-derived chondrocytes. Articular cartilage progenitors (ACPs) undergo chondrogenic differentiation with reduced hypertrophy marker expression in hyperoxia but have not been studied in physioxia. This study sought to delineate the effects of physioxic culture on both cell types undergoing chondrogenesis. MSCs were isolated from human bone marrow aspirates and ACP clones were isolated from healthy human cartilage. Cells were differentiated in pellet culture in physioxia (2 % oxygen) or hyperoxia (20 % oxygen) over 14 days. Chondrogenesis was characterized by biochemical assays and gene and protein expression analysis. MSC preparations and ACP clones of high intrinsic chondrogenicity (termed high-GAG) produced abundant matrix in hyperoxia and physioxia. Poorly chondrogenic cells (low-GAG) demonstrated a significant fold-change matrix increase in physioxia. Both high-GAG and low-GAG groups of MSCs and ACPs significantly upregulated chondrogenic genes; however, only high-GAG groups had a concomitant decrease in hypertrophy-related genes. High-GAG MSCs upregulated many common hypoxia-responsive genes in physioxia while low-GAG cells downregulated most of these genes. In physioxia, high-GAG MSCs and ACPs produced comparable type II collagen but less type I collagen than those in hyperoxia. Type X collagen was detectable in some ACP pellets in hyperoxia but reduced or absent in physioxia. In contrast, type X collagen was detectable in all MSC preparations in hyperoxia and physioxia. MSC preparations and ACP clones had a wide range of chondrogenicity between donors. Physioxia significantly enhanced the chondrogenic potential of both ACPs and MSCs compared with hyperoxia, but the magnitude of response was inversely related to intrinsic chondrogenic potential. Discrepancies in the literature regarding MSC hypertrophy in physioxia can be explained by the use of low numbers of preparations of variable chondrogenicity. Physioxic differentiation of MSC preparations of high chondrogenicity significantly decreased hypertrophy-related genes but still produced type X collagen protein. Highly chondrogenic ACP clones had significantly lower hypertrophic gene levels, and there was little to no type X collagen protein in physioxia, emphasizing the potential advantage of these cells.

Comparison of Osteogenic and Chondrogenic Differentiation Ability of Buccal Fat Pad Derived Mesenchymal Stem Cells and Gingival Derived Cells.

PubMed

Ghaderi, Hamid; Razmkhah, Mahboobeh; Kiany, Farin; Chenari, Nooshafarin; Haghshenas, Mohammad Reza; Ghaderi, Abbas

2018-06-01

One major goal of tissue engineering and regenerative medicine is to find an appropriate source of mesenchymal stem cells (MSCs) with higher differentiation ability. In this experimental study, the osteogenic and chondrogenic differentiation ability of buccal fat pad derived MSCs (BFP-MSCs) with gingival derived cells (GDCs) were compared. BFP-MSCs and GDCs were cultured enzymatically and expanded. The expanded cells were analyzed for membrane-associated markers, using flow cytometry. Then the ability of these cells to differentiate into osteocyte and chondrocyte was assessed morphologically and by mRNA expression of collagen I (COLL), BGLA and bone morphogenetic protein 2 (BMP2) using qRT-PCR. Flow cytometry analysis showed that both BFP-MSCs and GDCs expressed the characteristic stem cell markers such as CD73, CD44, and CD90, whereas they did not express hematopoietic markers. Mineralized calcium deposition was observed apparently in BFP-MSCs cultured in osteogenic medium but GDCs showed fewer mineralized nodules. The mRNA expression levels of BGLA and BMP2 showed 7×105 and 733-fold more mRNA expression in BFP-MSCs treated with differentiation media compared to the control group. In chondrogenic differentiation, BFP-MSCs transformed from a spindle to a cuboidal shape while GDCs showed only a slight transformation. In addition, mRNA expression of COLL showed 282-fold higher expression in BFP-MSCs in comparison to the control group. Such significant difference in mRNA expression of BGLA, BMP2, and COLL was not observed in GDCs compared to their corresponding controls. Based on the present results, BFP yields a greater proportion of stem cells compared to gingiva. Therefore, this tissue can be introduced as an easily available source for the treatment of periodontal defects and other maxillofacial injuries.

Evaluation of the maintenance of stemness, viability, and differentiation potential of gingiva-derived stem-cell spheroids.

PubMed

Lee, Sung-Il; Ko, Youngkyung; Park, Jun-Beom

2017-05-01

Gingiva-derived stem cells have been applied for tissue-engineering purposes and may be considered a favorable source of mesenchymal stem cells as harvesting stem cells from the mandible or maxilla may be performed with ease under local anesthesia. The present study was performed to fabricate stem-cell spheroids using concave microwells and to evaluate the maintenance of stemness, viability, and differentiation potential. Gingiva-derived stem cells were isolated, and the stem cells of 4×10 5 (group A) or 8×10 5 (group B) cells were seeded into polydimethylsiloxane-based, concave micromolds with 600 µm diameters. The morphology of the microspheres and the change of the diameters of the spheroids were evaluated. The viability of spheroids was qualitatively analyzed via Live/Dead kit assay. A cell viability analysis was performed on days 1, 3, 6, and 12 with Cell Counting Kit-8. The maintenance of stemness was evaluated with immunocytochemical staining using SSEA-4, TRA-1-60(R) (positive markers), and SSEA-1 (negative marker). Osteogenic, adipogenic, and chondrogenic differentiation potential was evaluated by incubating spheroids in osteogenic, adipogenic and chondrogenic induction medium, respectively. The gingiva-derived stem cells formed spheroids in the concave microwells. The diameters of the spheroids were larger in group A than in group B. The majority of cells in the spheroids emitted green fluorescence, indicating the presence of live cells at day 6. At day 12, the majority of cells in the spheroids emitted green fluorescence, and a small portion of red fluorescence was also noted, which indicated the presence of dead cells. The spheroids were positive for the stem-cell markers SSEA-4 and TRA-1-60(R) and were negative for SSEA-1, suggesting that these spheroids primarily contained undifferentiated human stem cells. Osteogenic, adipogenic, and chondrogenic differentiation was more evident with an increase of incubation time: Mineralized extracellular deposits were observed following Alizarin Red S staining at days 14 and 21; oil globules were increased at day 18 when compared with day 6; and Alcian blue staining was more evident at day 18 when compared with day 6. Within the limits of this study, stem-cell spheroids from gingival cells maintained the stemness, viability, and differentiation potential during the experimental periods. This method may be applied for a promising strategy for stem-cell therapy.

Chondrogenesis, osteogenesis and adipogenesis of canine mesenchymal stem cells: a biochemical, morphological and ultrastructural study.

PubMed

Csaki, C; Matis, U; Mobasheri, A; Ye, H; Shakibaei, M

2007-12-01

Musculoskeletal diseases with osteochondrotic articular cartilage defects, such as osteoarthritis, are an increasing problem for humans and companion animals which necessitates the development of novel and improved therapeutic strategies. Canine mesenchymal stem cells (cMSCs) offer significant promise as a multipotent source for cell-based therapies and could form the basis for the differentiation and cultivation of tissue grafts to replace damaged tissue. However, no comprehensive analysis has been undertaken to characterize the ultrastructure of in vitro differentiated cMSCs. The main goal of this paper was to focus on cMSCs and to analyse their differentiation capacity. To achieve this aim, bone marrow cMSCs from three canine patients were isolated, expanded in monolayer culture and characterized with respect to their ability for osteogenic, adipogenic and chondrogenic differentiation capacities. cMSCs showed proliferative potential and were capable of osteogenic, adipogenic and chondrogenic differentiation. cMSCs treated with the osteogenic induction medium differentiated into osteoblasts, produced typical bone matrix components, beta1-integrins and upregulated the osteogenic specific transcription factor Cbfa-1. cMSCs treated with the adipogenic induction medium showed typical adipocyte morphology, produced adiponectin, collagen type I and beta1-integrins, and upregulated the adipogenic specific transcription factor PPAR-gamma. cMSCs treated with the chondrogenic induction medium exhibited a round to oval shape, produced a cartilage-specific extracellular matrix, beta1-integrins and upregulated the chondrogenic specific transcription factor Sox9. These results demonstrate, at the biochemical, morphological and ultrastructural levels, the multipotency of cMSCs and thus highlight their potential therapeutic value for cell-based tissue engineering.

The Effects of Cyclic Hydrostatic Pressure on Chondrogenesis and Viability of Human Adipose- and Bone Marrow-Derived Mesenchymal Stem Cells in Three-Dimensional Agarose Constructs

PubMed Central

Puetzer, Jennifer; Williams, John; Gillies, Allison; Bernacki, Susan

2013-01-01

This study investigates the effects of cyclic hydrostatic pressure (CHP) on chondrogenic differentiation of human adipose-derived stem cells (hASCs) in three-dimensional (3-D) agarose constructs maintained in a complete growth medium without soluble chondrogenic inducing factors. hASCs were seeded in 2% agarose hydrogels and exposed to 7.5 MPa CHP for 4 h per day at a frequency of 1 Hz for up to 21 days. On days 0, 7, 14, and 21, the expression levels of collagen II, Sox9, aggrecan, and cartilage oligomeric matrix protein (COMP) were examined by real-time reverse transcriptase–polymerase chain reaction analysis. Gene expression analysis found collagen II mRNA expression in only the CHP-loaded construct at day 14 and at no other time during the study. CHP-loaded hASCs exhibited upregulated mRNA expression of Sox9, aggrecan, and COMP at day 7 relative to unloaded controls, suggesting that CHP initiated chondrogenic differentiation of hASCs in a manner similar to human bone marrow-derived mesenchymal stem cells (hMSC). By day 14, however, loaded hASC constructs exhibited significantly lower mRNA expression of the chondrogenic markers than unloaded controls. Additionally, by day 21, the samples exhibited little measurable mRNA expression at all, suggesting a decreased viability. Histological analysis validated the lack of mRNA expression at day 21 for both the loaded and unloaded control samples with a visible decrease in the cell number and change in morphology. A comparative study with hASCs and hMSCs further examined long-term cell viability in 3-D agarose constructs of both cell types. Decreased cell metabolic activity was observed throughout the 21-day experimental period in both the CHP-loaded and control constructs of both hMSCs and hASCs, suggesting a decrease in cell metabolic activity, alluding to a decrease in cell viability. This suggests that a 2% agarose hydrogel may not optimally support hASC or hMSC viability in a complete growth medium in the absence of soluble chondrogenic inducing factors over long culture durations. This is the first study to examine the ability of mechanical stimuli alone, in the absence of chondrogenic factors transforming growth factor beta (TGF-β)3, TGF-β1 and/or bone morphogenetic protein 6 (BMP6) to induce hASC chondrogenic differentiation. The findings of this study suggest that CHP initiates hASC chondrogenic differentiation, even in the absence of soluble chondrogenic inductive factors, confirming the importance of considering both mechanical stimuli and appropriate 3-D culture for cartilage tissue engineering using hASCs. PMID:22871265

Isolation of adipose derived stem cells and their induction to a chondrogenic phenotype

PubMed Central

Estes, Bradley T.; Diekman, Brian O.; Gimble, Jeffrey M.; Guilak, Farshid

2011-01-01

Summary The ability to isolate, expand, and differentiate adult stem cells into a chondrogenic lineage is an important step in the development of tissue engineering approaches for cartilage repair or regeneration for the treatment of joint injury or osteoarthritis, or for application in plastic or reconstructive surgery. Adipose-derived stem cells (ASCs) provide an abundant and easily accessible source of adult stem cells for use in such regenerative approaches. This protocol describes the isolation of ASCs from liposuction aspirate, as well as cell culture conditions for growth factor based induction of ASCs into chondrocyte-like cells. These methods are similar to those used for bone marrow mesenchymal stem cells but distinct due to the unique properties of ASCs. Investigators can expect consistent ASC differentiation, allowing for slight variation due to donor and serum lot effects. Approximately 10–12 weeks are needed for ASC isolation and the characterization of chondrocyte-like cells, which is also described. PMID:20595958

Chondrogenic properties of collagen type XI, a component of cartilage extracellular matrix.

PubMed

Li, Ang; Wei, Yiyong; Hung, Clark; Vunjak-Novakovic, Gordana

2018-08-01

Cartilage extracellular matrix (ECM) has been used for promoting tissue engineering. However, the exact effects of ECM on chondrogenesis and the acting mechanisms are not well understood. In this study, we investigated the chondrogenic effects of cartilage ECM on human mesenchymal stem cells (MSCs) and identified the contributing molecular components. To this end, a preparation of articular cartilage ECM was supplemented to pellets of chondrogenically differentiating MSCs, pellets of human chondrocytes, and bovine articular cartilage explants to evaluate the effects on cell proliferation and the production of cartilaginous matrix. Selective enzymatic digestion and screening of ECM components were conducted to identify matrix molecules with chondrogenic properties. Cartilage ECM promoted MSC proliferation, production of cartilaginous matrix, and maturity of chondrogenic differentiation, and inhibited the hypertrophic differentiation of MSC-derived chondrocytes. Selective digestion of ECM components revealed a contributory role of collagens in promoting chondrogenesis. The screening of various collagen subtypes revealed strong chondrogenic effect of collagen type XI. Finally, collagen XI was found to promote production and inhibit degradation of cartilage matrix in human articular chondrocyte pellets and bovine articular cartilage explants. Our results indicate that cartilage ECM promotes chondrogenesis and inhibits hypertrophic differentiation in MSCs. Collagen type XI is the ECM component that has the strongest effects on enhancing the production and inhibiting the degradation of cartilage matrix. Copyright © 2018 Elsevier Ltd. All rights reserved.

Dose-response of superparamagnetic iron oxide labeling on mesenchymal stem cells chondrogenic differentiation: a multi-scale in vitro study.

PubMed

Roeder, Emilie; Henrionnet, Christel; Goebel, Jean Christophe; Gambier, Nicolas; Beuf, Olivier; Grenier, Denis; Chen, Bailiang; Vuissoz, Pierre-André; Gillet, Pierre; Pinzano, Astrid

2014-01-01

The aim of this work was the development of successful cell therapy techniques for cartilage engineering. This will depend on the ability to monitor non-invasively transplanted cells, especially mesenchymal stem cells (MSCs) that are promising candidates to regenerate damaged tissues. MSCs were labeled with superparamagnetic iron oxide particles (SPIO). We examined the effects of long-term labeling, possible toxicological consequences and the possible influence of progressive concentrations of SPIO on chondrogenic differentiation capacity. No influence of various SPIO concentrations was noted on human bone marrow MSC viability or proliferation. We demonstrated long-term (4 weeks) in vitro retention of SPIO by human bone marrow MSCs seeded in collagenic sponges under TGF-β1 chondrogenic conditions, detectable by Magnetic Resonance Imaging (MRI) and histology. Chondrogenic differentiation was demonstrated by molecular and histological analysis of labeled and unlabeled cells. Chondrogenic gene expression (COL2A2, ACAN, SOX9, COL10, COMP) was significantly altered in a dose-dependent manner in labeled cells, as were GAG and type II collagen staining. As expected, SPIO induced a dramatic decrease of MRI T2 values of sponges at 7T and 3T, even at low concentrations. This study clearly demonstrates (1) long-term in vitro MSC traceability using SPIO and MRI and (2) a deleterious dose-dependence of SPIO on TGF-β1 driven chondrogenesis in collagen sponges. Low concentrations (12.5-25 µg Fe/mL) seem the best compromise to optimize both chondrogenesis and MRI labeling.

rFN/Cad-11-Modified Collagen Type II Biomimetic Interface Promotes the Adhesion and Chondrogenic Differentiation of Mesenchymal Stem Cells

PubMed Central

Guo, Hongfeng; Zhang, Yuan; Li, Zhengsheng; Kang, Fei; Yang, Bo; Kang, Xia; Wen, Can; Yan, Yanfei; Jiang, Bo; Fan, Yujiang

2013-01-01

Properties of the cell-material interface are determining factors in the successful function of cells for cartilage tissue engineering. Currently, cell adhesion is commonly promoted through the use of polypeptides; however, due to their lack of complementary or modulatory domains, polypeptides must be modified to improve their ability to promote adhesion. In this study, we utilized the principle of matrix-based biomimetic modification and a recombinant protein, which spans fragments 7–10 of fibronectin module III (heterophilic motif ) and extracellular domains 1–2 of cadherin-11 (rFN/Cad-11) (homophilic motif ), to modify the interface of collagen type II (Col II) sponges. We showed that the designed material was able to stimulate cell proliferation and promote better chondrogenic differentiation of rabbit mesenchymal stem cells (MSCs) in vitro than both the FN modified surfaces and the negative control. Further, the Col II/rFN/Cad-11-MSCs composite stimulated cartilage formation in vivo; the chondrogenic effect of Col II alone was much less significant. These results suggested that the rFN/Cad-11-modified collagen type II biomimetic interface has dual biological functions of promoting adhesion and stimulating chondrogenic differentiation. This substance, thus, may serve as an ideal scaffold material for cartilage tissue engineering, enhancing repair of injured cartilage in vivo. PMID:23919505

Investigation of the optimal timing for chondrogenic priming of MSCs to enhance osteogenic differentiation in vitro as a bone tissue engineering strategy.

PubMed

Freeman, F E; Haugh, M G; McNamara, L M

2016-04-01

Recent in vitro tissue engineering approaches have shown that chondrogenic priming of human bone marrow mesenchymal stem cells (MSCs) can have a positive effect on osteogenesis in vivo. However, whether chondrogenic priming is an effective in vitro bone regeneration strategy is not yet known. In particular, the appropriate timing for chondrogenic priming in vitro is unknown albeit that in vivo cartilage formation persists for a specific period before bone formation. The objective of this study is to determine the optimum time for chondrogenic priming of MSCs to enhance osteogenic differentiation by MSCs in vitro. Pellets derived from murine and human MSCs were cultured in six different media groups: two control groups (chondrogenic and osteogenic) and four chondrogenic priming groups (10, 14, 21 and 28 days priming). Biochemical analyses (Hoechst, sulfate glycosaminoglycan (sGAG), Alkaline Phosphate (ALP), calcium), histology (Alcian Blue, Alizarin Red) and immunohistochemistry (collagen types I, II and X) were performed on the samples at specific times. Our results show that after 49 days the highest amount of sGAG production occurred in MSCs chondrogenically primed for 21 days and 28 days. Moreover we found that chondrogenic priming of MSCs in vitro for specific amounts of time (14 days, 21 days) can have optimum influence on their mineralization capacity and can produce a construct that is mineralized throughout the core. Determining the optimum time for chondrogenic priming to enhance osteogenic differentiation in vitro provides information that might lead to a novel regenerative treatment for large bone defects, as well as addressing the major limitation of core degradation and construct failure. Copyright © 2013 John Wiley & Sons, Ltd.

* Hypoxia for Mesenchymal Stem Cell Expansion and Differentiation: The Best Way for Enhancing TGFß-Induced Chondrogenesis and Preventing Calcifications in Alginate Beads.

PubMed

Henrionnet, Christel; Liang, Gai; Roeder, Emilie; Dossot, Manuel; Wang, Hui; Magdalou, Jacques; Gillet, Pierre; Pinzano, Astrid

2017-09-01

We examined the respective influence of a sequential or a continuous hypoxia during expansion and transforming growth factor beta 1-driven chondrogenic differentiation of human bone marrow mesenchymal stem cells (MSCs). The differentiation was performed within alginate beads, a classical tool for the implantation of MSCs within the joint. The standard normoxic 2D (expansion) and 3D (differentiation) MSCs cultures served as reference. To determine the quality of chondrogenesis, we analyzed typical markers such as type II and X collagens, SOX9, COMP, versican, and aggrecan mRNAs using polymerase chain reaction and we assessed the production of type II collagen and hypoxia-inducible factor (HIF)-1α by histological stainings. We simultaneously assessed the expression of osteogenic mRNAs (Alkaline Phosphatase, RUNX2, and Osteocalcin) and the presence of micro-calcifications by Alizarin red and Raman spectroscopy. Chondrogenic differentiation is clearly improved by hypoxia in 3D. Best results were obtained when the entire process, that is, 2D expansion and 3D differentiation, was performed under continuous 5% hypoxic condition. In addition, no calcification (hydroxyapatite, proved by RAMAN) was observed after 2D hypoxic expansion even in the case of a normoxic differentiation, in contrast with controls. Finally, a better chondrogenic differentiation of human MSCs is achieved when a reduced oxygen tension is applied during both expansion and differentiation times, avoiding in vitro osteogenic commitment of cells and subsequently the calcification deposition.

Mesenchymal stem cell differentiation in an experimental cartilage defect: restriction of hypertrophy to bone-close neocartilage.

PubMed

Steck, Eric; Fischer, Jennifer; Lorenz, Helga; Gotterbarm, Tobias; Jung, Martin; Richter, Wiltrud

2009-09-01

Mesenchymal stem cells (MSCs) are promising for the treatment of articular cartilage defects; however, common protocols for in vitro chondrogenesis induce typical features of hypertrophic chondrocytes reminiscent of endochondral bone formation. Aim of the study was to compare chondrogenic differentiation of MSCs in vitro and in vivo in experimental full-thickness cartilage defects, asking whether MSCs can differentiate into collagen type X-negative chondrocytes in an orthotopic environment. Cartilage defects in knees of minipigs were covered with a collagen type I/III membrane, and half of them received transplantation of expanded autologous MSCs. At 1, 3, and 8 weeks, morphological and molecular aspects of repair were assessed. The orthotopic environment triggered a spatially organized repair tissue with upper fibrous, intermediate chondrogenic, and low layer hypertrophic differentiation of cells and a trend to more safranin-O and collagen type II-positive samples after MSC transplantation at 8 weeks. Compared to in vitro chondrogenesis, significant lower COL10A1/COL2A1 and MMP13/COL2A1 ratios were obtained for in vivo differentiation. This indicates that, as opposed to in vitro chondrogenic induction of MSCs, the in vivo signaling molecules and biomechanical stimuli provide an appropriate environment for progenitor cells to differentiate into collagen type X-negative chondrocytes. Thus, until better in vitro induction protocols become available for chondrogenesis of MSCs, their predifferentiation before transplantation may be unfavorable.

Interleukin-6 inhibits early differentiation of ATDC5 chondrogenic progenitor cells.

PubMed

Nakajima, Shoko; Naruto, Takuya; Miyamae, Takako; Imagawa, Tomoyuki; Mori, Masaaki; Nishimaki, Shigeru; Yokota, Shumpei

2009-08-01

Interleukin (IL)-6 is a causative agent of systemic juvenile idiopathic arthritis (sJIA), a chronic inflammatory disease complicated with severe growth impairment. Recent trials of anti-IL-6 receptor monoclonal antibody, tocilizumab, indicated that tocilizumab blocks IL-6/IL-6 receptor-mediated inflammation, and induces catch-up growth in children with sJIA. This study evaluates the effects of IL-6 on chondrogenesis by ATDC5 cells, a clonal murine chondrogenic cell line that provides an excellent model for studying endochondral ossification at growth plate. ATDC5 cells were examined for the expression of IL-6 receptor and gp130 by fluorescence-activated cell sorting analysis. Recombinant murine IL-6 was added to ATDC5 cultures to observe cell differentiation, using a quantitative RT-PCR for the chondrogenic differentiation markers type II collagen, aggrecan, and type X collagen. To block IL-6, the anti-mouse IL-6 receptor monoclonal antibody MR16-1 was added. As a result, the cells expressed IL-6 receptor and gp130. The expression of chondrogenic differentiation marker gene was reduced by IL-6, but this was abrogated by MR16-1. We conclude that IL-6 inhibits early chondrogenesis of ATDC5 cells suggesting that IL-6 may affect committed stem cells at a cellular level during chondrogenic differentiation of growth plate chondrocytes, and that IL-6 may be a cellular-level factor in growth impairment in sJIA.

Murine pluripotent stem cells derived scaffold-free cartilage grafts from a micro-cavitary hydrogel platform.

PubMed

He, Pengfei; Fu, Jiayin; Wang, Dong-An

2016-04-15

By means of appropriate cell type and scaffold, tissue-engineering approaches aim to construct grafts for cartilage repair. Pluripotent stem cells especially induced pluripotent stem cells (iPSCs) are of promising cell candidates due to the pluripotent plasticity and abundant cell source. We explored three dimensional (3D) culture and chondrogenesis of murine iPSCs (miPSCs) on an alginate-based micro-cavity hydrogel (MCG) platform in pursuit of fabricating synthetic-scaffold-free cartilage grafts. Murine embryonic stem cells (mESCs) were employed in parallel as the control. Chondrogenesis was fulfilled using a consecutive protocol via mesoderm differentiation followed by chondrogenic differentiation; subsequently, miPSC and mESC-seeded constructs were further respectively cultured in chondrocyte culture (CC) medium. Alginate phase in the constructs was then removed to generate a graft only comprised of induced chondrocytic cells and cartilaginous extracellular matrix (ECMs). We found that from the mESC-seeded constructs, formation of intact grafts could be achieved in greater sizes with relatively fewer chondrocytic cells and abundant ECMs; from miPSC-seeded constructs, relatively smaller sized cartilaginous grafts could be formed by cells with chondrocytic phenotype wrapped by abundant and better assembled collagen type II. This study demonstrated successful creation of pluripotent stem cells-derived cartilage/chondroid graft from a 3D MCG interim platform. By the support of materials and methodologies established from this study, particularly given the autologous availability of iPSCs, engineered autologous cartilage engraftment may be potentially fulfilled without relying on the limited and invasive autologous chondrocytes acquisition. In this study, we explored chondrogenic differentiation of pluripotent stem cells on a 3D micro-cavitary hydrogel interim platform and creation of pluripotent stem cells-derived cartilage/chondroid graft via a consecutive procedure. Our results demonstrated chondrogenic differentiation could be realized on the platform via mesoderm differentiation. The mESCs/miPSCs derived chondrocytic cells were further cultured to finally generate a pluripotent stem cells-derived scaffold-free construct based on the micro-cavitary hydrogel platform, in which alginate hydrogel could be removed finally. Our results showed that miPSC-derived graft could be formed by cells with chondrocytic phenotype wrapped by abundant and assembled collagen type II. To our knowledge, this study is the first study that initials from pluripotent stem cell seeding on 3D scaffold environment and ends with a scaffold-free chondrogenic micro-tissue. By the support of materials and methodologies established from this study, engineered autologous iPSC-derived cartilage engraftment may be potentially developed instead of autologous chondrocytes grafts that have limited source. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

In vitro chondrogenic commitment of human Wharton's jelly stem cells by co-culture with human articular chondrocytes.

PubMed

Pereira, R C; Costa-Pinto, A R; Frias, A M; Neves, N M; Azevedo, H S; Reis, R L

2017-06-01

Wharton's jelly stem cells (WJSCs) are a potential source of transplantable stem cells in cartilage-regenerative strategies, due to their highly proliferative and multilineage differentiation capacity. We hypothesized that a non-direct co-culture system with human articular chondrocytes (hACs) could enhance the potential chondrogenic phenotype of hWJSCs during the expansion phase compared to those expanded in monoculture conditions. Primary hWJSCs were cultured in the bottom of a multiwell plate separated by a porous transwell membrane insert seeded with hACs. No statistically significant differences in hWJSCs duplication number were observed under either of the culture conditions during the expansion phase. hWJSCs under co-culture conditions show upregulations of collagen type I and II, COMP, TGFβ1 and aggrecan, as well as of the main cartilage transcription factor, SOX9, when compared to those cultured in the absence of chondrocytes. Chondrogenic differentiation of hWJSCs, previously expanded in co-culture and monoculture conditions, was evaluated for each cellular passage using the micromass culture model. Cells expanded in co-culture showed higher accumulation of glycosaminoglycans (GAGs) compared to cells in monoculture, and immunohistochemistry for localization of collagen type I revealed a strong detection signal when hWJSCs were expanded under monoculture conditions. In contrast, type II collagen was detected when cells were expanded under co-culture conditions, where numerous round-shaped cell clusters were observed. Using a micromass differentiation model, hWJSCs, previously exposed to soluble factors secreted by hACs, were able to express higher levels of chondrogenic genes with deposition of cartilage extracellular matrix components, suggesting their use as an alternative cell source for treating degenerated cartilage. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

A preliminary study comparing the use of allogenic chondrogenic pre-differentiated and undifferentiated mesenchymal stem cells for the repair of full thickness articular cartilage defects in rabbits.

PubMed

Dashtdar, Havva; Rothan, Hussin A; Tay, Terence; Ahmad, Raja Elina; Ali, Razif; Tay, Liang Xin; Chong, Pan Pan; Kamarul, Tunku

2011-09-01

Chondrogenic differentiated mesenchymal stem cells (CMSCs) have been shown to produce superior chondrogenic expression markers in vitro. However, the use of these cells in vivo has not been fully explored. In this study, in vivo assessment of cartilage repair potential between allogenic-derived chondrogenic pre-differentiated mesenchymal stem cells and undifferentiated MSCs (MSCs) were compared. Bilateral full thickness cartilage defects were created on the medial femoral condyles of 12 rabbits (n = 12). Rabbits were divided into two groups. In one group, the defects in the right knees were repaired using alginate encapsulated MSCs while in the second group, CMSCs were used. The animals were sacrificed and the repaired and control knees were assessed at 3 and 6 months after implantation. Quantitative analysis was performed by measuring the Glycosaminoglycans (GAGs)/total protein content. The mean Brittberg score was higher in the transplanted knees as compared to the untreated knee at 6 months (p < 0.05). Quantitative analysis of GAGs was consistent with these results. Histological and immunohistochemical analysis demonstrated hyaline-like cartilage regeneration in the transplanted sites. Significant differences between the histological scores based on O'Driscoll histological grading were observed between contralateral knees at both 3 and 6 months (p < 0.05). No significant differences were observed between the Britberg, O'Driscoll scores, and GAGs/total protein content when comparing defect sites treated with MSC and CMSC (p > 0.05). This study demonstrates that the use of either MSC or CMSC produced superior healing when compared to cartilage defects that were untreated. However, both cells produced comparable treatment outcomes. Copyright © 2011 Orthopaedic Research Society.

Enhancement of mesenchymal stem cell chondrogenesis with short-term low intensity pulsed electromagnetic fields.

PubMed

Parate, Dinesh; Franco-Obregón, Alfredo; Fröhlich, Jürg; Beyer, Christian; Abbas, Azlina A; Kamarul, Tunku; Hui, James H P; Yang, Zheng

2017-08-25

Pulse electromagnetic fields (PEMFs) have been shown to recruit calcium-signaling cascades common to chondrogenesis. Here we document the effects of specified PEMF parameters over mesenchymal stem cells (MSC) chondrogenic differentiation. MSCs undergoing chondrogenesis are preferentially responsive to an electromagnetic efficacy window defined by field amplitude, duration and frequency of exposure. Contrary to conventional practice of administering prolonged and repetitive exposures to PEMFs, optimal chondrogenic outcome is achieved in response to brief (10 minutes), low intensity (2 mT) exposure to 6 ms bursts of magnetic pulses, at 15 Hz, administered only once at the onset of chondrogenic induction. By contrast, repeated exposures diminished chondrogenic outcome and could be attributed to calcium entry after the initial induction. Transient receptor potential (TRP) channels appear to mediate these aspects of PEMF stimulation, serving as a conduit for extracellular calcium. Preventing calcium entry during the repeated PEMF exposure with the co-administration of EGTA or TRP channel antagonists precluded the inhibition of differentiation. This study highlights the intricacies of calcium homeostasis during early chondrogenesis and the constraints that are placed on PEMF-based therapeutic strategies aimed at promoting MSC chondrogenesis. The demonstrated efficacy of our optimized PEMF regimens has clear clinical implications for future regenerative strategies for cartilage.

Spontaneous In Vivo Chondrogenesis of Bone Marrow-Derived Mesenchymal Progenitor Cells by Blocking Vascular Endothelial Growth Factor Signaling.

PubMed

Marsano, Anna; Medeiros da Cunha, Carolina M; Ghanaati, Shahram; Gueven, Sinan; Centola, Matteo; Tsaryk, Roman; Barbeck, Mike; Stuedle, Chiara; Barbero, Andrea; Helmrich, Uta; Schaeren, Stefan; Kirkpatrick, James C; Banfi, Andrea; Martin, Ivan

2016-12-01

: Chondrogenic differentiation of bone marrow-derived mesenchymal stromal/stem cells (MSCs) can be induced by presenting morphogenetic factors or soluble signals but typically suffers from limited efficiency, reproducibility across primary batches, and maintenance of phenotypic stability. Considering the avascular and hypoxic milieu of articular cartilage, we hypothesized that sole inhibition of angiogenesis can provide physiological cues to direct in vivo differentiation of uncommitted MSCs to stable cartilage formation. Human MSCs were retrovirally transduced to express a decoy soluble vascular endothelial growth factor (VEGF) receptor-2 (sFlk1), which efficiently sequesters endogenous VEGF in vivo, seeded on collagen sponges and immediately implanted ectopically in nude mice. Although naïve cells formed vascularized fibrous tissue, sFlk1-MSCs abolished vascular ingrowth into engineered constructs, which efficiently and reproducibly developed into hyaline cartilage. The generated cartilage was phenotypically stable and showed no sign of hypertrophic evolution up to 12 weeks. In vitro analyses indicated that spontaneous chondrogenic differentiation by blockade of angiogenesis was related to the generation of a hypoxic environment, in turn activating the transforming growth factor-β pathway. These findings suggest that VEGF blockade is a robust strategy to enhance cartilage repair by endogenous or grafted mesenchymal progenitors. This article outlines the general paradigm of controlling the fate of implanted stem/progenitor cells by engineering their ability to establish specific microenvironmental conditions rather than directly providing individual morphogenic cues. Chondrogenic differentiation of mesenchymal stromal/stem cells (MSCs) is typically targeted by morphogen delivery, which is often associated with limited efficiency, stability, and robustness. This article proposes a strategy to engineer MSCs with the capacity to establish specific microenvironmental conditions, supporting their own targeted differentiation program. Sole blockade of angiogenesis mediated by transduction for sFlk-1, without delivery of additional morphogens, is sufficient for inducing MSC chondrogenic differentiation. The findings represent a relevant step forward in the field because the method allowed reducing interdonor variability in MSC differentiation efficiency and, importantly, onset of a stable, nonhypertrophic chondrocyte phenotype. ©AlphaMed Press.

Molecular Validation of Chondrogenic Differentiation and Hypoxia Responsiveness of Platelet-Lysate Expanded Adipose Tissue-Derived Human Mesenchymal Stromal Cells.

PubMed

Galeano-Garces, Catalina; Camilleri, Emily T; Riester, Scott M; Dudakovic, Amel; Larson, Dirk R; Qu, Wenchun; Smith, Jay; Dietz, Allan B; Im, Hee-Jeong; Krych, Aaron J; Larson, A Noelle; Karperien, Marcel; van Wijnen, Andre J

2017-07-01

To determine the optimal environmental conditions for chondrogenic differentiation of human adipose tissue-derived mesenchymal stromal/stem cells (AMSCs). In this investigation we specifically investigate the role of oxygen tension and 3-dimensional (3D) culture systems. Both AMSCs and primary human chondrocytes were cultured for 21 days in chondrogenic media under normoxic (21% oxygen) or hypoxic (2% oxygen) conditions using 2 distinct 3D culture methods (high-density pellets and poly-ε-caprolactone [PCL] scaffolds). Histologic analysis of chondro-pellets and the expression of chondrocyte-related genes as measured by reverse transcriptase quantitative polymerase chain reaction were used to evaluate the efficiency of differentiation. AMSCs are capable of expressing established cartilage markers including COL2A1, ACAN, and DCN when grown in chondrogenic differentiation media as determined by gene expression and histologic analysis of cartilage markers. Expression of several cartilage-related genes was enhanced by low oxygen tension, including ACAN and HAPLN1. The pellet culture environment also promoted the expression of hypoxia-inducible cartilage markers compared with cells grown on 3D scaffolds. Cell type-specific effects of low oxygen and 3D environments indicate that mesenchymal cell fate and differentiation potential is remarkably sensitive to oxygen. Genetic programming of AMSCs to a chondrocytic phenotype is effective under hypoxic conditions as evidenced by increased expression of cartilage-related biomarkers and biosynthesis of a glycosaminoglycan-positive matrix. Lower local oxygen levels within cartilage pellets may be a significant driver of chondrogenic differentiation.

Parathyroid Hormone-Related Protein Gradients Affect the Progression of Mesenchymal Stem Cell Chondrogenesis and Hypertrophy.

PubMed

Fahy, Niamh; Gardner, Oliver F W; Alini, Mauro; Stoddart, Martin J

2018-05-01

Mesenchymal stem cells (MSCs) are considered a promising cell source for cartilage repair strategies due to their chondrogenic differentiation potential. However, their in vitro tendency to progress toward hypertrophy limits their clinical use. This unfavorable result may be due to the fact that MSCs used in tissue engineering approaches are all at the same developmental stage, and have lost crucial spatial and temporal signaling cues. In this study, we sought to investigate the effect of a spatial parathyroid hormone-related protein (PTHrP) signaling gradient on the chondrogenic differentiation of MSCs and progression to hypertrophy. Human bone marrow-derived MSCs were transduced with adenoviral vectors overexpressing PTHrP and seeded into fibrin-poly(ester-urethane) scaffolds. To investigate the effect of a spatial PTHrP signaling gradient, scaffolds were seeded with PTHrP-overexpressing MSCs positioned on top of the scaffold, with untransduced MSCs seeded evenly within. Scaffolds were cultured with or without 2 ng/mL transforming growth factor (TGF)-β1 for 28 days. PTHrP overexpression increased glycosaminoglycan (GAG) production by MSCs irrespective of TGF-β1 treatment, and exerted differential effects on chondrogenic and hypertrophic gene expression when MSCs were cultured in the presence of a PTHrP signaling gradient. Furthermore, PTHrP-overexpressing MSCs were associated with an increase of endogenous TGF-β1 production and reduced total MMP-13 secretion compared to controls. The presence of a spatial PTHrP signaling gradient may support chondrogenic differentiation of MSCs and promote the formation of a more stable cartilage phenotype in tissue engineering applications.

Dynamic modelling of microRNA regulation during mesenchymal stem cell differentiation.

PubMed

Weber, Michael; Sotoca, Ana M; Kupfer, Peter; Guthke, Reinhard; van Zoelen, Everardus J

2013-11-12

Network inference from gene expression data is a typical approach to reconstruct gene regulatory networks. During chondrogenic differentiation of human mesenchymal stem cells (hMSCs), a complex transcriptional network is active and regulates the temporal differentiation progress. As modulators of transcriptional regulation, microRNAs (miRNAs) play a critical role in stem cell differentiation. Integrated network inference aimes at determining interrelations between miRNAs and mRNAs on the basis of expression data as well as miRNA target predictions. We applied the NetGenerator tool in order to infer an integrated gene regulatory network. Time series experiments were performed to measure mRNA and miRNA abundances of TGF-beta1+BMP2 stimulated hMSCs. Network nodes were identified by analysing temporal expression changes, miRNA target gene predictions, time series correlation and literature knowledge. Network inference was performed using NetGenerator to reconstruct a dynamical regulatory model based on the measured data and prior knowledge. The resulting model is robust against noise and shows an optimal trade-off between fitting precision and inclusion of prior knowledge. It predicts the influence of miRNAs on the expression of chondrogenic marker genes and therefore proposes novel regulatory relations in differentiation control. By analysing the inferred network, we identified a previously unknown regulatory effect of miR-524-5p on the expression of the transcription factor SOX9 and the chondrogenic marker genes COL2A1, ACAN and COL10A1. Genome-wide exploration of miRNA-mRNA regulatory relationships is a reasonable approach to identify miRNAs which have so far not been associated with the investigated differentiation process. The NetGenerator tool is able to identify valid gene regulatory networks on the basis of miRNA and mRNA time series data.

Combinatorial Analysis of Growth Factors Reveals the Contribution of Bone Morphogenetic Proteins to Chondrogenic Differentiation of Human Periosteal Cells.

PubMed

Mendes, Luis Filipe; Tam, Wai Long; Chai, Yoke Chin; Geris, Liesbet; Luyten, Frank P; Roberts, Scott J

2016-05-01

Successful application of cell-based strategies in cartilage and bone tissue engineering has been hampered by the lack of robust protocols to efficiently differentiate mesenchymal stem cells into the chondrogenic lineage. The development of chemically defined culture media supplemented with growth factors (GFs) has been proposed as a way to overcome this limitation. In this work, we applied a fractional design of experiment (DoE) strategy to screen the effect of multiple GFs (BMP2, BMP6, GDF5, TGF-β1, and FGF2) on chondrogenic differentiation of human periosteum-derived mesenchymal stem cells (hPDCs) in vitro. In a micromass culture (μMass) system, BMP2 had a positive effect on glycosaminoglycan deposition at day 7 (p < 0.001), which in combination with BMP6 synergistically enhanced cartilage-like tissue formation that displayed in vitro mineralization capacity at day 14 (p < 0.001). Gene expression of μMasses cultured for 7 days with a medium formulation supplemented with 100 ng/mL of BMP2 and BMP6 and a low concentration of GDF5, TGF-β1, and FGF2 showed increased expression of Sox9 (1.7-fold) and the matrix molecules aggrecan (7-fold increase) and COL2A1 (40-fold increase) compared to nonstimulated control μMasses. The DoE analysis indicated that in GF combinations, BMP2 was the strongest effector for chondrogenic differentiation of hPDCs. When transplanted ectopically in nude mice, the in vitro-differentiated μMasses showed maintenance of the cartilaginous phenotype after 4 weeks in vivo. This study indicates the power of using the DoE approach for the creation of new medium formulations for skeletal tissue engineering approaches.

An In Vitro Comparison of the Incorporation, Growth, and Chondrogenic Potential of Human Bone Marrow versus Adipose Tissue Mesenchymal Stem Cells in Clinically Relevant Cell Scaffolds Used for Cartilage Repair

PubMed Central

Kohli, Nupur; Wright, Karina T.; Sammons, Rachel L.; Jeys, Lee; Snow, Martyn

2015-01-01

Aim To compare the incorporation, growth, and chondrogenic potential of bone marrow (BM) and adipose tissue (AT) mesenchymal stem cells (MSCs) in scaffolds used for cartilage repair. Methods Human BM and AT MSCs were isolated, culture expanded, and characterised using standard protocols, then seeded into 2 different scaffolds, Chondro-Gide or Alpha Chondro Shield. Cell adhesion, incorporation, and viable cell growth were assessed microscopically and following calcein AM/ethidium homodimer (Live/Dead) staining. Cell-seeded scaffolds were treated with chondrogenic inducers for 28 days. Extracellular matrix deposition and soluble glycosaminoglycan (GAG) release into the culture medium was measured at day 28 by histology/immunohistochemistry and dimethylmethylene blue assay, respectively. Results A greater number of viable MSCs from either source adhered and incorporated into Chondro-Gide than into Alpha Chondro Shield. In both cell scaffolds, this incorporation represented less than 2% of the cells that were seeded. There was a marked proliferation of BM MSCs, but not AT MSCs, in Chondro-Gide. MSCs from both sources underwent chondrogenic differentiation following induction. However, cartilaginous extracellular matrix deposition was most marked in Chondro-Gide seeded with BM MSCs. Soluble GAG secretion increased in chondrogenic versus control conditions. There was no marked difference in GAG secretion by MSCs from either cell source. Conclusion Chondro-Gide and Alpha Chondro Shield were permissive to the incorporation and chondrogenic differentiation of human BM and AT MSCs. Chondro-Gide seeded with BM MSCs demonstrated the greatest increase in MSC number and deposition of a cartilaginous tissue. PMID:26425263

Evaluation and comparison of the in vitro characteristics and chondrogenic capacity of four adult stem/progenitor cells for cartilage cell-based repair.

PubMed

Shafiee, Abbas; Kabiri, Mahboubeh; Langroudi, Lida; Soleimani, Masoud; Ai, Jafar

2016-03-01

Cell-based therapy is being considered as a promising approach to regenerate damaged cartilage. Though, autologous chondrocyte implantation is the most effective strategy currently in use, but is hampered by some drawbacks seeking comprehensive research to surmount existing limitations or introducing alternative cell sources. In this study, we aimed to evaluate and compare the in vitro characteristics and chondrogenic capacity of some easily available adult cell sources for use in cartilage repair which includes: bone marrow-derived mesenchymal stem cells (MSC), adipose tissue-derived MSC, articular chondrocyte progenitors, and nasal septum-derived progenitors. Human stem/progenitor cells were isolated and expanded. Cell's immunophenotype, biosafety, and cell cycle status were evaluated. Also, cells were seeded onto aligned electrospun poly (l-lactic acid)/poly (ε-caprolactone) nanofibrous scaffolds and their proliferation rate as well as chondrogenic potential were assessed. Cells were almost phenotypically alike as they showed similar cell surface marker expression pattern. The aligned nanofibrous hybrid scaffolds could support the proliferation and chondrogenic differentiation of all cell types. However, nasal cartilage progenitors showed a higher proliferation potential and a higher chondrogenic capacity. Though, mostly similar in the majority of the studied features, nasal septum progenitors demonstrated a higher chondrogenic potential that in combination with their higher proliferation rate and easier access to the source tissue, introduces it as a promising cell source for cartilage tissue engineering and regenerative medicine. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 600-610, 2016. © 2015 Wiley Periodicals, Inc.

Colonization of collagen scaffolds by adipocytes derived from mesenchymal stem cells of the common marmoset monkey

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bernemann, Inga, E-mail: bernemann@imp.uni-hannover.de; Mueller, Thomas; Blasczyk, Rainer

Highlights: {yields} Marmoset bone marrow-derived MSCs differentiate in suspension into adipogenic, osteogenic and chondrogenic lineages. {yields} Marmoset MSCs integrate in collagen type I scaffolds and differentiate excellently into adipogenic cells. {yields} Common marmoset monkey is a suitable model for soft tissue engineering in human regenerative medicine. -- Abstract: In regenerative medicine, human cell replacement therapy offers great potential, especially by cell types differentiated from immunologically and ethically unproblematic mesenchymal stem cells (MSCs). In terms of an appropriate carrier material, collagen scaffolds with homogeneous pore size of 65 {mu}m were optimal for cell seeding and cultivating. However, before clinical application andmore » transplantation of MSC-derived cells in scaffolds, the safety and efficiency, but also possible interference in differentiation due to the material must be preclinically tested. The common marmoset monkey (Callithrix jacchus) is a preferable non-human primate animal model for this aim due to its genetic and physiological similarities to the human. Marmoset bone marrow-derived MSCs were successfully isolated, cultured and differentiated in suspension into adipogenic, osteogenic and chondrogenic lineages by defined factors. The differentiation capability could be determined by FACS. Specific marker genes for all three cell types could be detected by RT-PCR. Furthermore, MSCs seeded on collagen I scaffolds differentiated in adipogenic lineage showed after 28 days of differentiation high cell viability and homogenous distribution on the material which was validated by calcein AM and EthD staining. As proof of adipogenic cells, the intracellular lipid vesicles in the cells were stained with Oil Red O. The generation of fat vacuoles was visibly extensive distinguishable and furthermore determined on the molecular level by expression of specific marker genes. The results of the study proved both the differential potential of marmoset MSCs in adipogenic, osteogenic and chondrogenic lineages and the suitability of collagen scaffolds as carrier material undisturbing differentiation of primate mesenchymal stem cells.« less

Synovium-derived stem cells: a tissue-specific stem cell for cartilage engineering and regeneration.

PubMed

Jones, Brendan A; Pei, Ming

2012-08-01

Articular cartilage is difficult to heal once injury or disease occurs. Autologous chondrocyte transplantation is a biological treatment with good prognosis, but donor site morbidity and limited cell source are disadvantages. Currently, mesenchymal stem cells (MSCs) are a promising approach for cartilage regeneration. Despite there being various sources, the best candidate for cartilage regeneration is the one with the greatest chondrogenic potential and the least hypertrophic differentiation. These properties are able to insure that the regenerated tissue is hyaline cartilage of high quality. This review article will summarize relevant literature to justify synovium-derived stem cells (SDSCs) as a tissue-specific stem cell for chondrogenesis by comparing synovium and cartilage with respect to anatomical location and functional structure, comparing the growth characterization and chondrogenic capacity of SDSCs and MSCs, evaluating the application of SDSCs in regenerative medicine and diseases, and discussing potential future directions.

Chm-1 gene-modified bone marrow mesenchymal stem cells maintain the chondrogenic phenotype of tissue-engineered cartilage.

PubMed

Chen, Zhuoyue; Wei, Jing; Zhu, Jun; Liu, Wei; Cui, Jihong; Li, Hongmin; Chen, Fulin

2016-05-05

Marrow mesenchymal stem cells (MSCs) can differentiate into specific phenotypes, including chondrocytes, and have been widely used for cartilage tissue engineering. However, cartilage grafts from MSCs exhibit phenotypic alternations after implantation, including matrix calcification and vascular ingrowth. We compared chondromodulin-1 (Chm-1) expression between chondrocytes and MSCs. We found that chondrocytes expressed a high level of Chm-1. We then adenovirally transduced MSCs with Chm-1 and applied modified cells to engineer cartilage in vivo. A gross inspection and histological observation indicated that the chondrogenic phenotype of the tissue-engineered cartilage graft was well maintained, and the stable expression of Chm-1 was detected by immunohistological staining in the cartilage graft derived from the Chm-1 gene-modified MSCs. Our findings defined an essential role for Chm-1 in maintaining chondrogenic phenotype and demonstrated that Chm-1 gene-modified MSCs may be used in cartilage tissue engineering.

Analysis of the Effects of Five Factors Relevant to In Vitro Chondrogenesis of Human Mesenchymal Stem Cells Using Factorial Design and High Throughput mRNA-Profiling

PubMed Central

Jakobsen, Rune B.; Østrup, Esben; Zhang, Xiaolan; Mikkelsen, Tarjei S.; Brinchmann, Jan E.

2014-01-01

The in vitro process of chondrogenic differentiation of mesenchymal stem cells for tissue engineering has been shown to require three-dimensional culture along with the addition of differentiation factors to the culture medium. In general, this leads to a phenotype lacking some of the cardinal features of native articular chondrocytes and their extracellular matrix. The factors used vary, but regularly include members of the transforming growth factor β superfamily and dexamethasone, sometimes in conjunction with fibroblast growth factor 2 and insulin-like growth factor 1, however the use of soluble factors to induce chondrogenesis has largely been studied on a single factor basis. In the present study we combined a factorial quality-by-design experiment with high-throughput mRNA profiling of a customized chondrogenesis related gene set as a tool to study in vitro chondrogenesis of human bone marrow derived mesenchymal stem cells in alginate. 48 different conditions of transforming growth factor β 1, 2 and 3, bone morphogenetic protein 2, 4 and 6, dexamethasone, insulin-like growth factor 1, fibroblast growth factor 2 and cell seeding density were included in the experiment. The analysis revealed that the best of the tested differentiation cocktails included transforming growth factor β 1 and dexamethasone. Dexamethasone acted in synergy with transforming growth factor β 1 by increasing many chondrogenic markers while directly downregulating expression of the pro-osteogenic gene osteocalcin. However, all factors beneficial to the expression of desirable hyaline cartilage markers also induced undesirable molecules, indicating that perfect chondrogenic differentiation is not achievable with the current differentiation protocols. PMID:24816923

The Effects of the WNT-Signaling Modulators BIO and PKF118-310 on the Chondrogenic Differentiation of Human Mesenchymal Stem Cells.

PubMed

Huang, Xiaobin; Zhong, Leilei; Hendriks, Jan; Post, Janine N; Karperien, Marcel

2018-02-13

Mesenchymal stem cells (MSCs) are multipotent cells, mainly from bone marrow, and an ideal source of cells in bone and cartilage tissue engineering. A study of the chondrogenic differentiation of MSCs is of particular interest for MSCs-based cartilage regeneration. In this study, we aimed to optimize the conditions for the chrondogenic differentiation of MSCs by regulating WNT signaling using the small molecule WNT inhibitor PKF118-310 and activator BIO. Human mesenchymal stem cells (hMSCs) were isolated from bone marrow aspirates and cultured in hMSCs proliferation medium. Pellet culture was subsequently established for three-dimensional chondrogenic differentiation of 5 weeks. WNT signaling was increased by the small molecule glycogen synthase kinase-3 inhibitor 6-bromoindirubin-3-oxim (BIO) and decreased by the WNT inhibitor PKF118-310 (PKF). The effects of BIO and PKF on the chondrogenesis of hMSCs was examined by real-time PCR, histological methods, and ELISA. We found that activation of canonical WNT-signaling by BIO significantly downregulated the expression of cartilage-specific genes SOX9 , COL2A1, and ACAN , and matrix metalloproteinase genes MMP1/3/9/13, but increased ADAMTS 4/5 . Inhibition of WNT signaling by PKF increased the expression of SOX9 , COL2A1 , ACAN , and MMP9, but decreased MMP13 and ADAMTS4/5 . In addition, a high level of WNT signaling induced the expression of hypertrophic markers COL10A1, ALPL , and RUNX2, the dedifferentiation marker COL1A1 , and glycolysis genes GULT1 and PGK1 . Deposition of glycosaminoglycan (GAG) and collagen type II in the pellet matrix was significantly lost in the BIO-treated group and increased in the PKF-treated group. The protein level of COL10A1 was also highly induced in the BIO group. Interestingly, BIO decreased the number of apoptotic cells while PKF significantly induced apoptosis during chondrogenesis. The natural WNT antagonist DKK1 and the protein level of MMP1 in the pellet culture medium were decreased after PKF treatment. All of these chondrogenic effects appeared to be mediated through the canonical WNT signaling pathway, since the target gene Axin2 and other WNT members, such as TCF4 and β-catenin , were upregulated by BIO and downregulated by PKF, respectively, and BIO induced nuclear translocation of β-catenin while PKF inhibited β-catenin translocation into the nucleus. We concluded that addition of BIO to a chondrogenic medium of hMSCs resulted in a loss of cartilage formation, while PKF induced chondrogenic differentiation and cartilage matrix deposition and inhibited hypertrophic differentiation. However, BIO promoted cell survival by inhibiting apoptosis while PKF induced cell apoptosis. This result indicates that either an overexpression or overinhibition of WNT signaling to some extent causes harmful effects on chondrogenic differentiation. Cartilage tissue engineering could benefit from the adjustment of the critical level of WNT signaling during chondrogenesis of hMSC.

Improved properties of bone and cartilage tissue from 3D inkjet-bioprinted human mesenchymal stem cells by simultaneous deposition and photocrosslinking in PEG-GelMA.

PubMed

Gao, Guifang; Schilling, Arndt F; Hubbell, Karen; Yonezawa, Tomo; Truong, Danh; Hong, Yi; Dai, Guohao; Cui, Xiaofeng

2015-11-01

Bioprinting of bone and cartilage suffers from low mechanical properties. Here we have developed a unique inkjet bioprinting approach of creating mechanically strong bone and cartilage tissue constructs using poly(ethylene glycol) dimethacrylate, gelatin methacrylate, and human MSCs. The printed hMSCs were evenly distributed in the polymerized PEG-GelMA scaffold during layer-by-layer assembly. The procedure showed a good biocompatibility with >80% of the cells surviving the printing process and the resulting constructs provided strong mechanical support to the embedded cells. The printed mesenchymal stem cells showed an excellent osteogenic and chondrogenic differentiation capacity. Both osteogenic and chondrogenic differentiation as determined by specific gene and protein expression analysis (RUNX2, SP7, DLX5, ALPL, Col1A1, IBSP, BGLAP, SPP1, Col10A1, MMP13, SOX9, Col2A1, ACAN) was improved by PEG-GelMA in comparison to PEG alone. These observations were consistent with the histological evaluation. Inkjet bioprinted-hMSCs in simultaneously photocrosslinked PEG-GelMA hydrogel scaffolds demonstrated an improvement of mechanical properties and osteogenic and chondrogenic differentiation, suggesting its promising potential for usage in bone and cartilage tissue engineering.

Multilayered dense collagen-silk fibroin hybrid: a platform for mesenchymal stem cell differentiation towards chondrogenic and osteogenic lineages.

PubMed

Ghezzi, Chiara E; Marelli, Benedetto; Donelli, Ilaria; Alessandrino, Antonio; Freddi, Giuliano; Nazhat, Showan N

2017-07-01

Type I collagen is a major structural and functional protein in connective tissues. However, collagen gels exhibit unstable geometrical properties, arising from extensive cell-mediated contraction. In an effort to stabilize collagen-based hydrogels, plastic compression was used to hybridize dense collagen (DC) with electrospun silk fibroin (SF) mats, generating multilayered DC-SF-DC constructs. Seeded mesenchymal stem cell (MSC)-mediated DC-SF-DC contraction, as well as growth and differentiation under chondrogenic and osteogenic supplements, were compared to those seeded in DC and on SF alone. The incorporation of SF within DC prevented extensive cell-mediated collagen gel contraction. The effect of the multilayered hybrid on MSC remodelling capacity was also evident at the transcription level, where the expression of matrix metalloproteinases and their inhibitor (MMP1, MMP2, MMP3, MMP13 and Timp1) by MSCs within DC-SF-DC were comparable to those on SF and significantly downregulated in comparison to DC, except for Timp1. Chondrogenic supplements stimulated extracellular matrix production within the construct, stabilizing its multilayered structure and promoting MSC chondrogenic differentiation, as indicated by the upregulation of the genes Col2a1 and Agg and the production of collagen type II. In osteogenic medium there was an upregulation in ALP and OP along with the presence of an apatitic phase, indicating MSC osteoblastic differentiation and matrix mineralization. In sum, these results have implications on the modulation of three-dimensional collagen-based gel structural stability and on the stimulation and maintenance of the MSC committed phenotype inherent to the in vitro formation of chondral tissue and bone, as well as on potential multilayered complex tissues. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Temporal activation of β-catenin signaling in the chondrogenic process of mesenchymal stem cells affects the phenotype of the cartilage generated.

PubMed

Yang, Zheng; Zou, Yu; Guo, Xi Min; Tan, Hwee San; Denslin, Vinitha; Yeow, Chen Hua; Ren, Xia Fei; Liu, Tong Ming; Hui, James Hp; Lee, Eng Hin

2012-07-20

Adult mesenchymal stem cells (MSCs) are an attractive cell source for cartilage tissue engineering. In vitro predifferentiation of MSCs has been explored as a means to enhance MSC-based articular cartilage repair. However, there remain challenges to control and prevent the premature progression of MSC-derived chondrocytes to the hypertrophy. This study investigated the temporal effect of transforming growth factor (TGF)-β and β-catenin signaling co-activation during MSC chondrogenic differentiation and evaluated the influence of these predifferentiation conditions to subsequent phenotypic development of the cartilage. MSCs were differentiated in chondrogenic medium that contained either TGFβ alone, TGFβ with transient β-catenin coactivation, or TGFβ with continuous β-catenin coactivation. After in vitro differentiation, the pellets were transplanted into SCID mice. Both coactivation protocols resulted in the enhancement of chondrogenic differentiation of MSCs. Compared with TGFβ activation, transient coactivation of TGFβ-induction with β-catenin activation resulted in heightened hypertrophy and formed highly ossified tissues with marrow-like hematopoietic tissue in vivo. The continuous coactivation of the 2 signaling pathways, however, resulted in inhibition of progression to hypertrophy, marked by the suppression of type X collagen, Runx2, and alkaline phosphatase expression, and did not result in ossified tissue in vivo. Chondrocytes of the continuous co-activation samples secreted significantly more parathyroid hormone-related protein (PTHrP) and expressed cyclin D1. Our results suggest that temporal co-activation of the TGFβ signaling pathway with β-catenin can yield cartilage of different phenotype, represents a potential MSC predifferentiation protocol before clinical implantation, and has potential applications for the engineering of cartilage tissue.

Allogeneic Umbilical Cord-Derived Mesenchymal Stem Cells as a Potential Source for Cartilage and Bone Regeneration: An In Vitro Study

PubMed Central

Mattia, S.; Castoldi, F.; Barbero, A.; Bonasia, D. E.; Bruzzone, M.; Dettoni, F.; Scurati, R.

2017-01-01

Umbilical cord (UC) may represent an attractive cell source for allogeneic mesenchymal stem cell (MSC) therapy. The aim of this in vitro study is to investigate the chondrogenic and osteogenic potential of UC-MSCs grown onto tridimensional scaffolds, to identify a possible clinical relevance for an allogeneic use in cartilage and bone reconstructive surgery. Chondrogenic differentiation on scaffolds was confirmed at 4 weeks by the expression of sox-9 and type II collagen; low oxygen tension improved the expression of these chondrogenic markers. A similar trend was observed in pellet culture in terms of matrix (proteoglycan) production. Osteogenic differentiation on bone-graft-substitute was also confirmed after 30 days of culture by the expression of osteocalcin and RunX-2. Cells grown in the hypertrophic medium showed at 5 weeks safranin o-positive stain and an increased CbFa1 expression, confirming the ability of these cells to undergo hypertrophy. These results suggest that the UC-MSCs isolated from minced umbilical cords may represent a valuable allogeneic cell population, which might have a potential for orthopaedic tissue engineering such as the on-demand cell delivery using chondrogenic, osteogenic, and endochondral scaffold. This study may have a clinical relevance as a future hypothetical option for allogeneic single-stage cartilage repair and bone regeneration. PMID:29358953

Allogeneic Umbilical Cord-Derived Mesenchymal Stem Cells as a Potential Source for Cartilage and Bone Regeneration: An In Vitro Study.

PubMed

Marmotti, A; Mattia, S; Castoldi, F; Barbero, A; Mangiavini, L; Bonasia, D E; Bruzzone, M; Dettoni, F; Scurati, R; Peretti, G M

2017-01-01

Umbilical cord (UC) may represent an attractive cell source for allogeneic mesenchymal stem cell (MSC) therapy. The aim of this in vitro study is to investigate the chondrogenic and osteogenic potential of UC-MSCs grown onto tridimensional scaffolds, to identify a possible clinical relevance for an allogeneic use in cartilage and bone reconstructive surgery. Chondrogenic differentiation on scaffolds was confirmed at 4 weeks by the expression of sox-9 and type II collagen; low oxygen tension improved the expression of these chondrogenic markers. A similar trend was observed in pellet culture in terms of matrix (proteoglycan) production. Osteogenic differentiation on bone-graft-substitute was also confirmed after 30 days of culture by the expression of osteocalcin and RunX-2. Cells grown in the hypertrophic medium showed at 5 weeks safranin o-positive stain and an increased CbFa1 expression, confirming the ability of these cells to undergo hypertrophy. These results suggest that the UC-MSCs isolated from minced umbilical cords may represent a valuable allogeneic cell population, which might have a potential for orthopaedic tissue engineering such as the on-demand cell delivery using chondrogenic, osteogenic, and endochondral scaffold. This study may have a clinical relevance as a future hypothetical option for allogeneic single-stage cartilage repair and bone regeneration.

Co-overexpression of TGF-β and SOX9 via rAAV gene transfer modulates the metabolic and chondrogenic activities of human bone marrow-derived mesenchymal stem cells.

PubMed

Tao, Ke; Frisch, Janina; Rey-Rico, Ana; Venkatesan, Jagadeesh K; Schmitt, Gertrud; Madry, Henning; Lin, Jianhao; Cucchiarini, Magali

2016-02-01

Articular cartilage has a limited potential for self-healing. Transplantation of genetically modified progenitor cells like bone marrow-derived mesenchymal stem cells (MSCs) is an attractive strategy to improve the intrinsic repair capacities of damaged articular cartilage. In this study, we examined the potential benefits of co-overexpressing the pleiotropic transformation growth factor beta (TGF-β) with the cartilage-specific transcription factor SOX9 via gene transfer with recombinant adeno-associated virus (rAAV) vectors upon the biological activities of human MSCs (hMSCs). Freshly isolated hMSCs were transduced over time with separate rAAV vectors carrying either TGF-β or sox9 in chondrogenically-induced aggregate cultures to evaluate the efficacy and duration of transgene expression and to monitor the effects of rAAV-mediated genetic modification upon the cellular activities (proliferation, matrix synthesis) and chondrogenic differentiation potency compared with control conditions (lacZ treatment, sequential transductions). Significant, prolonged TGF-β/sox9 co-overexpression was achieved in chondrogenically-induced hMSCs upon co-transduction via rAAV for up to 21 days, leading to enhanced proliferative, biosynthetic, and chondrogenic activities relative to control treatments, especially when co-applying the candidate vectors at the highest vector doses tested. Optimal co-administration of TGF-β with sox9 also advantageously reduced hypertrophic differentiation of the cells in the conditions applied here. The present findings demonstrate the possibility of modifying MSCs by combined therapeutic gene transfer as potent future strategies for implantation in clinically relevant animal models of cartilage defects in vivo.

Can one generate stable hyaline cartilage from adult mesenchymal stem cells? A developmental approach.

PubMed

Hellingman, Catharine A; Koevoet, Wendy; van Osch, Gerjo J V M

2012-11-01

Chondrogenically differentiating bone marrow-derived mesenchymal stem cells (BMSCs) display signs of chondrocyte hypertrophy, such as production of collagen type X, MMP13 and alkaline phosphatase (ALPL). For cartilage reconstructions this is undesirable, as terminally differentiated cartilage produced by BMSCs mineralizes when implanted in vivo. Terminal differentiation is not restricted to BMSCs but is also encountered in chondrogenic differentiation of adipose-derived mesenchymal stem cells (MSCs) as well as embryonic stem cells, which by definition should be able to generate all types of tissues, including stable cartilage. Therefore, we propose that the currently used culture conditions may drive the cells towards terminal differentiation. In this manuscript we aim to review the literature, supplemented by our own data to answer the question, is it possible to generate stable hyaline cartilage from adult MSCs? We demonstrate that recently published methods for inhibiting terminal differentiation (through PTHrP, MMP13 or blocking phosphorylation of Smad1/5/8) result in cartilage formation with reduction of hypertrophic markers, although this does not reach the low level of stable chondrocytes. A set of hypertrophy markers should be included in future studies to characterize the phenotype more precisely. Finally, we used what is currently known in developmental biology about the differential development of hyaline and terminally differentiated cartilage to provide thought and insights to change current culture models for creating hyaline cartilage. Inhibiting terminal differentiation may not result in stable hyaline cartilage if the right balance of signals has not been created from the start of culture onwards. Copyright © 2011 John Wiley & Sons, Ltd.

Chondrogenic differentiation of human mesenchymal stem cells on fish scale collagen.

PubMed

Hsu, Han-Hsiu; Uemura, Toshimasa; Yamaguchi, Isamu; Ikoma, Toshiyuki; Tanaka, Junzo

2016-08-01

Fish collagen has recently been reported to be a novel biomaterial for cell and tissue culture as an alternative to conventional mammalian collagens such as bovine and porcine collagens. Fish collagen could overcome the risk of zoonosis, such as from bovine spongiform encephalopathy. Among fish collagens, tilapia collagen, the denaturing temperature of which is near 37°C, is appropriate for cell and tissue culture. In this study, we investigated chondrogenic differentiation of human mesenchymal stem cells (hMSCs) cultured on tilapia scale collagen fibrils compared with porcine collagen and non-coated dishes. The collagen fibrils were observed using a scanning electronic microscope. Safranin O staining, glycosaminoglycans (GAG) expression, and real-time PCR were examined to evaluate chondrogenesis of hMSCs on each type of collagen fibril. The results showed that hMSCs cultured on tilapia scale collagen showed stronger Safranin O staining and higher GAG expression at day 6. Results of real-time PCR indicated that hMSCs cultured on tilapia collagen showed earlier SOX9 expression on day 4 and higher AGGRECAN and COLLAGEN II expression on day 6 compared with on porcine collagen and non-coated dishes. Furthermore, low mRNA levels of bone gamma-carboxyglutamate, a specific marker of osteogenesis, showed that tilapia collagen fibrils specifically enhanced chondrogenic differentiation of hMSCs in chondrogenic medium, as well as porcine collagen. Accordingly, tilapia scale collagen may provide an appropriate collagen source for hMSC chondrogenesis in vitro. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

The Effect of Hydrostatic Pressure on Three-Dimensional Chondroinduction of Human Adipose–Derived Stem Cells

PubMed Central

Mizuno, Shuichi; Murphy, George F.; Orgill, Dennis P.

2009-01-01

Background The optimal production of three-dimensional cartilage in vitro requires both inductive factors and specified culture conditions (e.g., hydrostatic pressure [HP], gas concentration, and nutrient supply) to promote cell viability and maintain phenotype. In this study, we optimized the conditions for human cartilage induction using human adipose–derived stem cells (ASCs), collagen scaffolds, and cyclic HP treatment. Methods Human ASCs underwent primary culture and three passages before being seeded into collagen scaffolds. These constructs were incubated for 1 week in an automated bioreactor using cyclic HP at 0–0.5 MPa, 0.5 Hz, and compared to constructs exposed to atmospheric pressure. In both groups, chondrogenic differentiation medium including transforming growth factor-β1 was employed. One, 2, 3, and 4 weeks after incubation, the cell constructs were harvested for histological, immunohistochemical, and gene expression evaluation. Results In histological and immunohistochemical analyzes, pericellular and extracellular metachromatic matrix was observed in both groups and increased over 4 weeks, but accumulated at a higher rate in the HP group. Cell number was maintained in the HP group over 4 weeks but decreased after 2 weeks in the atmospheric pressure group. Chondrogenic-specific gene expression of type II and X collagen, aggrecan, and SRY-box9 was increased in the HP group especially after 2 weeks. Conclusion Our results demonstrate chondrogenic differentiation of ASCs in a three-dimensional collagen scaffolds with treatment of a cyclic HP. Cyclic HP was effective in enhancing accumulation of extracellular matrix and expression of genes indicative of chondrogenic differentiation. PMID:19290804

Oxygen tension regulates the osteogenic, chondrogenic and endochondral phenotype of bone marrow derived mesenchymal stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sheehy, Eamon J.; Buckley, Conor T.; Kelly, Daniel J., E-mail: kellyd9@tcd.ie

2012-01-06

Highlights: Black-Right-Pointing-Pointer Expansion in low oxygen enhances MSC proliferation and osteogenesis. Black-Right-Pointing-Pointer Differentiation in low oxygen enhances chondrogenesis and suppresses hypertrophy. Black-Right-Pointing-Pointer Oxygen can regulate the MSC phenotype for use in tissue engineering applications. -- Abstract: The local oxygen tension is a key regulator of the fate of mesenchymal stem cells (MSCs). The objective of this study was to investigate the effect of a low oxygen tension during expansion and differentiation on the proliferation kinetics as well as the subsequent osteogenic and chondrogenic potential of MSCs. We first hypothesised that expansion in a low oxygen tension (5% pO{sub 2}) wouldmore » improve both the subsequent osteogenic and chondrogenic potential of MSCs compared to expansion in a normoxic environment (20% pO{sub 2}). Furthermore, we hypothesised that chondrogenic differentiation in a low oxygen environment would suppress hypertrophy of MSCs cultured in both pellets and hydrogels used in tissue engineering strategies. MSCs expanded at 5% pO{sub 2} proliferated faster forming larger colonies, resulting in higher cell yields. Expansion at 5% pO{sub 2} also enhanced subsequent osteogenesis of MSCs, whereas differentiation at 5% pO{sub 2} was found to be a more potent promoter of chondrogenesis than expansion at 5% pO{sub 2}. Greater collagen accumulation, and more intense staining for collagen types I and X, was observed in pellets maintained at 20% pO{sub 2} compared to 5% pO{sub 2}. Both pellets and hydrogels stained more intensely for type II collagen when undergoing chondrogenesis in a low oxygen environment. Differentiation at 5% pO{sub 2} also appeared to inhibit hypertrophy in both pellets and hydrogels, as demonstrated by reduced collagen type X and Alizarin Red staining and alkaline phosphatase activity. This study demonstrates that the local oxygen environment can be manipulated in vitro to either stabilise a chondrogenic phenotype for use in cartilage repair therapies or to promote hypertrophy of cartilaginous grafts for endochondral bone repair strategies.« less

Differentiation within autologous fibrin scaffolds of porcine dermal cells with the mesenchymal stem cell phenotype

DOE Office of Scientific and Technical Information (OSTI.GOV)

Puente, Pilar de la, E-mail: pilardelapuentegarcia@gmail.com; Ludeña, Dolores; López, Marta

2013-02-01

Porcine mesenchymal stem cells (pMSCs) are an attractive source of cells for tissue engineering because their properties are similar to those of human stem cells. pMSCs can be found in different tissues but their dermal origin has not been studied in depth. Additionally, MSCs differentiation in monolayer cultures requires subcultured cells, and these cells are at risk of dedifferentiation when implanting them into living tissue. Following this, we attempted to characterize the MSCs phenotype of porcine dermal cells and to evaluate their cellular proliferation and differentiation in autologous fibrin scaffolds (AFSs). Dermal biopsies and blood samples were obtained from 12more » pigs. Dermal cells were characterized by flow cytometry. Frozen autologous plasma was used to prepare AFSs. pMSC differentiation was studied in standard structures (monolayers and pellets) and in AFSs. The pMSCs expressed the CD90 and CD29 markers of the mesenchymal lineage. AFSs afforded adipogenic, osteogenic and chondrogenic differentiation. The porcine dermis can be proposed to be a good source of MSCs with adequate proliferative capacity and a suitable expression of markers. The pMSCs also showed optimal proliferation and differentiation in AFSs, such that these might serve as a promising autologous and implantable material for use in tissue engineering. -- Highlights: ► Low fibrinogen concentration provides a suitable matrix for cell migration and differentiation. ► Autologous fibrin scaffolds is a promising technique in tissue engineering. ► Dermal cells are an easily accessible mesenchymal stem cell source. ► Fibrin scaffolds afforded adipogenic, osteogenic and chondrogenic differentiation.« less

Poly(N-isopropylacrylamide) hydrogel/chitosan scaffold hybrid for three-dimensional stem cell culture and cartilage tissue engineering.

PubMed

Mellati, Amir; Kiamahalleh, Meisam Valizadeh; Madani, S Hadi; Dai, Sheng; Bi, Jingxiu; Jin, Bo; Zhang, Hu

2016-11-01

Providing a controllable and definable three-dimensional (3D) microenvironment for chondrogenic differentiation of mesenchymal stem cells (MSCs) remains a great challenge for cartilage tissue engineering. In this work, poly(N-isopropylacrylamide) (PNIPAAm) polymers with the degrees of polymerization of 100 and 400 (NI100 and NI400) were prepared and the polymer solutions were introduced into the preprepared chitosan porous scaffolds (CS) to form hybrids (CSNI100 and CSNI400, respectively). SEM images indicated that the PNIPAAm gel partially occupied chitosan pores while the interconnected porous structure of chitosan was preserved. MSCs were incorporated within the hybrid and cell proliferation and chondrogenic differentiation were monitored. After 7-day incubation of the cell-laden constructs in a growth medium, the cell viability in CSNI100 and CSNI400 were 54 and 108% higher than that in CS alone, respectively. Glycosaminoglycan and total collagen contents increased 2.6- and 2.5-fold after 28-day culture of cell-laden CSNI400 in the chondrogenic medium. These results suggest that the hybrid structure composed of the chitosan porous scaffold and the well-defined PNIPAAm hydrogel, in particular CSNI400, is suitable for 3D stem cell culture and cartilage tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2764-2774, 2016. © 2016 Wiley Periodicals, Inc.

Chondrogenic Differentiation Increases Antidonor Immune Response to Allogeneic Mesenchymal Stem Cell Transplantation

PubMed Central

Ryan, Aideen E; Lohan, Paul; O'Flynn, Lisa; Treacy, Oliver; Chen, Xizhe; Coleman, Cynthia; Shaw, Georgina; Murphy, Mary; Barry, Frank; Griffin, Matthew D; Ritter, Thomas

2014-01-01

Allogeneic mesenchymal stem cells (allo-MSCs) have potent regenerative and immunosuppressive potential and are being investigated as a therapy for osteoarthritis; however, little is known about the immunological changes that occur in allo-MSCs after ex vivo induced or in vivo differentiation. Three-dimensional chondrogenic differentiation was induced in an alginate matrix, which served to immobilize and potentially protect MSCs at the site of implantation. We show that allogeneic differentiated MSCs lost the ability to inhibit T-cell proliferation in vitro, in association with reduced nitric oxide and prostaglandin E2 secretion. Differentiation altered immunogenicity as evidenced by induced proliferation of allogeneic T cells and increased susceptibility to cytotoxic lysis by allo-specific T cells. Undifferentiated or differentiated allo-MSCs were implanted subcutaneously, with and without alginate encapsulation. Increased CD3+ and CD68+ infiltration was evident in differentiated and splenocyte encapsulated implants only. Without encapsulation, increased local memory T-cell responses were detectable in recipients of undifferentiated and differentiated MSCs; however, only differentiated MSCs induced systemic memory T-cell responses. In recipients of encapsulated allogeneic cells, only differentiated allo-MSCs induced memory T-cell responses locally and systemically. Systemic alloimmune responses to differentiated MSCs indicate immunogenicity regardless of alginate encapsulation and may require immunosuppressive therapy for therapeutic use. PMID:24184966

In vitro expansion impaired the stemness of early passage mesenchymal stem cells for treatment of cartilage defects

PubMed Central

Jiang, Tongmeng; Xu, Guojie; Wang, Qiuyan; Yang, Lihui; Zheng, Li; Zhao, Jinmin; Zhang, Xingdong

2017-01-01

In vitro cultured autologous mesenchymal stem cells (MSCs) within passage 5 have been approved for clinical application in stem cell-based treatment of cartilage defects. However, their chondrogenic potential has not yet been questioned or verified. In this study, the chondrogenic potential of bone marrow MSCs at passage 3 (P3 BMSCs) was investigated both in cartilage repair and in vitro, with freshly isolated bone marrow mononuclear cells (BMMNCs) as controls. The results showed that P3 BMSCs were inferior to BMMNCs not only in their chondrogenic differentiation ability but also as candidates for long-term repair of cartilage defects. Compared with BMMNCs, P3 BMSCs presented a decay in telomerase activity and a change in chromosomal morphology with potential anomalous karyotypes, indicating senescence. In addition, interindividual variability in P3 BMSCs is much higher than in BMMNCs, demonstrating genomic instability. Interestingly, remarkable downregulation in cell cycle, DNA replication and mismatch repair (MMR) pathways as well as in multiple genes associated with telomerase activity and chromosomal stability were found in P3 BMSCs. This result indicates that telomerase and chromosome anomalies might originate from expansion, leading to impaired stemness and pluripotency of stem cells. In vitro culture and expansion are not recommended for cell-based therapy, and fresh BMMNCs are the first choice. PMID:28569773

Osteochondral tissue formation through adipose-derived stromal cell differentiation on biomimetic polycaprolactone nanofibrous scaffolds with graded insulin and Beta-glycerophosphate concentrations.

PubMed

Erisken, Cevat; Kalyon, Dilhan M; Wang, Hongjun; Ornek-Ballanco, Ceren; Xu, Jiahua

2011-05-01

The ability to fabricate tissue engineering scaffolds containing systematic gradients in the distributions of stimulators provides additional means for the mimicking of the important gradients observed in native tissues. Here the concentration distributions of two bioactive agents were varied concomitantly for the first time (one increasing, whereas the other decreasing monotonically) in between the two sides of a nanofibrous scaffold. This was achieved via the application of a new processing method, that is, the twin-screw extrusion and electrospinning method, to generate gradients of insulin, a stimulator of chondrogenic differentiation, and β-glycerophosphate (β-GP), for mineralization. The graded poly(ɛ-caprolactone) mesh was seeded with human adipose-derived stromal cells and cultured over 8 weeks. The resulting tissue constructs were analyzed for and revealed indications of selective differentiation of human adipose-derived stromal cells toward chondrogenic lineage and mineralization as functions of position as a result of the corresponding concentrations of insulin and β-GP. Chondrogenic differentiation of the stem cells increased at insulin-rich locations and mineralization increased at β-GP-rich locations.

In vitro cartilage construct generation from silk fibroin- chitosan porous scaffold and umbilical cord blood derived human mesenchymal stem cells in dynamic culture condition.

PubMed

Agrawal, Parinita; Pramanik, Krishna; Biswas, Amit; Ku Patra, Ranjan

2018-02-01

Cartilage construct generation includes a scaffold with appropriate composition to mimic matrix of the damaged tissue on which the stem cells grow and differentiate. In this study, umbilical cord blood (UCB) derived human mesenchymal stem cells (hMSCs) were seeded on freeze dried porous silk-fibroin (SF)/chitosan (CS) scaffolds. Influence of static and dynamic (spinner flask bioreactor) culture conditions on the developing cartilage construct were studied by in-vitro characterization for viability, proliferation, distribution, and chondrogenic differentiation of hMSCs over the scaffold. Constructs developed in spinner flask consisted of 62% live cells, and exhibited 543% more cell density at the core than constructs cultured in static system. Quantification of DNA and glycosaminoglycans accumulation after 21 days showed the progression of chondrogenic differentiation of hMSCs was higher in dynamic culture compared to static one. In constructs generated under dynamic condition, histology staining for proteoglycan matrix, and fluorescence staining for collagen-II and aggrecan showed positive correlation between early and late stage chondrogenic markers, which was further confirmed by quantitative PCR analysis, showing low collagen-I expression and highly expressed Sox9, collagen-II and aggrecan. The present study demonstrated that construct generated by combining 3D SF/CS scaffold with UCB-hMSCs under dynamic condition using spinner flask bioreactor can be used for cartilage tissue regeneration for future medical treatments. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 397-407, 2018. © 2017 Wiley Periodicals, Inc.

Repair of injured articular and growth plate cartilage using mesenchymal stem cells and chondrogenic gene therapy.

PubMed

Xian, Cory J; Foster, Bruce K

2006-05-01

Injuries to the articular cartilage and growth plate are significant clinical problems due to their limited ability to regenerate themselves. Despite progress in orthopedic surgery and some success in development of chondrocyte transplantation treatment and in early tissue-engineering work, cartilage regeneration using a biological approach still remains a great challenge. In the last 15 years, researchers have made significant advances and tremendous progress in exploring the potentials of mesenchymal stem cells (MSCs) in cartilage repair. These include (a) identifying readily available sources of and devising appropriate techniques for isolation and culture expansion of MSCs that have good chondrogenic differentiation capability, (b) discovering appropriate growth factors (such as TGF-beta, IGF-I, BMPs, and FGF-2) that promote MSC chondrogenic differentiation, (c) identifying or engineering biological or artificial matrix scaffolds as carriers for MSCs and growth factors for their transplantation and defect filling. In addition, representing another new perspective for cartilage repair is the successful demonstration of gene therapy with chondrogenic growth factors or inflammatory inhibitors (either individually or in combination), either directly to the cartilage tissue or mediated through transducing and transplanting cultured chondrocytes, MSCs or other mesenchymal cells. However, despite these rapid pre-clinical advances and some success in engineering cartilage-like tissue and in repairing articular and growth plate cartilage, challenges of their clinical translation remain. To achieve clinical effectiveness, safety, and practicality of using MSCs for cartilage repair, one critical investigation will be to examine the optimal combination of MSC sources, growth factor cocktails, and supporting carrier matrixes. As more insights are acquired into the critical factors regulating MSC migration, proliferation and chondrogenic differentiation both ex vivo and in vivo, it will be possible clinically to orchestrate desirable repair of injured articular and growth plate cartilage, either by transplanting ex vivo expanded MSCs or MSCs with genetic modifications, or by mobilising endogenous MSCs from adjacent source tissues such as synovium, bone marrow, or trabecular bone.

Proteomic Analysis Profile of Engineered Articular Cartilage with Chondrogenic Differentiated Adipose Tissue-Derived Stem Cells Loaded Polyglycolic Acid Mesh for Weight-Bearing Area Defect Repair

PubMed Central

Gong, Lunli; Zhou, Xiao; Wu, Yaohao; Zhang, Yun; Wang, Chen; Zhou, Heng; Guo, Fangfang

2014-01-01

The present study was designed to investigate the possibility of full-thickness defects repair in porcine articular cartilage (AC) weight-bearing area using chondrogenic differentiated autologous adipose-derived stem cells (ASCs) with a follow-up of 3 and 6 months, which is successive to our previous study on nonweight-bearing area. The isolated ASCs were seeded onto the phosphoglycerate/polylactic acid (PGA/PLA) with chondrogenic induction in vitro for 2 weeks as the experimental group prior to implantation in porcine AC defects (8 mm in diameter, deep to subchondral bone), with PGA/PLA only as control. With follow-up time being 3 and 6 months, both neo-cartilages of postimplantation integrated well with the neighboring normal cartilage and subchondral bone histologically in experimental group, whereas only fibrous tissue in control group. Immunohistochemical and toluidine blue staining confirmed similar distribution of COL II and glycosaminoglycan in the regenerated cartilage to the native one. A vivid remolding process with repair time was also witnessed in the neo-cartilage as the compressive modulus significantly increased from 70% of the normal cartilage at 3 months to nearly 90% at 6 months, which is similar to our former research. Nevertheless, differences of the regenerated cartilages still could be detected from the native one. Meanwhile, the exact mechanism involved in chondrogenic differentiation from ASCs seeded on PGA/PLA is still unknown. Therefore, proteome is resorted leading to 43 proteins differentially identified from 20 chosen two-dimensional spots, which do help us further our research on some committed factors. In conclusion, the comparison via proteome provided a thorough understanding of mechanisms implicating ASC differentiation toward chondrocytes, which is further substantiated by the present study as a perfect supplement to the former one in nonweight-bearing area. PMID:24044689

Continuing differentiation of human mesenchymal stem cells and induced chondrogenic and osteogenic lineages in electrospun PLGA nanofiber scaffold

PubMed Central

Xin, Xuejun; Hussain, Mohammad; Mao, Jeremy J.

2010-01-01

Nanofibers have recently gained substantial interest for potential applications in tissue engineering. The objective of this study was to determine whether electrospun nanofibers accommodate the viability, growth, and differentiation of human mesenchymal stem cells (hMSCs) as well as their osteogenic (hMSC-Ob) and chondrogenic (hMSC-Ch) derivatives. Poly(D,L-lactide-co-glycolide) (PLGA) beads with a PLA:PGA ratio of 85:15 were electrospun into non-woven fibers with an average diameter of 760±210 nm. The average Young’s modulus of electrospun PLGA nanofibers was 42±26 kPa, per nanoindentation with atomic force microscopy (AFM). Human MSCs were seeded 1–4 weeks at a density of 2×106 cells/mL in PLGA nanofiber sheets. After 2 week culture on PLGA nanofiber scaffold, hMSCs remained as precursors upon immunoblotting with hKL12 antibody. SEM taken up to 7 days after cell seeding revealed that hMSCs, hMSC-Ob and hMSC-Ch apparently attached to PLGA nanofibers. The overwhelming majority of hMSCs was viable and proliferating in PLGA nanofiber scaffolds up to the tested 14 days, as assayed live/dead tests, DNA assay and BrdU. In a separate experiment, hMSCs seeded in PLGA nanofiber scaffolds were differentiated into chodrogenic and osteogenic cells. Histological assays revealed that hMSCs continuously differentiated into chondrogenic cells and osteogenic cells after 2 week incubation in PLGA nanofibers. Taken together, these data represent an original investigation of continuous differentiation of hMSCs into chondrogenic and osteogenic cells in PLGA nanofiber scaffold. Consistent with previous work, these findings also suggest that nanofibers may serve as accommodative milieu for not only hMSCs, but also as a 3D carrier vehicle for lineage specific cells. PMID:17010425

[In vitro differentiation of synovial-derived mesenchymal stem cells infected by adenovirus vector mediated by bone morphogenetic protein 2/7 genes into fibrocartilage cells in rabbits].

PubMed

Fu, Peiliang; Zhang, Lei; Wu, Haishan; Cong, Ruijun; Chen, Song; Ding, Zheru; Hu, Kaimen

2013-03-01

To investigate the feasibility of rabbit synovial-derived mesenchymal stem cells (SMSCs) differentiating into fibrocartilage cells by the recombinant adenovirus vector mediated by bone morphogenetic protein 2/7 (BMP-2/7) genes in vitro. SMSCs were isolated and purified from 3-month-old New Zealand white rabbits [male or female, weighing (2.1 +/- 0.3) kg]; the morphology was observed; the cells were identified with immunocytological fluorescent staining, flow cytometry, and cell cycles. The adipogenic, osteogenic, and chondrogenic differentiations were detected. The recombinant plasmid of pAdTrack-BMP-2-internal ribosome entry site (IRES)-BMP-7 was constructed and then was used to infect SMSCs. The cell DNA content and the oncogenicity were tested to determine the safety. Then infected SMSCs were cultured in incomplete chondrogenic medium in vitro. Chondrogenic differentiation of infected SMSCs was detected by RT-PCR, immunofluorescent staining, and toluidine blue staining. SMSCs expressed surface markers of stem cells, and had multi-directional potential. The transfection efficiency of SMSCs infected by recombinant plasmid of pAdTrack-BMP-2-IRES-BMP-7 was about 70%. The safety results showed that infected SMSCs had normal double time, normal chromosome number, and normal DNA content and had no oncogenicity. At 21 days after cultured in incomplete chondrocyte medium, RT-PCR results showed SMSCs had increased expressions of collegan type I and collegan type II, particularly collegan type II; the expressions of RhoA and Sox-9 increased obviously. Immunofluorescent staining and toluidine blue staining showed differentiation of SMSCs into fibrocartilage cells. It is safe to use pAdTrack-BMP-2-IRES-BMP-7 for infecting SMSCs. SMSCs infected by pAdTrack-BMP-2-IRES-BMP-7 can differentiate into fibrocartilage cells spontaneously in vitro.

Hydrostatic pressure promotes the proliferation and osteogenic/chondrogenic differentiation of mesenchymal stem cells: The roles of RhoA and Rac1.

PubMed

Zhao, Yin-Hua; Lv, Xin; Liu, Yan-Li; Zhao, Ying; Li, Qiang; Chen, Yong-Jin; Zhang, Min

2015-05-01

Our previous studies have shown that hydrostatic pressure can serve as an active regulator for bone marrow mesenchymal stem cells (BMSCs). The current work further investigates the roles of cytoskeletal regulatory proteins Ras homolog gene family member A (RhoA) and Ras-related C3 botulinum toxin substrate 1 (Rac1) in hydrostatic pressure-related effects on BMSCs. Flow cytometry assays showed that the hydrostatic pressure promoted cell cycle initiation in a RhoA- and Rac1-dependent manner. Furthermore, fluorescence assays confirmed that RhoA played a positive and Rac1 displayed a negative role in the hydrostatic pressure-induced F-actin stress fiber assembly. Western blots suggested that RhoA and Rac1 play central roles in the pressure-inhibited ERK phosphorylation, and Rac1 but not RhoA was involved in the pressure-promoted JNK phosphorylation. Finally, real-time polymerase chain reaction (PCR) experiments showed that pressure promoted the expression of osteogenic marker genes in BMSCs at an early stage of osteogenic differentiation through the up-regulation of RhoA activity. Additionally, the PCR results showed that pressure enhanced the expression of chondrogenic marker genes in BMSCs during chondrogenic differentiation via the up-regulation of Rac1 activity. Collectively, our results suggested that RhoA and Rac1 are critical to the pressure-induced proliferation and differentiation, the stress fiber assembly, and MAPK activation in BMSCs. Copyright © 2015. Published by Elsevier B.V.

Osteoarthritis-derived chondrocytes are a potential source of multipotent progenitor cells for cartilage tissue engineering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oda, Tomoyuki; Sakai, Tadahiro; Hiraiwa, Hideki

The natural healing capacity of damaged articular cartilage is poor, rendering joint surface injuries a prime target for regenerative medicine. While autologous chondrocyte or mesenchymal stem cell (MSC) implantation can be applied to repair cartilage defects in young patients, no appropriate long-lasting treatment alternative is available for elderly patients with osteoarthritis (OA). Multipotent progenitor cells are reported to present in adult human articular cartilage, with a preponderance in OA cartilage. These facts led us to hypothesize the possible use of osteoarthritis-derived chondrocytes as a cell source for cartilage tissue engineering. We therefore analyzed chondrocyte- and stem cell-related markers, cell growthmore » rate, and multipotency in OA chondrocytes (OACs) and bone marrow-derived MSCs, along with normal articular chondrocytes (ACs) as a control. OACs demonstrated similar phenotype and proliferation rate to MSCs. Furthermore, OACs exhibited multilineage differentiation ability with a greater chondrogenic differentiation ability than MSCs, which was equivalent to ACs. We conclude that chondrogenic capacity is not significantly affected by OA, and OACs could be a potential source of multipotent progenitor cells for cartilage tissue engineering. - Highlights: • Osteoarthritis chondrocytes (OACs) have multilineage differentiation capacity. • Articular chondrocytes (ACs) and OACs have similar gene expression profiles. • OACs have high chondrogenic potential. • OACs could be a cell resource for cartilage tissue engineering.« less

Mangiferin Reduces the Inhibition of Chondrogenic Differentiation by IL-1β in Mesenchymal Stem Cells from Subchondral Bone and Targets Multiple Aspects of the Smad and SOX9 Pathways

PubMed Central

Huh, Jeong-Eun; Koh, Pil-Seong; Seo, Byung-Kwan; Park, Yeon-Chul; Baek, Yong-Hyun; Lee, Jae-Dong; Park, Dong-Suk

2014-01-01

Mangiferin is a natural immunomodulator found in plants including mango trees. The effects of mangiferin on chondrogenesis and cartilage repair have not yet been reported. This study was designed to determine the effect of mangiferin on chondrogenic differentiation in IL-1β-stimulated mesenchymal stem cells (MSCs) from subchondral bone and to explore the mechanisms underlying these effects. MSCs were isolated from the subchondral bone of rabbit and treated with mangiferin alone and/or interleukin-1β (IL-1β). Mangiferin induced chondrogenic differentiation in MSCs by upregulating transforming growth factor (TGF)-β, bone morphogenetic protein (BMP)-2, and BMP-4 and several key markers of chondrogenesis, including sex-determining region Y–box (SRY-box) containing gene 9 (SOX9), type 2α1 collagen (Col2α1), cartilage link protein, and aggrecan. In IL-1β-stimulated MSCs, mangiferin significantly reversed the production of TGF-β, BMP-2, BMP-4, SOX9, Col2α1, cartilage link protein, and aggrecan, as well as matrix metalloproteinase (MMP)-1, MMP-13, and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS5). Mangiferin upregulated the phosphorylation of Smad 2, Smad 3, Smad 1/5/8, and SOX9 in IL-1β-stimulated MSCs. In the presence of mangiferin, SOX9 siRNA suppressed the activation of Smad 2, Smad 3, Smad 1/5/8, aggrecan, and Col2α1 expression. In conclusion, mangiferin exhibits both chondrogenic and chondroprotective effects on damaged MSCs and mediates these effects by targeting multiple aspects of the Smad and SOX9 signaling pathways. PMID:25216336

Mangiferin reduces the inhibition of chondrogenic differentiation by IL-1β in mesenchymal stem cells from subchondral bone and targets multiple aspects of the Smad and SOX9 pathways.

PubMed

Huh, Jeong-Eun; Koh, Pil-Seong; Seo, Byung-Kwan; Park, Yeon-Chul; Baek, Yong-Hyun; Lee, Jae-Dong; Park, Dong-Suk

2014-09-11

Mangiferin is a natural immunomodulator found in plants including mango trees. The effects of mangiferin on chondrogenesis and cartilage repair have not yet been reported. This study was designed to determine the effect of mangiferin on chondrogenic differentiation in IL-1β-stimulated mesenchymal stem cells (MSCs) from subchondral bone and to explore the mechanisms underlying these effects. MSCs were isolated from the subchondral bone of rabbit and treated with mangiferin alone and/or interleukin-1β (IL-1β). Mangiferin induced chondrogenic differentiation in MSCs by upregulating transforming growth factor (TGF)-β, bone morphogenetic protein (BMP)-2, and BMP-4 and several key markers of chondrogenesis, including sex-determining region Y-box (SRY-box) containing gene 9 (SOX9), type 2α1 collagen (Col2α1), cartilage link protein, and aggrecan. In IL-1β-stimulated MSCs, mangiferin significantly reversed the production of TGF-β, BMP-2, BMP-4, SOX9, Col2α1, cartilage link protein, and aggrecan, as well as matrix metalloproteinase (MMP)-1, MMP-13, and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS5). Mangiferin upregulated the phosphorylation of Smad 2, Smad 3, Smad 1/5/8, and SOX9 in IL-1β-stimulated MSCs. In the presence of mangiferin, SOX9 siRNA suppressed the activation of Smad 2, Smad 3, Smad 1/5/8, aggrecan, and Col2α1 expression. In conclusion, mangiferin exhibits both chondrogenic and chondroprotective effects on damaged MSCs and mediates these effects by targeting multiple aspects of the Smad and SOX9 signaling pathways.

Clonal analysis of synovial fluid stem cells to characterize and identify stable mesenchymal stromal cell/mesenchymal progenitor cell phenotypes in a porcine model: a cell source with enhanced commitment to the chondrogenic lineage.

PubMed

Ando, Wataru; Kutcher, Josh J; Krawetz, Roman; Sen, Arindom; Nakamura, Norimasa; Frank, Cyril B; Hart, David A

2014-06-01

Previous studies have demonstrated that porcine synovial membrane stem cells can adhere to a cartilage defect in vivo through the use of a tissue-engineered construct approach. To optimize this model, we wanted to compare effectiveness of tissue sources to determine whether porcine synovial fluid, synovial membrane, bone marrow and skin sources replicate our understanding of synovial fluid mesenchymal stromal cells or mesenchymal progenitor cells from humans both at the population level and the single-cell level. Synovial fluid clones were subsequently isolated and characterized to identify cells with a highly characterized optimal phenotype. The chondrogenic, osteogenic and adipogenic potentials were assessed in vitro for skin, bone marrow, adipose, synovial fluid and synovial membrane-derived stem cells. Synovial fluid cells then underwent limiting dilution analysis to isolate single clonal populations. These clonal populations were assessed for proliferative and differentiation potential by use of standardized protocols. Porcine-derived cells demonstrated the same relationship between cell sources as that demonstrated previously for humans, suggesting that the pig may be an ideal preclinical animal model. Synovial fluid cells demonstrated the highest chondrogenic potential that was further characterized, demonstrating the existence of a unique clonal phenotype with enhanced chondrogenic potential. Porcine stem cells demonstrate characteristics similar to those in human-derived mesenchymal stromal cells from the same sources. Synovial fluid-derived stem cells contain an inherent phenotype that may be optimal for cartilage repair. This must be more fully investigated for future use in the in vivo tissue-engineered construct approach in this physiologically relevant preclinical porcine model. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

The influence of ferucarbotran on the chondrogenesis of human mesenchymal stem cells

PubMed Central

Henning, Tobias D; Sutton, Elizabeth J; Kim, Anne; Golovko, Daniel; Horvai, Andrew; Ackerman, Larry; Sennino, Barbara; McDonald, Donald; Lotz, Jeffrey; Daldrup-Link, Heike E

2010-01-01

For in vivo applications of magnetically labeled stem cells, biological effects of the labeling procedure have to be precluded. This study evaluates the effect of different Ferucarbotran cell labeling protocols on chondrogenic differentiation of human mesenchymal stem cells (hMSC) as well as their implications for MR imaging. hMSC were labeled with Ferucarbotran using various protocols: Cells were labeled with 100μg Fe/ml for 4h and 18h and additional samples were cultured for 6 or 12 days after the 18-hour labeling. Supplementary samples were labeled by transfection with protamine sulfate. Iron uptake was quantified by ICP-spectrometry and labeled cells were investigated by transmission electron microscopy and by immunostaining for ferucarbotran. The differentiation potential of labeled cells was compared to unlabeled controls by staining with alcian blue and hematoxylin & eosin, then quantified by measurements of glucosaminoglycans (GAG). Contrast agent effect at 3T was investigated on day 1 and day 14 of chondrogenic differentiation by measuring signal-to-noise ratios on T2-SE and T2*-GE-sequences. Iron uptake was significant for all labeling protocols (p< 0.05). The uptake was highest after transfection with protamine sulfate (25.65 ± 3.96 pg/cell) and lowest at an incubation time of 4h without transfection (3.21 ± 0.21 pg/cell). While chondrogenic differentiation was decreased using all labeling protocols, the decrease in GAG synthesis was not significant after labeling for 4h without transfection. After labeling by simple incubation, chondrogenesis was found to be dose-dependent. MR imaging showed markedly lower SNR values of all labeled cells compared to the unlabeled controls. This contrast agent effect persisted for 14 days and the duration of differentiation. Magnetic labeling of hMSC with ferucarbotran inhibits chondrogenesis in a dose-dependent manner when using simple incubation techniques. When decreasing the incubation time to 4h, inhibition of chondrogenesis was not significant. PMID:19670250

Cartilage fragments from osteoarthritic knee promote chondrogenesis of mesenchymal stem cells without exogenous growth factor induction.

PubMed

Chen, Chia-Chun; Liao, Cheng-Hao; Wang, Yao-Horng; Hsu, Yuan-Ming; Huang, Shih-Horng; Chang, Chih-Hung; Fang, Hsu-Wei

2012-03-01

Extracellular matrix (ECM) is thought to participate significantly in guiding the differentiation process of mesenchymal stem cells (MSCs). In this study, we hypothesized that cartilage fragments from osteoarthritic knee could promote chondrogenesis of MSCs. Nonworn parts of cartilage tissues were obtained during total knee arthroplasty (TKA) surgery. Cartilage fragments and MSCs were wrapped into fibrin glue; and the constructs were implanted subcutaneously into nude mice. Histological analysis showed neocartilage-like structure with positive Alcian blue staining in the cartilage fragment-fibrin-MSC constructs. However, constructs with only MSCs in fibrin showed condensed appearance like MSCs in the pellet culture. Gene expression of type II collagen in the constructs with 60 mg cartilage fragments were significantly elevated after 4 weeks of implantation. Conversely, the constructs without cartilage fragments failed to express type II collagen, which indicated MSCs did not differentiate into a chondrogenic lineage. In conclusion, we demonstrated the effect of cartilage fragments from osteoarthritic knee in promoting chondrogenic differentiation of MSCs. This may be a favorable strategy for MSC chondrogenesis without exogenous growth factor induction. Copyright © 2011 Orthopaedic Research Society.

Genome-wide map of quantified epigenetic changes during in vitro chondrogenic differentiation of primary human mesenchymal stem cells.

PubMed

Herlofsen, Sarah R; Bryne, Jan Christian; Høiby, Torill; Wang, Li; Issner, Robbyn; Zhang, Xiaolan; Coyne, Michael J; Boyle, Patrick; Gu, Hongcang; Meza-Zepeda, Leonardo A; Collas, Philippe; Mikkelsen, Tarjei S; Brinchmann, Jan E

2013-02-15

For safe clinical application of engineered cartilage made from mesenchymal stem cells (MSCs), molecular mechanisms for chondrogenic differentiation must be known in detail. Changes in gene expression and extracellular matrix synthesis have been extensively studied, but the epigenomic modifications underlying these changes have not been described. To this end we performed whole-genome chromatin immunoprecipitation and deep sequencing to quantify six histone modifications, reduced representation bisulphite sequencing to quantify DNA methylation and mRNA microarrays to quantify gene expression before and after 7 days of chondrogenic differentiation of MSCs in an alginate scaffold. To add to the clinical relevance of our observations, the study is based on primary bone marrow-derived MSCs from four donors, allowing us to investigate inter-individual variations. We see two levels of relationship between epigenetic marking and gene expression. First, a large number of genes ontogenetically linked to MSC properties and the musculoskeletal system are epigenetically prepatterned by moderate changes in H3K4me3 and H3K9ac near transcription start sites. Most of these genes remain transcriptionally unaltered. Second, transcriptionally upregulated genes, more closely associated with chondrogenesis, are marked by H3K36me3 in gene bodies, highly increased H3K4me3 and H3K9ac on promoters and 5' end of genes, and increased H3K27ac and H3K4me1 marking in at least one enhancer region per upregulated gene. Within the 7-day time frame, changes in promoter DNA methylation do not correlate significantly with changes in gene expression. Inter-donor variability analysis shows high level of similarity between the donors for this data set. Histone modifications, rather than DNA methylation, provide the primary epigenetic control of early differentiation of MSCs towards the chondrogenic lineage.

Differential Effects of Small Molecule WNT Agonists on the Multilineage Differentiation Capacity of Human Mesenchymal Stem Cells.

PubMed

Narcisi, Roberto; Arikan, Ozan H; Lehmann, Johannes; Ten Berge, Derk; van Osch, Gerjo J V M

2016-11-01

Human bone marrow-derived mesenchymal stem cells (MSCs) are promising candidates for cell-based therapies, but loss of expansion and differentiation potential in vitro limits their applicability. Recently we showed that WNT3A protein promoted MSC proliferation and enhanced their chondrogenic potential, while simultaneously suppressing the propensity of the cartilage to undergo hypertrophic maturation. Since WNT3A protein is costly and rapidly loses its activity in culture, we investigated the possibility of replacing it with cheaper commercially available WNT agonists, specifically lithium chloride (LiCl), CHIR99021 (CHIR), SKL2001, and AMBMP. Of these, we found that only CHIR and LiCl stimulated MSC proliferation. Moreover, CHIR enhanced the chondrogenic capacity of MSCs, whereas LiCl predominantly increased the osteo- and adipogenic capacity. The different WNT agonists also differentially impacted the surface marker profile of the MSCs, possibly explaining the observed differences. Moreover, CHIR suppressed the hypertrophic propensity of the MSC-derived cartilage after in vivo implantation to an extent approaching that of WNT3A protein. These results indicate that CHIR may be a promising alternative for WNT3A protein for certain applications of human bone marrow-derived MSCs.

Homing of mesenchymal stem cells: mechanistic or stochastic? Implications for targeted delivery in arthritis.

PubMed

Eseonu, Onyedikachi I; De Bari, Cosimo

2015-02-01

Mesenchymal stem cells (MSCs) are multipotent cells with the capacity to undergo chondrogenic differentiation. Systemically administered MSCs have been shown to preferentially accumulate at sites of tissue damage and inflammation, thus MSC-based therapy holds great promise for the treatment of inflammatory diseases such as RA. Modulation of MSC homing may allow targeted delivery of systemically administered MSCs to damaged articular cartilage, where they can suppress immune-mediated cartilage destruction and contribute to cartilage repair via a combination of chondrogenic differentiation and paracrine stimulation of intrinsic residual repair. To harness the potential of MSC homing, a thorough understanding of the mechanism is key. This review discusses current knowledge of the mechanism of MSC homing to injured/inflamed tissue and its implications for targeted MSC-based therapy in arthritis. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Hydrophilic polyurethane matrix promotes chondrogenesis of mesenchymal stem cells☆

PubMed Central

Nalluri, Sandeep M.; Krishnan, G. Rajesh; Cheah, Calvin; Arzumand, Ayesha; Yuan, Yuan; Richardson, Caley A.; Yang, Shuying; Sarkar, Debanjan

2016-01-01

Segmental polyurethanes exhibit biphasic morphology and can control cell fate by providing distinct matrix guided signals to increase the chondrogenic potential of mesenchymal stem cells (MSCs). Polyethylene glycol (PEG) based hydrophilic polyurethanes can deliver differential signals to MSCs through their matrix phases where hard segments are cell-interactive domains and PEG based soft segments are minimally interactive with cells. These coordinated communications can modulate cell–matrix interactions to control cell shape and size for chondrogenesis. Biphasic character and hydrophilicity of polyurethanes with gel like architecture provide a synthetic matrix conducive for chondrogenesis of MSCs, as evidenced by deposition of cartilage-associated extracellular matrix. Compared to monophasic hydrogels, presence of cell interactive domains in hydrophilic polyurethanes gels can balance cell–cell and cell–matrix interactions. These results demonstrate the correlation between lineage commitment and the changes in cell shape, cell–matrix interaction, and cell–cell adhesion during chondrogenic differentiation which is regulated by polyurethane phase morphology, and thus, represent hydrophilic polyurethanes as promising synthetic matrices for cartilage regeneration. PMID:26046282

Seven diverse human embryonic stem cell-derived chondrogenic clonal embryonic progenitor cell lines display site-specific cell fates.

PubMed

Sternberg, Hal; Kidd, Jennifer; Murai, James T; Jiang, Jianjie; Rinon, Ariel; Erickson, Isaac E; Funk, Walter D; Wang, Qian; Chapman, Karen B; Vangsness, C Thomas; West, Michael D

2013-03-01

The transcriptomes of seven diverse clonal human embryonic progenitor cell lines with chondrogenic potential were compared with that of bone marrow-derived mesenchymal stem cells (MSCs). The cell lines 4D20.8, 7PEND24, 7SMOO32, E15, MEL2, SK11 and SM30 were compared with MSCs using immunohistochemical methods, gene expression microarrays and quantitative real-time PCR. In the undifferentiated progenitor state, each line displayed unique combinations of site-specific markers, including AJAP1, ALDH1A2, BMP5, BARX1, HAND2, HOXB2, LHX1, LHX8, PITX1, TBX15 and ZIC2, but none of the lines expressed the MSC marker CD74. The lines showed diverse responses when differentiated in the presence of combinations of TGF-β3, BMP2, 4, 6 and 7 and GDF5, with the lines 4D20.8, SK11, SM30 and MEL2 showing osteogenic markers in some differentiation conditions. The line 7PEND24 showed evidence of regenerating articular cartilage and, in some conditions, markers of tendon differentiation. The scalability of site-specific clonal human embryonic stem cell-derived embryonic progenitor cell lines may provide novel models for the study of differentiation and methods for preparing purified and identified cells types for use in therapy.

SMAD3 and SMAD4 have a more dominant role than SMAD2 in TGFβ-induced chondrogenic differentiation of bone marrow-derived mesenchymal stem cells

PubMed Central

de Kroon, Laurie M. G.; Narcisi, Roberto; van den Akker, Guus G. H.; Vitters, Elly L.; Blaney Davidson, Esmeralda N.; van Osch, Gerjo J. V. M.; van der Kraan, Peter M.

2017-01-01

To improve cartilage formation by bone marrow-derived mesenchymal stem cells (BMSCs), the signaling mechanism governing chondrogenic differentiation requires better understanding. We previously showed that the transforming growth factor-β (TGFβ) receptor ALK5 is crucial for chondrogenesis induced by TGFβ. ALK5 phosphorylates SMAD2 and SMAD3 proteins, which then form complexes with SMAD4 to regulate gene transcription. By modulating the expression of SMAD2, SMAD3 and SMAD4 in human BMSCs, we investigated their role in TGFβ-induced chondrogenesis. Activation of TGFβ signaling, represented by SMAD2 phosphorylation, was decreased by SMAD2 knockdown and highly increased by SMAD2 overexpression. Moreover, TGFβ signaling via the alternative SMAD1/5/9 pathway was strongly decreased by SMAD4 knockdown. TGFβ-induced chondrogenesis of human BMSCs was strongly inhibited by SMAD4 knockdown and only mildly inhibited by SMAD2 knockdown. Remarkably, both knockdown and overexpression of SMAD3 blocked chondrogenic differentiation. Chondrogenesis appears to rely on a delicate balance in the amount of SMAD3 and SMAD4 as it was not enhanced by SMAD4 overexpression and was inhibited by SMAD3 overexpression. Furthermore, this study reveals that TGFβ-activated phosphorylation of SMAD2 and SMAD1/5/9 depends on the abundance of SMAD4. Overall, our findings suggest a more dominant role for SMAD3 and SMAD4 than SMAD2 in TGFβ-induced chondrogenesis of human BMSCs. PMID:28240243

Hypoxia Is a Critical Parameter for Chondrogenic Differentiation of Human Umbilical Cord Blood Mesenchymal Stem Cells in Type I/III Collagen Sponges

PubMed Central

Gómez-Leduc, Tangni; Desancé, Mélanie; Hervieu, Magalie; Legendre, Florence; Ollitrault, David; de Vienne, Claire; Herlicoviez, Michel; Galéra, Philippe; Demoor, Magali

2017-01-01

Umbilical cord blood (UCB) is an attractive alternative to bone marrow for isolation of mesenchymal stem cells (MSCs) to treat articular cartilage defects. Here, we set out to determine the growth factors (bone morphogenetic protein 2 (BMP-2) and transforming growth factor-β (TGF-β1)) and oxygen tension effects during chondrogenesis of human UCB-MSCs for cartilage engineering. Chondrogenic differentiation was induced using 3D cultures in type I/III collagen sponges with chondrogenic factors in normoxia (21% O2) or hypoxia (<5% O2) for 7, 14 and 21 days. Our results show that UCB-MSCs can be committed to chondrogenesis in the presence of BMP-2+TGF-β1. Normoxia induced the highest levels of chondrocyte-specific markers. However, hypoxia exerted more benefit by decreasing collagen X and matrix metalloproteinase-13 (MMP13) expression, two chondrocyte hypertrophy markers. However, a better chondrogenesis was obtained by switching oxygen conditions, with seven days in normoxia followed by 14 days in hypoxia, since these conditions avoid hypertrophy of hUCB-MSC-derived chondrocytes while maintaining the expression of chondrocyte-specific markers observed in normoxia. Our study demonstrates that oxygen tension is a key factor for chondrogenesis and suggests that UBC-MSCs 3D-culture should begin in normoxia to obtain a more efficient chondrocyte differentiation before placing them in hypoxia for chondrocyte phenotype stabilization. UCB-MSCs are therefore a reliable source for cartilage engineering. PMID:28885597

SMAD3 and SMAD4 have a more dominant role than SMAD2 in TGFβ-induced chondrogenic differentiation of bone marrow-derived mesenchymal stem cells.

PubMed

de Kroon, Laurie M G; Narcisi, Roberto; van den Akker, Guus G H; Vitters, Elly L; Blaney Davidson, Esmeralda N; van Osch, Gerjo J V M; van der Kraan, Peter M

2017-02-27

To improve cartilage formation by bone marrow-derived mesenchymal stem cells (BMSCs), the signaling mechanism governing chondrogenic differentiation requires better understanding. We previously showed that the transforming growth factor-β (TGFβ) receptor ALK5 is crucial for chondrogenesis induced by TGFβ. ALK5 phosphorylates SMAD2 and SMAD3 proteins, which then form complexes with SMAD4 to regulate gene transcription. By modulating the expression of SMAD2, SMAD3 and SMAD4 in human BMSCs, we investigated their role in TGFβ-induced chondrogenesis. Activation of TGFβ signaling, represented by SMAD2 phosphorylation, was decreased by SMAD2 knockdown and highly increased by SMAD2 overexpression. Moreover, TGFβ signaling via the alternative SMAD1/5/9 pathway was strongly decreased by SMAD4 knockdown. TGFβ-induced chondrogenesis of human BMSCs was strongly inhibited by SMAD4 knockdown and only mildly inhibited by SMAD2 knockdown. Remarkably, both knockdown and overexpression of SMAD3 blocked chondrogenic differentiation. Chondrogenesis appears to rely on a delicate balance in the amount of SMAD3 and SMAD4 as it was not enhanced by SMAD4 overexpression and was inhibited by SMAD3 overexpression. Furthermore, this study reveals that TGFβ-activated phosphorylation of SMAD2 and SMAD1/5/9 depends on the abundance of SMAD4. Overall, our findings suggest a more dominant role for SMAD3 and SMAD4 than SMAD2 in TGFβ-induced chondrogenesis of human BMSCs.

Nicotine-induced chondrogenic differentiation of human bone marrow stromal cells in vitro.

PubMed

Ying, Xiaozhou; Zhang, Wei; Cheng, Shaowen; Nie, Pengfei; Cheng, Xiaojie; Shen, Yue; Wang, Wei; Xue, Enxing; Chen, Qingyu; Kou, Dongquan; Peng, Lei; Zhang, Yu; Lu, Chuanzhu

2012-11-01

Nicotine has been reported that it has a dose-dependent effect on matrix mineralization by human bone marrow cells. However, there is no relevant research concerning on chondrogenic differentiation potential of bone marrow stromal stem cells (BMSCs) treated with nicotine in vitro. The aims of the study were to examine the effects of nicotine (0, 10(-7), 10(-6) and 10(-5) M) on the proliferation and chondrogenic differentiation of BMSCs from three healthy donors in vitro. BMSCs proliferation was analyzed by CCK8 assay and real-time polymerase chain reaction was used to assay the expression of type II collagen, aggrecan, type I collagen and type X collagen. The proteoglycan content was stained by Alcian blue, and the sulfated glycosaminoglycan (sGAG) content of BMSCs was quantified spectrofluorometrically using dimethylmethylene blue. The cell viability was not significantly impaired until up to a concentration of 10(-5) M nicotine. Nicotine promoted the proliferation and enhanced the expression of type II collagen at the level up to 10(-6) M (P < 0.05). The expression of aggrecan was reduced at the concentration of 10(-5) M nicotine at day 14 (P < 0.05), and there was no significant difference in aggrecan gene expression at 10(-7) and 10(-6) M nicotine levels compared to control group (n.s.). Also the fibroblastic and hypertrophic gene expressions were down-regulated in the chondrogenic medium with 10(-7)-10(-5) M nicotine (P < 0.05). It was implied that local application of nicotine at an appropriate concentration may be a promising approach for enhancing chondrogenic differentiation capacity of BMSCs in cell-based cartilage tissue engineering. Also these results indicate that nicotine maybe a potentially useful drug for the treatment of Osteoarthritis.

Biological and MRI characterization of biomimetic ECM scaffolds for cartilage tissue regeneration.

PubMed

Ravindran, Sriram; Kotecha, Mrignayani; Huang, Chun-Chieh; Ye, Allen; Pothirajan, Padmabharathi; Yin, Ziying; Magin, Richard; George, Anne

2015-12-01

Osteoarthritis is the most common joint disorder affecting millions of people. Most scaffolds developed for cartilage regeneration fail due to vascularization and matrix mineralization. In this study we present a chondrogenic extracellular matrix (ECM) incorporated collagen/chitosan scaffold (chondrogenic ECM scaffold) for potential use in cartilage regenerative therapy. Biochemical characterization showed that these scaffolds possess key pro-chondrogenic ECM components and growth factors. MRI characterization showed that the scaffolds possess mechanical properties and diffusion characteristics important for cartilage tissue regeneration. In vivo implantation of the chondrogenic ECM scaffolds with bone marrow derived mesenchymal stem cells (MSCs) triggered chondrogenic differentiation of the MSCs without the need for external stimulus. Finally, results from in vivo MRI experiments indicate that the chondrogenic ECM scaffolds are stable and possess MR properties on par with native cartilage. Based on our results, we envision that such ECM incorporated scaffolds have great potential in cartilage regenerative therapy. Additionally, our validation of MR parameters with histology and biochemical analysis indicates the ability of MRI techniques to track the progress of our ECM scaffolds non-invasively in vivo; highlighting the translatory potential of this technology. Copyright © 2015 Elsevier Ltd. All rights reserved.

Stem Cells in Aggregate Form to Enhance Chondrogenesis in Hydrogels

PubMed Central

Sridharan, BanuPriya; Lin, Staphany M.; Hwu, Alexander T.; Laflin, Amy D.; Detamore, Michael S.

2015-01-01

There are a variety of exciting hydrogel technologies being explored for cartilage regenerative medicine. Our overall goal is to explore whether using stem cells in an aggregate form may be advantageous in these applications. 3D stem cell aggregates hold great promise as they may recapitulate the in vivo skeletal tissue condensation, a property that is not typically observed in 2D culture. We considered two different stem cell sources, human umbilical cord Wharton’s jelly cells (hWJCs, currently being used in clinical trials) and rat bone marrow-derived mesenchymal stem cells (rBMSCs). The objective of the current study was to compare the influence of cell phenotype, aggregate size, and aggregate number on chondrogenic differentiation in a generic hydrogel (agarose) platform. Despite being differing cell sources, both rBMSC and hWJC aggregates were consistent in outperforming cell suspension control groups in biosynthesis and chondrogenesis. Higher cell density impacted biosynthesis favorably, and the number of aggregates positively influenced chondrogenesis. Therefore, we recommend that investigators employing hydrogels consider using cells in an aggregate form for enhanced chondrogenic performance. PMID:26719986

Cell type dependent morphological adaptation in polyelectrolyte hydrogels governs chondrogenic fate.

PubMed

Raghothaman, Deepak; Leong, Meng Fatt; Lim, Tze Chiun; Wan, Andrew C A; Ser, Zheng; Lee, Eng Hin; Yang, Zheng

2016-04-04

Repair of critical-size articular cartilage defects typically involves delivery of cells in biodegradable, 3D matrices. Differences in the developmental status of mesenchymal stem cells (MSCs) and terminally differentiated mature chondrocytes might be a critical factor in engineering appropriate 3D matrices for articular cartilage tissue engineering. This study examined the relationship between material-driven early cell morphological adaptations and chondrogenic outcomes, by studying the influence of aligned collagen type I (Col I) presentation on chondrocytes and MSC in interfacial polyelectrolyte complexation (IPC)-based hydrogels. In the absence of Col I, both chondrocytes and MSCs adopted rounded cell morphology and formed clusters, with chondrocyte clusters favoring the maintenance of hyaline phenotype, while MSC clusters differentiated to fibro-superficial zone-like chondrocytes. Encapsulated chondrocytes in IPC-Col I hydrogel adopted a fibroblastic morphology forming fibro-superficial zone-like phenotype, which could be reversed by inhibiting actin polymerization using cytochalasin D (CytD). In contrast, adoption of fibroblastic morphology by encapsulated MSCs in IPC-Col I facilitated superior chondrogenesis, generating a mature, hyaline neocartilage tissue. CytD treatment abrogated the elongation of MSCs and brought about a single cell-like state, resulting in insignificant chondrogenic differentiation, underscoring the essential requirement of providing matrix environments that are amenable to cell-cell interactions for robust MSC chondrogenic differentiation. Our study demonstrates that MSCs and culture-expanded chondrocytes favour differential microenvironmental niches and emphasizes the importance of designing biomaterials that meet cell type-specific requirements, in adopting chondrocyte or MSC-based approaches for regenerating hyaline, articular cartilage.

Decoding the Regulatory Landscape of Ageing in Musculoskeletal Engineered Tissues Using Genome-Wide DNA Methylation and RNASeq

PubMed Central

Peffers, Mandy Jayne; Goljanek-Whysall, Katarzyna; Collins, John; Fang, Yongxiang; Rushton, Michael; Loughlin, John; Proctor, Carole; Clegg, Peter David

2016-01-01

Mesenchymal stem cells (MSC) are capable of multipotent differentiation into connective tissues and as such are an attractive source for autologous cell-based regenerative medicine and tissue engineering. Epigenetic mechanisms, like DNA methylation, contribute to the changes in gene expression in ageing. However there was a lack of sufficient knowledge of the role that differential methylation plays during chondrogenic, osteogenic and tenogenic differentiation from ageing MSCs. This study undertook genome level determination of the effects of DNA methylation on expression in engineered tissues from chronologically aged MSCs. We compiled unique DNA methylation signatures from chondrogenic, osteogenic, and tenogenic engineered tissues derived from young; n = 4 (21.8 years ± 2.4 SD) and old; n = 4 (65.5 years±8.3SD) human MSCs donors using the Illumina HumanMethylation 450 Beadchip arrays and compared these to gene expression by RNA sequencing. Unique and common signatures of global DNA methylation were identified. There were 201, 67 and 32 chondrogenic, osteogenic and tenogenic age-related DE protein-coding genes respectively. Findings inferred the nature of the transcript networks was predominantly for ‘cell death and survival’, ‘cell morphology’, and ‘cell growth and proliferation’. Further studies are required to validate if this gene expression effect translates to cell events. Alternative splicing (AS) was dysregulated in ageing with 119, 21 and 9 differential splicing events identified in chondrogenic, osteogenic and tenogenic respectively, and enrichment in genes associated principally with metabolic processes. Gene ontology analysis of differentially methylated loci indicated age-related enrichment for all engineered tissue types in ‘skeletal system morphogenesis’, ‘regulation of cell proliferation’ and ‘regulation of transcription’ suggesting that dynamic epigenetic modifications may occur in genes associated with shared and distinct pathways dependent upon engineered tissue type. An altered phenotype in engineered tissues was observed with ageing at numerous levels. These changes represent novel insights into the ageing process, with implications for stem cell therapies in older patients. In addition we have identified a number of tissue-dependant pathways, which warrant further studies. PMID:27533049

Oxygen and Cell Fate Decisions

DTIC Science & Technology

2009-05-27

mesenchymal stem cells may still be able to commit to chondro- genic differentiation under hypoxic conditions in vivo. Surprisingly, Malladi et al. have...are induced to undergo differentiation in vitro at 2% O2 ( Malladi et al. 2006). Nonetheless, targeted deletion of HIF-1α in adipose-derived adult... Malladi et al. 2007). These dis- crepancies could potentially be appreciated from at least two perspectives. (1) Chondrogenic dif- ferentiation is

Cyclic compression maintains viability and induces chondrogenesis of human mesenchymal stem cells in fibrin gel scaffolds.

PubMed

Pelaez, Daniel; Huang, Chun-Yuh Charles; Cheung, Herman S

2009-01-01

Mechanical loading has long been shown to modulate cartilage-specific extracellular matrix synthesis. With joint motion, cartilage can experience mechanical loading in the form of compressive, tensile or shearing load, and hydrostatic pressure. Recent studies have demonstrated the capacity of unconfined cyclic compression to induce chondrogenic differentiation of human mesenchymal stem cell (hMSC) in agarose culture. However, the use of a nonbiodegradable material such as agarose limits the applicability of these constructs. Of the possible biocompatible materials available for tissue engineering, fibrin is a natural regenerative scaffold, which possesses several desired characteristics including a controllable degradation rate and low immunogenicity. The objective of the present study was to determine the capability of fibrin gels for supporting chondrogenesis of hMSCs under cyclic compression. To optimize the system, three concentrations of fibrin gel (40, 60, and 80 mg/mL) and three different stimulus frequencies (0.1, 0.5, and 1.0 Hz) were used to examine the effects of cyclic compression on viability, proliferation and chondrogenic differentiation of hMSCs. Our results show that cyclic compression (10% strain) at frequencies >0.5 Hz and gel concentration of 40 mg/mL fibrinogen appears to maintain cellular viability within scaffolds. Similarly, variations in gel component concentration and stimulus frequency can be modified such that a significant chondrogenic response can be achieved by hMSC in fibrin constructs after 8 h of compression spread out over 2 days. This study demonstrates the suitability of fibrin gel for supporting the cyclic compression-induced chondrogenesis of mesenchymal stem cells.

Chondrogenic potential of injectable κ-carrageenan hydrogel with encapsulated adipose stem cells for cartilage tissue-engineering applications.

PubMed

Popa, Elena G; Caridade, Sofia G; Mano, João F; Reis, Rui L; Gomes, Manuela E

2015-05-01

Due to the limited self-repair capacity of cartilage, regenerative medicine therapies for the treatment of cartilage defects must use a significant amount of cells, preferably applied using a hydrogel system that can promise their delivery and functionality at the specific site. This paper discusses the potential use of κ-carrageenan hydrogels for the delivery of stem cells obtained from adipose tissue in the treatment of cartilage tissue defects. The developed hydrogels were produced by an ionotropic gelation method and human adipose stem cells (hASCs) were encapsulated in 1.5% w/v κ-carrageenan solution at a cell density of 5 × 10(6) cells/ml. The results from the analysis of the cell-encapsulating hydrogels, cultured for up to 21 days, indicated that κ-carrageenan hydrogels support the viability, proliferation and chondrogenic differentiation of hASCs. Additionally, the mechanical analysis demonstrated an increase in stiffness and viscoelastic properties of κ-carrageenan gels with their encapsulated cells with increasing time in culture with chondrogenic medium. These results allowed the conclusion that κ-carrageenan exhibits properties that enable the in vitro functionality of encapsulated hASCs and thus may provide the basis for new successful approaches for the treatment of cartilage defects. Copyright © 2013 John Wiley & Sons, Ltd.

The promotion of cartilage defect repair using adenovirus mediated Sox9 gene transfer of rabbit bone marrow mesenchymal stem cells.

PubMed

Cao, Lei; Yang, Fei; Liu, Guangwang; Yu, Degang; Li, Huiwu; Fan, Qiming; Gan, Yaokai; Tang, Tingting; Dai, Kerong

2011-06-01

Although Sox9 is essential for chondrogenic differentiation and matrix production, its application in cartilage tissue engineering has been rarely reported. In this study, the chondrogenic effect of Sox9 on bone marrow mesenchymal stem cells (BMSCs) in vitro and its application in articular cartilage repair in vivo were evaluated. Rabbit BMSCs were transduced with adenoviral vector containing Sox9. Toluidine blue, safranin O staining and real-time PCR were performed to check chondrogenic differentiation. The results showed that Sox9 could induce chondrogenesis of BMSCs both in monolayer and on PGA scaffold effectively. The rabbit model with full-thickness cartilage defects was established and then repaired by PGA scaffold and rabbit BMSCs with or without Sox9 transduction. HE, safranin O staining and immunohistochemistry were used to assess the repair of defects by the complex. Better repair, including more newly-formed cartilage tissue and hyaline cartilage-specific extracellular matrix and greater expression of several chondrogenesis marker genes were observed in PGA scaffold and BMSCs with Sox9 transduction, compared to that without transduction. Our findings defined the important role of Sox9 in the repair of cartilage defects in vivo and provided evidence that Sox9 had the potential and advantage in the application of tissue engineering. Copyright © 2011 Elsevier Ltd. All rights reserved.

Platelet lysate 3D scaffold supports mesenchymal stem cell chondrogenesis: an improved approach in cartilage tissue engineering.

PubMed

Moroz, Andrei; Bittencourt, Renata Aparecida Camargo; Almeida, Renan Padron; Felisbino, Sérgio Luis; Deffune, Elenice

2013-01-01

Articular lesions are still a major challenge in orthopedics because of cartilage's poor healing properties. A major improvement in therapeutics was the development of autologous chondrocytes implantation (ACI), a biotechnology-derived technique that delivers healthy autologous chondrocytes after in vitro expansion. To obtain cartilage-like tissue, 3D scaffolds are essential to maintain chondrocyte differentiated status. Currently, bioactive 3D scaffolds are promising as they can deliver growth factors, cytokines, and hormones to the cells, giving them a boost to attach, proliferate, induce protein synthesis, and differentiate. Using mesenchymal stem cells (MSCs) differentiated into chondrocytes, one can avoid cartilage harvesting. Thus, we investigated the potential use of a platelet-lysate-based 3D bioactive scaffold to support chondrogenic differentiation and maintenance of MSCs. The MSCs from adult rabbit bone marrow (n = 5) were cultivated and characterized using three antibodies by flow cytometry. MSCs (1 × 10(5)) were than encapsulated inside 60 µl of a rabbit platelet-lysate clot scaffold and maintained in Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 supplemented with chondrogenic inductors. After 21 days, the MSCs-seeded scaffolds were processed for histological analysis and stained with toluidine blue. This scaffold was able to maintain round-shaped cells, typical chondrocyte metachromatic extracellular matrix deposition, and isogenous group formation. Cells accumulated inside lacunae and cytoplasm lipid droplets were other observed typical chondrocyte features. In conclusion, the usage of a platelet-lysate bioactive scaffold, associated with a suitable chondrogenic culture medium, supports MSCs chondrogenesis. As such, it offers an alternative tool for cartilage engineering research and ACI.

Curcumin mediated suppression of nuclear factor-κB promotes chondrogenic differentiation of mesenchymal stem cells in a high-density co-culture microenvironment.

PubMed

Buhrmann, Constanze; Mobasheri, Ali; Matis, Ulrike; Shakibaei, Mehdi

2010-01-01

Osteoarthritis (OA) and rheumatoid arthritis (RA) are characterised by joint inflammation and cartilage degradation. Although mesenchymal stem cell (MSC)-like progenitors are resident in the superficial zone of articular cartilage, damaged tissue does not possess the capacity for regeneration. The high levels of pro-inflammatory cytokines present in OA/RA joints may impede the chondrogenic differentiation of these progenitors. Interleukin (IL)-1β activates the transcription factor nuclear factor-κB (NF-κB), which in turn activates proteins involved in matrix degradation, inflammation and apoptosis. Curcumin is a phytochemical capable of inhibiting IL-1β-induced activation of NF-κB and expression of apoptotic and pro-inflammatory genes in chondrocytes. Therefore, the aim of the present study was to evaluate the influence of curcumin on IL-1β-induced NF-κB signalling pathway in MSCs during chondrogenic differentiation. MSCs were either cultured in a ratio of 1:1 with primary chondrocytes in high-density culture or cultured alone in monolayer with/without curcumin and/or IL-1β. We demonstrate that although curcumin alone does not have chondrogenic effects on MSCs, it inhibits IL-1β-induced activation of NF-κB, activation of caspase-3 and cyclooxygenase-2 in MSCs time and concentration dependently, as it does in chondrocytes. In IL-1β stimulated co-cultures, four-hour pre-treatment with curcumin significantly enhanced the production of collagen type II, cartilage specific proteoglycans (CSPGs), β1-integrin, as well as activating MAPKinase signaling and suppressing caspase-3 and cyclooxygenase-2. Curcumin treatment may help establish a microenvironment in which the effects of pro-inflammatory cytokines are antagonized, thus facilitating chondrogenesis of MSC-like progenitor cells in vivo. This strategy may support the regeneration of articular cartilage.

Sirtuin-1 (SIRT1) Is Required for Promoting Chondrogenic Differentiation of Mesenchymal Stem Cells

PubMed Central

Buhrmann, Constanze; Busch, Franziska; Shayan, Parviz; Shakibaei, Mehdi

2014-01-01

Sirtuin-1 (SIRT1), NAD+-dependent deacetylase, has been linked to anabolic effects in cartilage, although the mechanisms of SIRT1 signaling during differentiation of mesenchymal stem cells (MSCs) to chondrocytes are poorly understood. Therefore, we investigated the role of SIRT1-mediated signaling during chondrogenic differentiation of MSCs in vitro. High density and alginate cultures of MSCs were treated with chondrogenic induction medium with/without the SIRT1 inhibitor nicotinamide, antisense oligonucleotides against SIRT1 (SIRT1-ASO), IL-1β, and/or resveratrol. Transient transfection of MSCs with SIRT1-antisense oligonucleotides, nicotinamide, and IL-1β inhibited chondrogenesis-induced down-regulation of cartilage-specific proteins, cartilage-specific transcription factor Sox9, and enhanced NF-κB-regulated gene products involved in the inflammatory and degradative processes in cartilage (MMP-9, COX-2, and caspase-3), and NF-κB phosphorylation, acetylation, and activation of IκBα kinase. In contrast, the SIRT1 activator resveratrol or BMS-345541 (inhibitor of IKK) inhibited IL-1β- and NAM-induced suppression of cartilage-specific proteins, Sox9, and up-regulation of NF-κB-regulated gene products. Moreover, SIRT1 was found to interact directly with NF-κB and resveratrol-suppressed IL-1β and NAM but not SIRT1-ASO-induced NF-κB phosphorylation, acetylation, and activation of IκBα kinase. Knockdown of SIRT1 by mRNA abolished the inhibitory effects of resveratrol on inflammatory and apoptotic signaling and Sox9 expression, suggesting the essential role of this enzyme. Finally, the modulatory effects of resveratrol were found to be mediated at least in part by the association between SIRT1 and Sox9. These results indicate for the first time that SIRT1 supports chondrogenic development of MSCs at least in part through inhibition/deacetylation of NF-κB and activation of Sox9. PMID:24962570

Effect of chondrocyte-derived early extracellular matrix on chondrogenesis of placenta-derived mesenchymal stem cells.

PubMed

Park, Yong-Beom; Seo, Sinji; Kim, Jin-A; Heo, Jin-Chul; Lim, Young-Cheol; Ha, Chul-Won

2015-06-24

The extracellular matrix (ECM) surrounding cells contains a variety of proteins that provide structural support and regulate cellular functions. Previous studies have shown that decellularized ECM isolated from tissues or cultured cells can be used to improve cell differentiation in tissue engineering applications. In this study we evaluated the effect of decellularized chondrocyte-derived ECM (CDECM) on the chondrogenesis of human placenta-derived mesenchymal stem cells (hPDMSCs) in a pellet culture system. After incubation with or without chondrocyte-derived ECM in chondrogenic medium for 1 or 3 weeks, the sizes and wet masses of the cell pellets were compared with untreated controls (hPDMSCs incubated in chondrogenic medium without chondrocyte-derived ECM). In addition, histologic analysis of the cell pellets (Safranin O and collagen type II staining) and quantitative reverse transcription-PCR analysis of chondrogenic markers (aggrecan, collagen type II, and SOX9) were carried out. Our results showed that the sizes and masses of hPDMSC pellets incubated with chondrocyte-derived ECM were significantly higher than those of untreated controls. Differentiation of hPDMSCs (both with and without chondrocyte-derived ECM) was confirmed by Safranin O and collagen type II staining. Chondrogenic marker expression and glycosaminoglycan (GAG) levels were significantly higher in hPDMSC pellets incubated with chondrocyte-derived ECM compared with untreated controls, especially in cells precultured with chondrocyte-derived ECM for 7 d. Taken together, these results demonstrate that chondrocyte-derived ECM enhances the chondrogenesis of hPDMSCs, and this effect is further increased by preculture with chondrocyte-derived ECM. This preculture method for hPDMSC chondrogenesis represents a promising approach for cartilage tissue engineering.

Enhanced chondrogenesis of human bone marrow mesenchymal Stem Cell (BMSC) on nanofiber-based polyethersulfone (PES) scaffold.

PubMed

Mahboudi, Hossein; Kazemi, Bahram; Soleimani, Masoud; Hanaee-Ahvaz, Hana; Ghanbarian, Hossein; Bandehpour, Mojgan; Enderami, Seyed Ehsan; Kehtari, Mousa; Barati, Ghasem

2018-02-15

Mesenchymal stem cells (MSC) from bone marrow hold great potential as a cell source for cartilage repair. The objective of our study was differentiation of MSC toward chondrocyte by using Nanofiber-based polyethersulfone (PES) scaffold and also enhanced chondrogenic differentiation of BMSC in vitro. MSCs were harvested from bone marrow of human and PES scaffold was fabricated via Electrospinning. The isolated cells were cultured on the PES scaffold and scaffold free method. After 21days, Real-time PCR was performed to evaluate the cartilage-specific genes in the mRNA levels. Also, in order to confirm our results, we have done immunocytochemistry and SEM imaging. Flowcytometry confirmed the nature of the isolated adherent cells. Immunocytochemistry and SEM imaging confirmed the differentiation of MSC toward chondrocyte. Also, real time PCR showed a significant increased gene expression of collagen type II and aggrecan on the PES scaffold method when compared to the mRNA levels measured in scaffold free method. Down regulation of Collagen type I was observed in PES scaffold compared to scaffold free at day 21. Also, both methods showed a similar pattern of expression of SOX9. Our results showed that PES scaffold maintains BMSC proliferation and differentiation, and can significantly enhance chondrogenic differentiation of BMSC. PES scaffold seeded BMSC showed the highest capacity for differentiation into chondrocyte-like cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Introduction of N-cadherin-binding motif to alginate hydrogels for controlled stem cell differentiation.

PubMed

Lee, Jae Won; An, Hyoseok; Lee, Kuen Yong

2017-07-01

Control of stem cell fate and phenotype using biomimetic synthetic extracellular matrices (ECMs) is an important tissue engineering approach. Many studies have focused on improving cell-matrix interactions. However, proper control of cell-cell interactions using synthetic ECMs could be critical for tissue engineering, especially with undifferentiated stem cells. In this study, alginate hydrogels were modified with a peptide derived from the low-density lipoprotein receptor-related protein 5 (LRP5), which is known to bind to N-cadherin, as a cell-cell interaction motif. In vitro changes in the morphology and differentiation of mouse bone marrow stromal cells (D1 stem cells) cultured in LRP5-alginate hydrogels were investigated. LRP5-alginate gels successfully induced stem cell aggregation and enhanced chondrogenic differentiation of D1 stem cells, compared to RGD-alginate gels, at low cell density. This approach to tailoring synthetic biomimetic ECMs using cell-cell interaction motifs may be critical in tissue engineering approaches using stem cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Isolation and characterization of multipotent human periodontal ligament stem cells.

PubMed

Gay, I C; Chen, S; MacDougall, M

2007-08-01

Periodontal ligament (PDL) repair is thought to involve mesenchymal progenitor cells capable of forming fibroblasts, osteoblasts and cementoblasts. However, full characterization of PDL stem cell (SC) populations has not been achieved. To isolate and characterize PDLSC and assess their capability to differentiate into bone, cartilage and adipose tissue. Human PDL cells were stained for STRO-1, FACS sorted and expanded in culture. Human bone marrow SC (BMSC) served as a positive control. PDLSC and BMSC were cultured using standard conditions conducive for osteogenic, chondrogenic and adipogenic differentiation. Osteogenic induction was assayed using alizarine red S staining and expression of alkaline phosphatase (ALP) and bone sialoprotein (BSP). Adipogenic induction was assayed using Oil Red O staining and the expression of PPAR gamma 2 (early) and LPL (late) adipogenic markers. Chondrogenic induction was assayed by collagen type II expression and toluidine blue staining. Human PDL tissue contains about 27% STRO-1 positive cells with 3% strongly positive. In osteogenic cultures ALP was observed by day-7 in BMSC and day-14 in PDLSC. BSP expression was detectable by day-7; with more intense staining in PDLSC cultures. In adipogenic cultures both cell populations showed positive Oil Red O staining by day-25 with PPAR gamma 2 and LPL expression. By day-21, both BMSC and PDLSC chondrogenic induced cultures expressed collagen type II and glycosaminoglycans. The PDL contains SC that have the potential to differentiate into osteoblasts, chondrocytes and adipocytes, comparable with previously characterized BMSC. This adult PDLSC population can be utilized for potential therapeutic procedures related to PDL regeneration.

Effects of single and combined low frequency electromagnetic fields and simulated microgravity on gene expression of human mesenchymal stem cells during chondrogenesis

PubMed Central

Hammerschmid, Florian; Blum, Helmut; Krebs, Stefan; Redeker, Julia I.; Holzapfel, Boris M.; Jansson, Volkmar; Müller, Peter E.

2016-01-01

Introduction Low frequency electromagnetic fields (LF-EMF) and simulated microgravity (SMG) have been observed to affect chondrogenesis. A controlled bioreactor system was developed to apply LF-EMF and SMG singly or combined during chondrogenic differentiation of human mesenchymal stem cells (hMSCs) in 3D culture. Material and methods An external motor gear SMG bioreactor was combined with magnetic Helmholtz coils for EMF (5 mT; 15 Hz). Pellets of hMSCs (±TGF-β3) were cultured (P5) under SMG, LF-EMF, LF-EMF/SMG and control (1 g) conditions for 3 weeks. Sections were stained with safranin-O and collagen type II. Gene expression was evaluated by microarray and real-time polymerase chain reaction analysis. Results Simulated microgravity application significantly changed gene expression; specifically, COLXA1 but also COL2A1, which represents the chondrogenic potential, were reduced (p < 0.05). Low frequency electromagnetic fields application showed no gene expression changes on a microarray basis. LF-EMF/SMG application obtained significant different expression values from cultures obtained under SMG conditions with a re-increase of COL2A1, therefore rescuing the chondrogenic potential, which had been lowered by SMG. Conclusions Simulated microgravity lowered hypertrophy but also the chondrogenic potential of hMSCs. Combined LF-EMF/SMG provided a rescue effect of the chondrogenic potential of hMSCs although no LF-EMF effect was observed under optimal conditions. The study provides new insights into how LF-EMF and SMG affect chondrogenesis of hMSCs and how they generate interdependent effects. PMID:29765449

Creating an Animal Model of Tendinopathy by Inducing Chondrogenic Differentiation with Kartogenin.

PubMed

Yuan, Ting; Zhang, Jianying; Zhao, Guangyi; Zhou, Yiqin; Zhang, Chang-Qing; Wang, James H-C

2016-01-01

Previous animal studies have shown that long term rat treadmill running induces over-use tendinopathy, which manifests as proteoglycan accumulation and chondrocytes-like cells within the affected tendons. Creating this animal model of tendinopathy by long term treadmill running is however time-consuming, costly and may vary among animals. In this study, we used a new approach to develop an animal model of tendinopathy using kartogenin (KGN), a bio-compound that can stimulate endogenous stem/progenitor cells to differentiate into chondrocytes. KGN-beads were fabricated and implanted into rat Achilles tendons. Five weeks after implantation, chondrocytes and proteoglycan accumulation were found at the KGN implanted site. Vascularity as well as disorganization in collagen fibers were also present in the same site along with increased expression of the chondrocyte specific marker, collagen type II (Col. II). In vitro studies confirmed that KGN was released continuously from KGN-alginate in vivo beads and induced chondrogenic differentiation of tendon stem/progenitor cells (TSCs) suggesting that chondrogenesis after KGN-bead implantation into the rat tendons is likely due to the aberrant differentiation of TSCs into chondrocytes. Taken together, our results showed that KGN-alginate beads can be used to create a rat model of tendinopathy, which, at least in part, reproduces the features of over-use tendinopathy model created by long term treadmill running. This model is mechanistic (stem cell differentiation), highly reproducible and precise in creating localized tendinopathic lesions. It is expected that this model will be useful to evaluate the effects of various topical treatments such as NSAIDs and platelet-rich plasma (PRP) for the treatment of tendinopathy.

Hydrophilic polyurethane matrix promotes chondrogenesis of mesenchymal stem cells.

PubMed

Nalluri, Sandeep M; Krishnan, G Rajesh; Cheah, Calvin; Arzumand, Ayesha; Yuan, Yuan; Richardson, Caley A; Yang, Shuying; Sarkar, Debanjan

2015-09-01

Segmental polyurethanes exhibit biphasic morphology and can control cell fate by providing distinct matrix guided signals to increase the chondrogenic potential of mesenchymal stem cells (MSCs). Polyethylene glycol (PEG) based hydrophilic polyurethanes can deliver differential signals to MSCs through their matrix phases where hard segments are cell-interactive domains and PEG based soft segments are minimally interactive with cells. These coordinated communications can modulate cell-matrix interactions to control cell shape and size for chondrogenesis. Biphasic character and hydrophilicity of polyurethanes with gel like architecture provide a synthetic matrix conducive for chondrogenesis of MSCs, as evidenced by deposition of cartilage-associated extracellular matrix. Compared to monophasic hydrogels, presence of cell interactive domains in hydrophilic polyurethanes gels can balance cell-cell and cell-matrix interactions. These results demonstrate the correlation between lineage commitment and the changes in cell shape, cell-matrix interaction, and cell-cell adhesion during chondrogenic differentiation which is regulated by polyurethane phase morphology, and thus, represent hydrophilic polyurethanes as promising synthetic matrices for cartilage regeneration. Copyright © 2015 Elsevier B.V. All rights reserved.

A Closed Chondromimetic Environment within Magnetic-Responsive Liquified Capsules Encapsulating Stem Cells and Collagen II/TGF-β3 Microparticles.

PubMed

Correia, Clara R; Gil, Sara; Reis, Rui L; Mano, João F

2016-06-01

TGF-β3 is enzymatically immobilized by transglutaminase-2 action to poly(l-lactic acid) microparticles coated with collagen II. Microparticles are then encapsulated with stem cells inside liquified spherical compartments enfolded with a permselective shell through layer-by-layer adsorption. Magnetic nanoparticles are electrostatically bound to the multilayered shell, conferring magnetic-response ability. The goal of this study is to engineer a closed environment inside which encapsulated stem cells would undergo a self-regulated chondrogenesis. To test this hypothesis, capsules are cultured in chondrogenic differentiation medium without TGF-β3. Their biological outcome is compared with capsules encapsulating microparticles without TGF-β3 immobilization and cultured in normal chondrogenic differentiation medium containing soluble TGF-β3. Glycosaminoglycans quantification demosntrates that similar chondrogenesis levels are achieved. Moreover, collagen fibrils resembling the native extracellular matrix of cartilage can be observed. Importantly, the genetic evaluation of characteristic cartilage markers confirms the successful chondrogenesis, while hypertrophic markers are downregulated. In summary, the engineered capsules are able to provide a suitable and stable chondrogenesis environment for stem cells without the need of TGF-β3 supplementation. This kind of self-regulated capsules with softness, robustness, and magnetic responsive characteristics is expected to provide injectability and in situ fixation, which is of great advantage for minimal invasive strategies to regenerate cartilage. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Wnt5a Receptor, Receptor Tyrosine Kinase-Like Orphan Receptor 2, Is a Predictive Cell Surface Marker of Human Mesenchymal Stem Cells with an Enhanced Capacity for Chondrogenic Differentiation.

PubMed

Dickinson, Sally C; Sutton, Catherine A; Brady, Kyla; Salerno, Anna; Katopodi, Theoni; Williams, Rhys L; West, Christopher C; Evseenko, Denis; Wu, Ling; Pang, Suzanna; Ferro de Godoy, Roberta; Goodship, Allen E; Péault, Bruno; Blom, Ashley W; Kafienah, Wael; Hollander, Anthony P

2017-11-01

Multipotent mesenchymal stem cells (MSCs) have enormous potential in tissue engineering and regenerative medicine. However, until now, their development for clinical use has been severely limited as they are a mixed population of cells with varying capacities for lineage differentiation and tissue formation. Here, we identify receptor tyrosine kinase-like orphan receptor 2 (ROR2) as a cell surface marker expressed by those MSCs with an enhanced capacity for cartilage formation. We generated clonal human MSC populations with varying capacities for chondrogenesis. ROR2 was identified through screening for upregulated genes in the most chondrogenic clones. When isolated from uncloned populations, ROR2+ve MSCs were significantly more chondrogenic than either ROR2-ve or unfractionated MSCs. In a sheep cartilage-repair model, they produced significantly more defect filling with no loss of cartilage quality compared with controls. ROR2+ve MSCs/perivascular cells were present in developing human cartilage, adult bone marrow, and adipose tissue. Their frequency in bone marrow was significantly lower in patients with osteoarthritis (OA) than in controls. However, after isolation of these cells and their initial expansion in vitro, there was greater ROR2 expression in the population derived from OA patients compared with controls. Furthermore, osteoarthritis-derived MSCs were better able to form cartilage than MSCs from control patients in a tissue engineering assay. We conclude that MSCs expressing high levels of ROR2 provide a defined population capable of predictably enhanced cartilage production. Stem Cells 2017;35:2280-2291. © 2017 AlphaMed Press.

The Wnt5a Receptor, Receptor Tyrosine Kinase‐Like Orphan Receptor 2, Is a Predictive Cell Surface Marker of Human Mesenchymal Stem Cells with an Enhanced Capacity for Chondrogenic Differentiation

PubMed Central

Dickinson, Sally C.; Sutton, Catherine A.; Brady, Kyla; Salerno, Anna; Katopodi, Theoni; Williams, Rhys L.; West, Christopher C.; Evseenko, Denis; Wu, Ling; Pang, Suzanna; Ferro de Godoy, Roberta; Goodship, Allen E.; Péault, Bruno; Blom, Ashley W.; Kafienah, Wael

2017-01-01

Abstract Multipotent mesenchymal stem cells (MSCs) have enormous potential in tissue engineering and regenerative medicine. However, until now, their development for clinical use has been severely limited as they are a mixed population of cells with varying capacities for lineage differentiation and tissue formation. Here, we identify receptor tyrosine kinase‐like orphan receptor 2 (ROR2) as a cell surface marker expressed by those MSCs with an enhanced capacity for cartilage formation. We generated clonal human MSC populations with varying capacities for chondrogenesis. ROR2 was identified through screening for upregulated genes in the most chondrogenic clones. When isolated from uncloned populations, ROR2+ve MSCs were significantly more chondrogenic than either ROR2–ve or unfractionated MSCs. In a sheep cartilage‐repair model, they produced significantly more defect filling with no loss of cartilage quality compared with controls. ROR2+ve MSCs/perivascular cells were present in developing human cartilage, adult bone marrow, and adipose tissue. Their frequency in bone marrow was significantly lower in patients with osteoarthritis (OA) than in controls. However, after isolation of these cells and their initial expansion in vitro, there was greater ROR2 expression in the population derived from OA patients compared with controls. Furthermore, osteoarthritis‐derived MSCs were better able to form cartilage than MSCs from control patients in a tissue engineering assay. We conclude that MSCs expressing high levels of ROR2 provide a defined population capable of predictably enhanced cartilage production. Stem Cells 2017;35:2280–2291 PMID:28833807

Use of the second harmonic generation microscopy to evaluate chondrogenic differentiation of mesenchymal stem cells for cartilage repair

NASA Astrophysics Data System (ADS)

Bordeaux-Rego, P.; Baratti, M. O.; Duarte, A. S. S.; Ribeiro, T. B.; Andreoli-Risso, M. F.; Vidal, B.; Miranda, J. B.; Adur, J.; de Thomaz, A. A.; Pelegati, V. B.; Costa, F. F.; Carvalho, H. F.; Cesar, C. L.; Luzo, A.; Olalla Saad, S. T.

2012-03-01

Articular cartilage injury remains one of the major concerns in orthopedic surgery. Mesenchymal stem cell (MSC) transplantation has been introduced to avoid some of the side effects and complications of current techniques.. With the aim to evaluate chondrogenic differentiation of mesenchymal stem cells, we used Second Harmonic Generation (SHG) microscopy to analyze the aggregation and orientation of collagen fibrils in the hyaline cartilage of rabbit knees. The experiment was performed using implants with type II collagen hydrogel (a biomaterial that mimics the microenvironment of the cartilage), one implant containing MSC and one other without MSC (control). After 10 weeks, the rabbit knees were dissected and fibril collagen distribution and spatial organization in the extracellular matrix of the lesions were verified by SHG. The result showed significant differences, whereas in histological sections of the cartilaginous lesions with MSC the collagen fibers are organized and regular; in the control sections the collagen fibers are more irregular, with absence of cells. A macroscopic analysis of the lesions confirmed this difference, showing a greater percentage of lesions filling in knees treated with MSC than in the knees used as controls. This study demonstrates that SHG microscopy will be an excellent tool to help in the evaluation of the effectiveness of MSC-based cell therapy for cartilage repair.

Low Molecular Weight Fraction of Commercial Human Serum Albumin Induces Morphologic and Transcriptional Changes of Bone Marrow-Derived Mesenchymal Stem Cells.

PubMed

Bar-Or, David; Thomas, Gregory W; Rael, Leonard T; Gersch, Elizabeth D; Rubinstein, Pablo; Brody, Edward

2015-08-01

Osteoarthritis (OA) is the most common chronic disease of the joint; however, the therapeutic options for severe OA are limited. The low molecular weight fraction of commercial 5% human serum albumin (LMWF5A) has been shown to have anti-inflammatory properties that are mediated, in part, by a diketopiperazine that is present in the albumin preparation and that was demonstrated to be safe and effective in reducing pain and improving function when administered intra-articularly in a phase III clinical trial. In the present study, bone marrow-derived mesenchymal stem cells (BMMSCs) exposed to LMWF5A exhibited an elongated phenotype with diffuse intracellular F-actin, pronounced migratory leading edges, and filopodia-like projections. In addition, LMWF5A promoted chondrogenic condensation in "micromass" culture, concurrent with the upregulation of collagen 2α1 mRNA. Furthermore, the transcription of the CXCR4-CXCL12 axis was significantly regulated in a manner conducive to migration and homing. Several transcription factors involved in stem cell differentiation were also found to bind oligonucleotide response element probes following exposure to LMWF5A. Finally, a rapid increase in PRAS40 phosphorylation was observed following treatment, potentially resulting in the activation mTORC1. Proteomic analysis of synovial fluid taken from a preliminary set of patients indicated that at 12 weeks following administration of LMWF5A, a microenvironment exists in the knee conducive to stem cell infiltration, self-renewal, and differentiation, in addition to indications of remodeling with a reduction in inflammation. Taken together, these findings imply that LMWF5A treatment may prime stem cells for both mobilization and chondrogenic differentiation, potentially explaining some of the beneficial effects achieved in clinical trials. ©AlphaMed Press.

Angiogenic Potential of Human Bone Marrow‐Derived Mesenchymal Stem Cells in Chondrocyte Brick‐Enriched Constructs Promoted Stable Regeneration of Craniofacial Cartilage

PubMed Central

Li, Zhiye; Ba, Ruikai; Wang, Zhifa; Wei, Jianhua; Zhao, Yimin

2016-01-01

Abstract Craniofacial deformities caused by congenital defects or trauma remain challenges for clinicians, whereas current surgical interventions present limited therapeutic outcomes. Injection of bone marrow‐derived mesenchymal stem cells (BMSCs) into the defect is highly desirable because such a procedure is microinvasive and grafts are more flexible to fill the lesions. However, preventing hypertrophic transition and morphological contraction remain significant challenges. We have developed an “all host derived” cell transplantation system composed of chondrocyte brick (CB)‐enriched platelet‐rich plasma (P) gel and BMSCs (B). Without exogenous biomaterials or growth factors, such grafts regenerate cartilage efficiently and present great clinical promise. In immunodeficient mice, we compared performance of BMSCs and BMSCs lacking angiogenic potential in CB‐B‐P constructs and followed the cartilage maturation process by histology, immunostaining, micro‐computed tomography, and protein analysis. We determined that angiogenesis occurred quickly inside rudimentary cartilage derived from CB‐B‐P constructs after implantation, which improved tissue survival, tissue growth, and production of chondrogenic signals from chondrocytes. In contrast, silencing angiogenic potential of BMSCs led to poor chondrogenesis accompanied by necrosis. Chondrocyte bricks merged rapidly with angiogenesis, which constituted an enclosed chondrogenic niche and effectively inhibited runt‐related transcription factor‐2‐dependent hypertrophic transition of BMSCs as well as endochondral ossification; progressive chondrogenic differentiation of BMSCs resulted in vascularization regression, thus favoring persistent chondrogenesis and effectively augmenting nasal cartilage. In conclusion, these findings provided a novel, efficient approach to regenerating cartilage tissues in vivo. Chondrocyte bricks mixed with P provide transient vascularization and a persistently chondrogenic microenvironment for BMSCs; this provides a mini‐invasive approach for craniofacial cartilage reconstruction. Stem Cells Translational Medicine 2017;6:601–612 PMID:28191761

ABCG2 Is a Selectable Marker for Enhanced Multilineage Differentiation Potential in Periodontal Ligament Stem Cells

PubMed Central

Szepesi, Áron; Matula, Zsolt; Szigeti, Anna; Várady, György; Szabó, Gyula; Uher, Ferenc; Sarkadi, Balázs

2015-01-01

Periodontal ligament stem cells (PDLSCs) provide an important source for tissue regeneration and may become especially useful in the formation of osteogenic seeds. PDLSCs can be cultured, expanded, and differentiated in vitro; thus, they may be applied in the long-term treatment of the defects in the dental regions. Here we studied numerous potential markers allowing the selection of human PDLSCs with a maximum differentiation potential. We followed the expression of the ATP-binding cassette subfamily G member 2 (ABCG2) membrane transporter protein and isolated ABCG2-expressing cells by using a monoclonal antibody, recognizing the transporter at the cell surface in intact cells. The expression of the ABCG2 protein, corresponding to the so-called side-population phenotype in various tissue-derived stem cells, was found to be a useful marker for the selection of PDLSCs with enhanced osteogenic, chondrogenic, and adipogenic differentiation. These findings may have important applications in achieving efficient dental tissue regeneration by using stem cells from extracted teeth. PMID:25101689

IGF-1 Gene Transfer to Human Synovial MSCs Promotes Their Chondrogenic Differentiation Potential without Induction of the Hypertrophic Phenotype.

PubMed

Ikeda, Yasutoshi; Sakaue, Morito; Chijimatsu, Ryota; Hart, David A; Otsubo, Hidenori; Shimomura, Kazunori; Madry, Henning; Suzuki, Tomoyuki; Yoshikawa, Hideki; Yamashita, Toshihiko; Nakamura, Norimasa

2017-01-01

Mesenchymal stem cell- (MSC-) based therapy is a promising treatment for cartilage. However, repair tissue in general fails to regenerate an original hyaline-like tissue. In this study, we focused on increasing the expression levels for insulin-like growth factor-1 (IGF-1) to improve repair tissue quality. The IGF-1 gene was introduced into human synovial MSCs with a lentiviral vector and examined the levels of gene expression and morphological status of MSCs under chondrogenic differentiation condition using pellet cultures. The size of the pellets derived from IGF-1-MSCs were significantly larger than those of the control group. The abundance of glycosaminoglycan (GAG) was also significantly higher in the IGF-1-MSC group. The histology of the IGF-1-induced pellets demonstrated similarities to hyaline cartilage without exhibiting features of a hypertrophic chondrocyte phenotype. Expression levels for the Col2A1 gene and protein were significantly higher in the IGF-1 pellets than in the control pellets, but expression levels for Col10, MMP-13, ALP, and Osterix were not higher. Thus, IGF-1 gene transfer to human synovial MSCs led to an improved chondrogenic differentiation capacity without the detectable induction of a hypertrophic or osteogenic phenotype.

Projection Stereolithographic Fabrication of Human Adipose Stem Cell-Incorporated Biodegradable Scaffolds for Cartilage Tissue Engineering.

PubMed

Sun, Aaron X; Lin, Hang; Beck, Angela M; Kilroy, Evan J; Tuan, Rocky S

2015-01-01

The poor self-healing ability of cartilage necessitates the development of methods for cartilage regeneration. Scaffold construction with live stem cell incorporation and subsequent differentiation presents a promising route. Projection stereolithography (PSL) offers high resolution and processing speed as well as the ability to fabricate scaffolds that precisely fit the anatomy of cartilage defects using medical imaging as the design template. We report here the use of a visible-light-based PSL (VL-PSL) system to encapsulate human adipose-derived stem cells (hASCs) into a biodegradable polymer [poly-d,l-lactic acid/polyethylene glycol/poly-d,l-lactic acid (PDLLA-PEG)]/hyaluronic acid (HA) matrix to produce live cell constructs with customized architectures. After fabrication, hASCs showed high viability (84%) and were uniformly distributed throughout the constructs, which possessed high mechanical properties with a compressive modulus of 780 kPa. The hASC-seeded constructs were then cultured in control or TGF-β3-containing chondrogenic medium for up to 28 days. In chondrogenic medium-treated group (TGF-β3 group), hASCs maintained 77% viability and expressed chondrogenic genes Sox9, collagen type II, and aggrecan at 11, 232, and 2.29 × 10(5) fold increases, respectively compared to levels at day 0 in non-chondrogenic medium. The TGF-β3 group also produced a collagen type II and glycosaminoglycan-rich extracellular matrix, detected by immunohistochemistry, Alcian blue staining, and Safranin O staining suggesting robust chondrogenesis within the scaffold. Without chondroinductive addition (Control group), cell viability decreased with time (65% at 28 days) and showed poor cartilage matrix deposition. After 28 days, mechanical strength of the TGF-β3 group remained high at 240 kPa. Thus, the PSL and PDLLA-PEG/HA-based fabrication method using adult stem cells is a promising approach in producing mechanically competent engineered cartilage for joint cartilage resurfacing.

Transforming growth factor-beta1 promotes articular cartilage repair through canonical Smad and Hippo pathways in bone mesenchymal stem cells.

PubMed

Ying, Jun; Wang, Pinger; Zhang, Shanxing; Xu, Taotao; Zhang, Lei; Dong, Rui; Xu, Shibing; Tong, Peijian; Wu, Chengliang; Jin, Hongting

2018-01-01

Transforming growth factor-β1 (TGF-β1) is a chondrogenic factor and has been reported to be able to enhance chondrocyte differentiation from bone marrow mesenchymal stem cells (BMSCs). Here we investigate the molecular mechanism through which TGF-β1 chronically promotes the repair of cartilage defect and inhibit chondrocyte hypertrophy. Animal models of full thickness cartilage defects were divided into three groups: model group, BMSCs group (treated with BMSCs/calcium alginate gel) and BMSCs+TGF-β1 group (treated with Lentivirus-TGF-β1-EGFP transduced BMSCs/calcium alginate gel). 4 and 8weeks after treatment, macroscopic observation, histopathological study and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were done to analyze phenotypes of the animals. BMSCs were transduced with Lentivirus-TGF-β1-EGFP in vitro and Western blot analysis was performed. We found that TGF-β1-expressiing BMSCs improved the repair of the cartilage defect. The impaired cartilage contained higher amount of GAG and type II collagen and was integrated to the surrounding normal cartilage and higher content of GAG and type II collagen. The major events include increased expression of type II collagen following Smad2/3 phosphorylation, and inhibition of cartilage hypertrophy by increasing Yes-associated protein-1 (YAP-1) and inhibiting Runx2 and Col10 after the completion of chondrogenic differentiation. We conclude that TGF-β1 is beneficial to chondrogenic differentiation of BMSCs via canonical Smad pathway to promote early-repairing of cartilage defect. Furthermore, TGF-β1 inhibits chondrocyte hypertrophy by decreasing hypertrophy marker gene expression via Hippo signaling. Long-term rational use of TGF-β1 may be an alternative approach in clinic for cartilage repair and regeneration. Copyright © 2017. Published by Elsevier Inc.

Mechanical stimulation of mesenchymal stem cells: Implications for cartilage tissue engineering.

PubMed

Fahy, Niamh; Alini, Mauro; Stoddart, Martin J

2018-01-01

Articular cartilage is a load-bearing tissue playing a crucial mechanical role in diarthrodial joints, facilitating joint articulation, and minimizing wear. The significance of biomechanical stimuli in the development of cartilage and maintenance of chondrocyte phenotype in adult tissues has been well documented. Furthermore, dysregulated loading is associated with cartilage pathology highlighting the importance of mechanical cues in cartilage homeostasis. The repair of damaged articular cartilage resulting from trauma or degenerative joint disease poses a major challenge due to a low intrinsic capacity of cartilage for self-renewal, attributable to its avascular nature. Bone marrow-derived mesenchymal stem cells (MSCs) are considered a promising cell type for cartilage replacement strategies due to their chondrogenic differentiation potential. Chondrogenesis of MSCs is influenced not only by biological factors but also by the environment itself, and various efforts to date have focused on harnessing biomechanics to enhance chondrogenic differentiation of MSCs. Furthermore, recapitulating mechanical cues associated with cartilage development and homeostasis in vivo, may facilitate the development of a cellular phenotype resembling native articular cartilage. The goal of this review is to summarize current literature examining the effect of mechanical cues on cartilage homeostasis, disease, and MSC chondrogenesis. The role of biological factors produced by MSCs in response to mechanical loading will also be examined. An in-depth understanding of the impact of mechanical stimulation on the chondrogenic differentiation of MSCs in terms of endogenous bioactive factor production and signaling pathways involved, may identify therapeutic targets and facilitate the development of more robust strategies for cartilage replacement using MSCs. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:52-63, 2018. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Blocking VEGF with sFlt1 improves the chondrogenic potential of mouse skeletal muscle-derived stem cells

PubMed Central

Kubo, Seiji; Cooper, Gregory M.; Matsumoto, Tomoyuki; Phillippi, Julie A.; Corsi, Karin A.; Usas, Arvydas; Li, Guangheng; Fu, Freddie H.; Huard, Johnny

2010-01-01

Objective To investigate the effect of VEGF stimulation and the effect of blocking VEGF with its antagonist, sFlt1, on chondrogenesis using skeletal muscle-derived stem cells (MDSCs). Methods The direct effect of VEGF on the in vitro chondrogenic ability of mouse MDSCs was tested using a pellet culture system followed by quantitative real time PCR and histological analyses. Next, the effect of VEGF on chondrogenesis within the synovial joint was tested using genetically engineered MDSCs implanted into the rat osteochondral defect. In this model, MDSCs, transduced with a retroviral vector to express BMP4, were co-implanted with MDSCs transduced to express either VEGF or sFlt1 (a VEGF antagonist) to provide a gain- and loss-of VEGF function experimental design. Histological scoring was used to compare cartilage formation among the treatment groups. Results Hyaline-like cartilage matrix production was observed in both VEGF-treated and VEGF-blocked (sFlt1-treated) pellet cultures, but real-time PCR revealed that sFlt1 treatment improved the expression of chondrogenic genes in MDSCs that were stimulated to undergo chondrogenic differentiation with BMP4 and TGF-β3. In vivo testing of articular cartilage repair showed that VEGF-transduced MDSCs caused an arthritic change in the knee joint, and sFlt1 improved the MDSC-mediated repair of articular cartilage, compared to BMP4 alone. Conclusion sFlt1 gene therapy improved BMP4- and TGF-β3-induced chondrogenic gene expression of MDSCs in vitro, and improved the persistence of repaired articular cartilage by preventing vascularization and bone invasion into the repaired articular cartilage. PMID:19116905

Cyclic hydrostatic pressure promotes a stable cartilage phenotype and enhances the functional development of cartilaginous grafts engineered using multipotent stromal cells isolated from bone marrow and infrapatellar fat pad.

PubMed

Carroll, S F; Buckley, C T; Kelly, D J

2014-06-27

The objective of this study was to investigate how joint specific biomechanical loading influences the functional development and phenotypic stability of cartilage grafts engineered in vitro using stem/progenitor cells isolated from different source tissues. Porcine bone marrow derived multipotent stromal cells (BMSCs) and infrapatellar fat pad derived multipotent stromal cells (FPSCs) were seeded in agarose hydrogels and cultured in chondrogenic medium, while simultaneously subjected to 10MPa of cyclic hydrostatic pressure (HP). To mimic the endochondral phenotype observed in vivo with cartilaginous tissues engineered using BMSCs, the culture media was additionally supplemented with hypertrophic factors, while the loss of phenotype observed in vivo with FPSCs was induced by withdrawing transforming growth factor (TGF)-β3 from the media. The application of HP was found to enhance the functional development of cartilaginous tissues engineered using both BMSCs and FPSCs. In addition, HP was found to suppress calcification of tissues engineered using BMSCs cultured in chondrogenic conditions and acted to maintain a chondrogenic phenotype in cartilaginous grafts engineered using FPSCs. The results of this study point to the importance of in vivo specific mechanical cues for determining the terminal phenotype of chondrogenically primed multipotent stromal cells. Furthermore, demonstrating that stem or progenitor cells will appropriately differentiate in response to such biophysical cues might also be considered as an additional functional assay for evaluating their therapeutic potential. Copyright © 2013 Elsevier Ltd. All rights reserved.

The long non-coding RNA ROCR contributes to SOX9 expression and chondrogenic differentiation of human mesenchymal stem cells

PubMed Central

Hyatt, Sam; Cheung, Kat; Skelton, Andrew J.; Xu, Yaobo; Clark, Ian M.

2017-01-01

Long non-coding RNAs (lncRNAs) are expressed in a highly tissue-specific manner and function in various aspects of cell biology, often as key regulators of gene expression. In this study, we established a role for lncRNAs in chondrocyte differentiation. Using RNA sequencing we identified a human articular chondrocyte repertoire of lncRNAs from normal hip cartilage donated by neck of femur fracture patients. Of particular interest are lncRNAs upstream of the master chondrocyte transcription factor SOX9 locus. SOX9 is an HMG-box transcription factor that plays an essential role in chondrocyte development by directing the expression of chondrocyte-specific genes. Two of these lncRNAs are upregulated during chondrogenic differentiation of mesenchymal stem cells (MSCs). Depletion of one of these lncRNAs, LOC102723505, which we termed ROCR (regulator of chondrogenesis RNA), by RNA interference disrupted MSC chondrogenesis, concomitant with reduced cartilage-specific gene expression and incomplete matrix component production, indicating an important role in chondrocyte biology. Specifically, SOX9 induction was significantly ablated in the absence of ROCR, and overexpression of SOX9 rescued the differentiation of MSCs into chondrocytes. Our work sheds further light on chondrocyte-specific SOX9 expression and highlights a novel method of chondrocyte gene regulation involving a lncRNA. PMID:29084806

Platelet-Rich Plasma Preparation Types Show Impact on Chondrogenic Differentiation, Migration, and Proliferation of Human Subchondral Mesenchymal Progenitor Cells.

PubMed

Kreuz, Peter Cornelius; Krüger, Jan Philipp; Metzlaff, Sebastian; Freymann, Undine; Endres, Michaela; Pruss, Axel; Petersen, Wolf; Kaps, Christian

2015-10-01

To evaluate the chondrogenic potential of platelet concentrates on human subchondral mesenchymal progenitor cells (MPCs) as assessed by histomorphometric analysis of proteoglycans and type II collagen. Furthermore, the migratory and proliferative effect of platelet concentrates were assessed. Platelet-rich plasma (PRP) was prepared using preparation kits (Autologous Conditioned Plasma [ACP] Kit [Arthrex, Naples, FL]; Regen ACR-C Kit [Regen Lab, Le Mont-Sur-Lausanne, Switzerland]; and Dr.PRP Kit [Rmedica, Seoul, Republic of Korea]) by apheresis (PRP-A) and by centrifugation (PRP-C). In contrast to clinical application, freeze-and-thaw cycles were subsequently performed to activate platelets and to prevent medium coagulation by residual fibrinogen in vitro. MPCs were harvested from the cortico-spongious bone of femoral heads. Chondrogenic differentiation of MPCs was induced in high-density pellet cultures and evaluated by histochemical staining of typical cartilage matrix components. Migration of MPCs was assessed using a chemotaxis assay, and proliferation activity was measured by DNA content. MPCs cultured in the presence of 5% ACP, Regen, or Dr.PRP formed fibrous tissue, whereas MPCs stimulated with 5% PRP-A or PRP-C developed compact and dense cartilaginous tissue rich in type II collagen and proteoglycans. All platelet concentrates significantly (ACP, P = .00041; Regen, P = .00029; Dr.PRP, P = .00051; PRP-A, P < .0001; and PRP-C, P < .0001) stimulated migration of MPCs. All platelet concentrates but one (Dr.PRP, P = .63) showed a proliferative effect on MPCs, as shown by significant increases (ACP, P = .027; Regen, P = .0029; PRP-A, P = .00021; and PRP-C, P = .00069) in DNA content. Platelet concentrates obtained by different preparation methods exhibit different potentials to stimulate chondrogenic differentiation, migration, and proliferation of MPCs. Platelet concentrates obtained by commercially available preparation kits failed to induce chondrogenic differentiation of MPCs, whereas highly standardized PRP preparations did induce such differentiation. These findings suggest differing outcomes with PRP treatment in stem cell-based cartilage repair. Our findings may help to explain the variability of results in studies examining the use of PRP clinically. Copyright © 2015 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.

Angiopoietin-like protein 2 promotes chondrogenic differentiation during bone growth as a cartilage matrix factor.

PubMed

Tanoue, H; Morinaga, J; Yoshizawa, T; Yugami, M; Itoh, H; Nakamura, T; Uehara, Y; Masuda, T; Odagiri, H; Sugizaki, T; Kadomatsu, T; Miyata, K; Endo, M; Terada, K; Ochi, H; Takeda, S; Yamagata, K; Fukuda, T; Mizuta, H; Oike, Y

2018-01-01

Chondrocyte differentiation is crucial for long bone growth. Many cartilage extracellular matrix (ECM) proteins reportedly contribute to chondrocyte differentiation, indicating that mechanisms underlying chondrocyte differentiation are likely more complex than previously appreciated. Angiopoietin-like protein 2 (ANGPTL2) is a secreted factor normally abundantly produced in mesenchymal lineage cells such as adipocytes and fibroblasts, but its loss contributes to the pathogenesis of lifestyle- or aging-related diseases. However, the function of ANGPTL2 in chondrocytes, which are also differentiated from mesenchymal stem cells, remains unclear. Here, we investigate whether ANGPTL2 is expressed in or functions in chondrocytes. First, we evaluated Angptl2 expression during chondrocyte differentiation using chondrogenic ATDC5 cells and wild-type epiphyseal cartilage of newborn mice. We next assessed ANGPTL2 function in chondrogenic differentiation and associated signaling using Angptl2 knockdown ATDC5 cells and Angptl2 knockout mice. ANGPTL2 is expressed in chondrocytes, particularly those located in resting and proliferative zones, and accumulates in ECM surrounding chondrocytes. Interestingly, long bone growth was retarded in Angptl2 knockout mice from neonatal to adult stages via attenuation of chondrocyte differentiation. Both in vivo and in vitro experiments show that changes in ANGPTL2 expression can also alter p38 mitogen-activated protein kinase (MAPK) activity mediated by integrin α5β1. ANGPTL2 contributes to chondrocyte differentiation and subsequent endochondral ossification through α5β1 integrin and p38 MAPK signaling during bone growth. Our findings provide insight into molecular mechanisms governing communication between chondrocytes and surrounding ECM components in bone growth activities. Copyright © 2017. Published by Elsevier Ltd.

Multipotential differentiation of human urine-derived stem cells: potential for therapeutic applications in urology.

PubMed

Bharadwaj, Shantaram; Liu, Guihua; Shi, Yingai; Wu, Rongpei; Yang, Bin; He, Tongchuan; Fan, Yuxin; Lu, Xinyan; Zhou, Xiaobo; Liu, Hong; Atala, Anthony; Rohozinski, Jan; Zhang, Yuanyuan

2013-09-01

We sought to biologically characterize and identify a subpopulation of urine-derived stem cells (USCs) with the capacity for multipotent differentiation. We demonstrated that single USCs can expand to a large population with 60-70 population doublings. Nine of 15 individual USC clones expressed detectable levels of telomerase and have long telomeres. These cells expressed pericyte and mesenchymal stem cell markers. Upon induction with appropriate media in vitro, USCs differentiated into bladder-associated cell types, including functional urothelial and smooth muscle cell lineages. When the differentiated USCs were seeded onto a scaffold and subcutaneously implanted into nude mice, multilayered tissue-like structures formed consisting of urothelium and smooth muscle. Additionally, USCs were able to differentiate into endothelial, osteogenic, chondrogenic, adipogenic, skeletal myogenic, and neurogenic lineages but did not form teratomas during the 1-month study despite telomerase activity. USCs may be useful in cell-based therapies and tissue engineering applications, including urogenital reconstruction. © AlphaMed Press.

Paracrine and autocrine signals promoting full chondrogenic differentiation of a mesoblastic cell line.

PubMed

Locker, Morgane; Kellermann, Odile; Boucquey, Marie; Khun, Huot; Huerre, Michel; Poliard, Anne

2004-01-01

The pluripotent mesoblastic C1 cell line was used under serum-free culture conditions to investigate how paracrine and autocrine signals cooperate to drive chondrogenesis. Sequential addition of two systemic hormones, dexamethasone and triiodothyronine, permits full chondrogenic differentiation. The cell intrinsic activation of the BMP signaling pathway and Sox9 expression occurring on mesoblastic condensation is insufficient for recruitment of the progenitors. Dexamethasone-dependent Sox9 upregulation is essential for chondrogenesis. Differentiation of lineage stem cells relies on cell autonomous regulations modulated by external signals. We used the pluripotent mesoblastic C1 cell line under serum-free culture conditions to investigate how paracrine and autocrine signals cooperate to induce differentiation of a precursor clone along the chondrogenic lineage. C1 cells, cultured as aggregates, were induced toward chondrogenesis by addition of 10(-7) M dexamethasone in serum-free medium. After 30 days, dexamethasone was replaced by 10 nM triiodothyronine to promote final hypertrophic conversion. Mature and hypertrophic phenotypes were characterized by immunocytochemistry using specific antibodies against types II and X collagens, respectively. Type II collagen, bone morphogenetic proteins (BMPs), BMP receptors, Smads, and Sox9 expression were monitored by reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot, and/or Western blot analysis. Once C1 cells have formed nodules, sequential addition of two systemic hormones is sufficient to promote full chondrogenic differentiation. In response to dexamethasone, nearly 100% of the C1 precursors engage in chondrogenesis and convert within 30 days into mature chondrocytes, which triggers a typical cartilage matrix. On day 25, a switch in type II procollagen mRNA splicing acted as a limiting step in the acquisition of the mature chondrocyte phenotype. On day 30, substitution of dexamethasone with triiodothyronine triggers the final differentiation into hypertrophic chondrocytes within a further 15 days. The chondrogenic process is supported by intrinsic expression of Sox9 and BMP family genes. Similarly to the in vivo situation, activation of Sox9 expression and the BMP signaling pathway occurred on mesoblastic condensation. After induction, BMP-activated Smad nuclear translocation persisted throughout the process until the onset of hypertrophy. After dexamethasone addition, Sox9 expression was upregulated. Dexamethasone withdrawal reversed the increase in Sox9 expression and stopped differentiation. Thus, Sox9 seems to be a downstream mediator of dexamethasone action.

Melatonin rescued interleukin 1β-impaired chondrogenesis of human mesenchymal stem cells.

PubMed

Gao, Bo; Gao, Wenjie; Wu, Zizhao; Zhou, Taifeng; Qiu, Xianjian; Wang, Xudong; Lian, Chengjie; Peng, Yan; Liang, Anjing; Qiu, Jincheng; Zhu, Yuanxin; Xu, Caixia; Li, Yibing; Su, Peiqiang; Huang, Dongsheng

2018-06-14

Osteoarthritis (OA) is a widespread arthritic disease and a primary cause of disability. Increasing evidence suggests that inflammation has a pivotal part in its pathogenesis. Interleukin-1β (IL-1β) is a primary mediator of local inflammatory processes in OA. Current therapies for OA mainly focus on the symptoms of the advanced stage of the disease. The possible utilization of bone marrow mesenchymal stem cells (BMSCs) to regenerate cartilage is an appealing method, but in the case of OA requires chondrogenesis to take place within an inflamed environment. Our previous study showed that melatonin (MLT) can promote chondrogenic differentiation of MSCs, but whether MLT can rescue IL-1β-impaired chondrogenesis in human BMSCs has not yet been established. MLT, which can have anti-inflammatory and prochondrogenic effects, has demonstrated potential in defeating IL-1β-induced inhibition of chondrogenesis and further study should be conducted. Human bone marrow-derived MSCs were separated and cultured based on our system that was already documented. A high-density micromass culture system was used for the chondrogenic differentiation of human BMSCs, which was also described previously. Human BMSCs were induced for chondrogenesis for 7, 14, and 21 days with the treatment of IL-1β and MLT. The cultured cartilage pellets were then evaluated by morphology, extracellular matrix accumulation, and chondrogenic, metabolic, and apoptotic marker expression. Furthermore, cell apoptosis was assessed by TUNEL assay. The phosphorylation level P65 and IκBα of the NF-κB pathway activity was explored on day 21 of chondrogenic differentiation of BMSCs. The current evaluation showed that MLT can save IL-1β-impaired chondrogenesis of human BMSCs in different aspects. Firstly, MLT can restore the chondrogenic pellet size, and rescue matrix synthesis and accumulation. Secondly, MLT can upregulate chondrogenic marker COL2A1 expression at both mRNA and protein levels, and also regulate the expression levels of other chondrogenic markers like ACAN, SOX9, and COL10A1 in the presence of IL-1β. Thirdly, MLT can maintain the metabolic balance of the chondrogenic process by suppressing expression of catabolic genes, such as MMP, MMP13, and ADAMTS4. Furthermore, MLT can subdue IL-1β-induced cell apoptosis of BMSCs throughout chondrogenesis. Meanwhile, MLT suppressed the phosphorylation level of P65 and IκBα, which were elevated by IL-1β treatment, indicating that MLT can attenuate the IL-1β-induced activation of NF-κB signaling. The current evaluation showed that MLT can save IL-1β-impaired chondrogenesis of human BMSCs by restoring the pellet size and matrix accumulation, and maintaining the metabolic balance, reducing cell apoptosis. Our study also showed that MLT can attenuate the IL-1β-induced activation of the NF-κB signaling pathway, which is the most important pathway downstream of IL-1β, and plays a crucial role in inflammation, apoptosis, and metabolism. Thus, MLT has prospects for treating OA due to its multifaceted functions, such as mitigating inflammation, maintaining metabolic balance, and mitigating apoptosis.

In vitro and in vivo co-culture of chondrocytes and bone marrow stem cells in photocrosslinked PCL-PEG-PCL hydrogels enhances cartilage formation.

PubMed

Ko, Chao-Yin; Ku, Kuan-Lin; Yang, Shu-Rui; Lin, Tsai-Yu; Peng, Sydney; Peng, Yu-Shiang; Cheng, Ming-Huei; Chu, I-Ming

2016-10-01

Chondrocytes (CH) and bone marrow stem cells (BMSCs) are sources that can be used in cartilage tissue engineering. Co-culture of CHs and BMSCs is a promising strategy for promoting chondrogenic differentiation. In this study, articular CHs and BMSCs were encapsulated in PCL-PEG-PCL photocrosslinked hydrogels for 4 weeks. Various ratios of CH:BMSC co-cultures were investigated to identify the optimal ratio for cartilage formation. The results thus obtained revealed that co-culturing CHs and BMSCs in hydrogels provides an appropriate in vitro microenvironment for chondrogenic differentiation and cartilage matrix production. Co-culture with a 1:4 CH:BMSC ratio significantly increased the synthesis of GAGs and collagen. In vivo cartilage regeneration was evaluated using a co-culture system in rabbit models. The co-culture system exhibited a hyaline chondrocyte phenotype with excellent regeneration, resembling the morphology of native cartilage. This finding suggests that the co-culture of these two cell types promotes cartilage regeneration and that the system, including the hydrogel scaffold, has potential in cartilage tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2013 John Wiley & Sons, Ltd.

Regeneration of articular cartilage by adipose tissue derived mesenchymal stem cells: perspectives from stem cell biology and molecular medicine.

PubMed

Wu, Ling; Cai, Xiaoxiao; Zhang, Shu; Karperien, Marcel; Lin, Yunfeng

2013-05-01

Adipose-derived stem cells (ASCs) have been discovered for more than a decade. Due to the large numbers of cells that can be harvested with relatively little donor morbidity, they are considered to be an attractive alternative to bone marrow derived mesenchymal stem cells. Consequently, isolation and differentiation of ASCs draw great attention in the research of tissue engineering and regenerative medicine. Cartilage defects cause big therapeutic problems because of their low self-repair capacity. Application of ASCs in cartilage regeneration gives hope to treat cartilage defects with autologous stem cells. In recent years, a lot of studies have been performed to test the possibility of using ASCs to re-construct damaged cartilage tissue. In this article, we have reviewed the most up-to-date articles utilizing ASCs for cartilage regeneration in basic and translational research. Our topic covers differentiation of adipose tissue derived mesenchymal stem cells into chondrocytes, increased cartilage formation by co-culture of ASCs with chondrocytes and enhancing chondrogenic differentiation of ASCs by gene manipulation. Copyright © 2012 Wiley Periodicals, Inc.

Disruption of Cell-Cell Contact-mediated Notch Signaling via Hydrogel Encapsulation Reduces Mesenchymal Stem Cell Chondrogenic Potential

PubMed Central

Chen, Amanda X.; Hoffman, Michael D.; Chen, Caressa S.; Shubin, Andrew D.; Reynolds, Daniel S.; Benoit, Danielle S. W.

2015-01-01

Cell-cell contact-mediated Notch signaling is essential for mesenchymal stem cell (MSC) chondrogenesis during development. However, subsequent deactivation of Notch signaling is also required to allow for stem cell chondrogenic progression. Recent literature has shown that Notch signaling can also influence Wnt/β-catenin signaling, critical for MSC differentiation, through perturbations in cell-cell contacts. Traditionally, abundant cell-cell contacts, consistent with development, are emulated in vitro using pellet cultures for chondrogenesis. However, cells are often encapsulated within biomaterials-based scaffolds, such as hydrogels, to improve therapeutic cell localization in vivo. To explore the role of Notch and Wnt/β-catenin signaling in the context of hydrogel-encapsulated MSC chondrogenesis, we compared signaling and differentiation capacity of MSCs in both hydrogels and traditional pellet cultures. We demonstrate that encapsulation within poly(ethylene glycol) (PEG) hydrogels reduces cell-cell contacts, and both Notch (7.5-fold) and Wnt/β-catenin (84.7-fold) pathway activation. Finally, we demonstrate that following establishment of cell-cell contacts and transient Notch signaling in pellet cultures, followed by Notch signaling deactivation, resulted in a 1.5-fold increase in MSC chondrogenesis. Taken together, these findings support that cellular condensation, and the establishment of initial cell-cell contacts is critical for MSC chondrogenesis, and this process is inhibited by hydrogel encapsulation. PMID:25504509

A putative mesenchymal stem cells population isolated from adult human testes.

PubMed

Gonzalez, R; Griparic, L; Vargas, V; Burgee, K; Santacruz, P; Anderson, R; Schiewe, M; Silva, F; Patel, A

2009-08-07

Mesenchymal stem cells (MSCs) isolated from several adult human tissues are reported to be a promising tool for regenerative medicine. In order to broaden the array of tools for therapeutic application, we isolated a new population of cells from adult human testis termed gonadal stem cells (GSCs). GSCs express CD105, CD166, CD73, CD90, STRO-1 and lack hematopoietic markers CD34, CD45, and HLA-DR which are characteristic identifiers of MSCs. In addition, GSCs express pluripotent markers Oct4, Nanog, and SSEA-4. GSCs propagated for at least 64 population doublings and exhibited clonogenic capability. GSCs have a broad plasticity and the potential to differentiate into adipogenic, osteogenic, and chondrogenic cells. These studies demonstrate that GSCs are easily obtainable stem cells, have growth kinetics and marker expression similar to MSCs, and differentiate into mesodermal lineage cells. Therefore, GSCs may be a valuable tool for therapeutic applications.

Infrapatellar Fat Pad Stem Cells: From Developmental Biology to Cell Therapy.

PubMed

do Amaral, Ronaldo J F C; Almeida, Henrique V; Kelly, Daniel J; O'Brien, Fergal J; Kearney, Cathal J

2017-01-01

The ideal cell type to be used for cartilage therapy should possess a proven chondrogenic capacity, not cause donor-site morbidity, and should be readily expandable in culture without losing their phenotype. There are several cell sources being investigated to promote cartilage regeneration: mature articular chondrocytes, chondrocyte progenitors, and various stem cells. Most recently, stem cells isolated from joint tissue, such as chondrogenic stem/progenitors from cartilage itself, synovial fluid, synovial membrane, and infrapatellar fat pad (IFP) have gained great attention due to their increased chondrogenic capacity over the bone marrow and subcutaneous adipose-derived stem cells. In this review, we first describe the IFP anatomy and compare and contrast it with other adipose tissues, with a particular focus on the embryological and developmental aspects of the tissue. We then discuss the recent advances in IFP stem cells for regenerative medicine. We compare their properties with other stem cell types and discuss an ontogeny relationship with other joint cells and their role on in vivo cartilage repair. We conclude with a perspective for future clinical trials using IFP stem cells.

Infrapatellar Fat Pad Stem Cells: From Developmental Biology to Cell Therapy

PubMed Central

Almeida, Henrique V.; Kelly, Daniel J.; O'Brien, Fergal J.; Kearney, Cathal J.

2017-01-01

The ideal cell type to be used for cartilage therapy should possess a proven chondrogenic capacity, not cause donor-site morbidity, and should be readily expandable in culture without losing their phenotype. There are several cell sources being investigated to promote cartilage regeneration: mature articular chondrocytes, chondrocyte progenitors, and various stem cells. Most recently, stem cells isolated from joint tissue, such as chondrogenic stem/progenitors from cartilage itself, synovial fluid, synovial membrane, and infrapatellar fat pad (IFP) have gained great attention due to their increased chondrogenic capacity over the bone marrow and subcutaneous adipose-derived stem cells. In this review, we first describe the IFP anatomy and compare and contrast it with other adipose tissues, with a particular focus on the embryological and developmental aspects of the tissue. We then discuss the recent advances in IFP stem cells for regenerative medicine. We compare their properties with other stem cell types and discuss an ontogeny relationship with other joint cells and their role on in vivo cartilage repair. We conclude with a perspective for future clinical trials using IFP stem cells. PMID:29018484

A comparison of the functionality and in vivo phenotypic stability of cartilaginous tissues engineered from different stem cell sources.

PubMed

Vinardell, Tatiana; Sheehy, Eamon J; Buckley, Conor T; Kelly, Daniel J

2012-06-01

Joint-derived stem cells are a promising alternative cell source for cartilage repair therapies that may overcome many of the problems associated with the use of primary chondrocytes (CCs). The objective of this study was to compare the in vitro functionality and in vivo phenotypic stability of cartilaginous tissues engineered using bone marrow-derived stem cells (BMSCs) and joint tissue-derived stem cells following encapsulation in agarose hydrogels. Culture-expanded BMSCs, fat pad-derived stem cells (FPSCs), and synovial membrane-derived stem cells (SDSCs) were encapsulated in agarose and maintained in a chondrogenic medium supplemented with transforming growth factor-β3. After 21 days of culture, constructs were either implanted subcutaneously into the back of nude mice for an additional 28 days or maintained for a similar period in vitro in either chondrogenic or hypertrophic media formulations. After 49 days of in vitro culture in chondrogenic media, SDSC constructs accumulated the highest levels of sulfated glycosaminoglycan (sGAG) (∼2.8% w/w) and collagen (∼1.8% w/w) and were mechanically stiffer than constructs engineered using other cell types. After subcutaneous implantation in nude mice, sGAG content significantly decreased for all stem cell-seeded constructs, while no significant change was observed in the control constructs engineered using primary CCs, indicating that the in vitro chondrocyte-like phenotype generated in all stem cell-seeded agarose constructs was transient. FPSCs and SDSCs appeared to undergo fibrous dedifferentiation or resorption, as evident from increased collagen type I staining and a dramatic loss in sGAG content. BMSCs followed a more endochondral pathway with increased type X collagen expression and mineralization of the engineered tissue. In conclusion, while joint tissue-derived stem cells possess a strong intrinsic chondrogenic capacity, further studies are needed to identify the factors that will lead to the generation of a more stable chondrogenic phenotype.

Investigation of Migration and Differentiation of Human Mesenchymal Stem Cells on Five-Layered Collagenous Electrospun Scaffold Mimicking Native Cartilage Structure.

PubMed

Reboredo, Jenny W; Weigel, Tobias; Steinert, Andre; Rackwitz, Lars; Rudert, Maximilian; Walles, Heike

2016-09-01

Cartilage degeneration is the major cause of chronic pain, lost mobility, and reduced quality of life for over estimated 150 million osteoarthritis sufferers worldwide. Despite intensive research, none of the available therapies can restore the hyaline cartilage surface beyond just fibrous repair. To overcome these limitations, numerous cell-based approaches for cartilage repair are being explored that aim to provide an appropriate microenvironment for chondrocyte maintenance and differentiation of multipotent mesenchymal stem cells (MSCs) toward the chondrogenic lineage. Articular cartilage is composed of highly organized collagen network that entails the tissue into four distinct zones and each zone into three different regions based on differences in matrix morphology and biochemistry. Current cartilage implants cannot establish the hierarchical tissue organization that seems critical for normal cartilage function. Therefore, in this study, a structured, multilayered collagen scaffold designed for the replacement of damaged cartilage is presented that allows repopulation by host cells and synthesis of a new natural matrix. By using the electrospinning method, the potential to engineer a scaffold consisting of two different collagen types is obtained. With the developed collagen scaffold, a five-layered biomaterial is created that has the potency to induce the differentiation of human bone marrow derived MSCs toward the chondrogenic lineage. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization and use of Equine Bone Marrow Mesenchymal Stem Cells in Equine Cartilage Engineering. Study of their Hyaline Cartilage Forming Potential when Cultured under Hypoxia within a Biomaterial in the Presence of BMP-2 and TGF-ß1.

PubMed

Branly, Thomas; Bertoni, Lélia; Contentin, Romain; Rakic, Rodolphe; Gomez-Leduc, Tangni; Desancé, Mélanie; Hervieu, Magalie; Legendre, Florence; Jacquet, Sandrine; Audigié, Fabrice; Denoix, Jean-Marie; Demoor, Magali; Galéra, Philippe

2017-10-01

Articular cartilage presents a poor capacity for self-repair. Its structure-function are frequently disrupted or damaged upon physical trauma or osteoarthritis in humans. Similar musculoskeletal disorders also affect horses and are the leading cause of poor performance or early retirement of sport- and racehorses. To develop a therapeutic solution for horses, we tested the autologous chondrocyte implantation technique developed on human bone marrow (BM) mesenchymal stem cells (MSCs) on horse BM-MSCs. This technique involves BM-MSC chondrogenesis using a combinatory approach based on the association of 3D-culture in collagen sponges, under hypoxia in the presence of chondrogenic factors (BMP-2 + TGF-β 1 ) and siRNA to knockdown collagen I and HtrA1. Horse BM-MSCs were characterized before being cultured in chondrogenic conditions to find the best combination to enhance, stabilize, the chondrocyte phenotype. Our results show a very high proliferation of MSCs and these cells satisfy the criteria defining stem cells (pluripotency-surface markers expression). The combination of BMP-2 + TGF-β 1 strongly induces the chondrogenic differentiation of MSCs and prevents HtrA1 expression. siRNAs targeting Col1a1 and Htra1 were functionally validated. Ultimately, the combined use of specific culture conditions defined here with specific growth factors and a Col1a1 siRNAs (50 nM) association leads to the in vitro synthesis of a hyaline-type neocartilage whose chondrocytes present an optimal phenotypic index similar to that of healthy, differentiated chondrocytes. Our results lead the way to setting up pre-clinical trials in horses to better understand the reaction of neocartilage substitute and to carry out a proof-of-concept of this therapeutic strategy on a large animal model.

Rap1b Is an Effector of Axin2 Regulating Crosstalk of Signaling Pathways During Skeletal Development.

PubMed

Maruyama, Takamitsu; Jiang, Ming; Abbott, Alycia; Yu, H-M Ivy; Huang, Qirong; Chrzanowska-Wodnicka, Magdalena; Chen, Emily I; Hsu, Wei

2017-09-01

Recent identification and isolation of suture stem cells capable of long-term self-renewal, clonal expanding, and differentiating demonstrate their essential role in calvarial bone development, homeostasis, and injury repair. These bona fide stem cells express a high level of Axin2 and are able to mediate bone regeneration and repair in a cell autonomous fashion. The importance of Axin2 is further demonstrated by its genetic inactivation in mice causing skeletal deformities resembling craniosynostosis in humans. The fate determination and subsequent differentiation of Axin2+ stem cells are highly orchestrated by a variety of evolutionary conserved signaling pathways including Wnt, FGF, and BMP. These signals are often antagonistic of each other and possess differential effects on osteogenic and chondrogenic cell types. However, the mechanisms underlying the interplay of these signaling transductions remain largely elusive. Here we identify Rap1b acting downstream of Axin2 as a signaling interrogator for FGF and BMP. Genetic analysis reveals that Rap1b is essential for development of craniofacial and body skeletons. Axin2 regulates Rap1b through modulation of canonical BMP signaling. The BMP-mediated activation of Rap1b promotes chondrogenic fate and chondrogenesis. Furthermore, by inhibiting MAPK signaling, Rap1b mediates the antagonizing effect of BMP on FGF to repress osteoblast differentiation. Disruption of Rap1b in mice not only enhances osteoblast differentiation but also impairs chondrocyte differentiation during intramembranous and endochondral ossifications, respectively, leading to severe defects in craniofacial and body skeletons. Our findings reveal a dual role of Rap1b in development of the skeletogenic cell types. Rap1b is critical for balancing the signaling effects of BMP and FGF during skeletal development and disease. © 2017 American Society for Bone and Mineral Research. © 2017 American Society for Bone and Mineral Research.

Nonstimulated human uncommitted mesenchymal stem cells express cell markers of mesenchymal and neural lineages.

PubMed

Minguell, José J; Fierro, Fernando A; Epuñan, María J; Erices, Alejandro A; Sierralta, Walter D

2005-08-01

Ex vivo cultures of human bone marrow-derived mesenchymal stem cells (MSCs) contain subsets of progenitors exhibiting dissimilar properties. One of these subsets comprises uncommitted progenitors displaying distinctive features, such as morphology, a quiescent condition, growth factor production, and restricted tissue biodistribution after transplantation. In this study, we assessed the competence of these cells to express, in the absence of differentiation stimuli, markers of mesoderm and ectodermic (neural) cell lineages. Fluorescence microscopy analysis showed a unique pattern of expression of osteogenic, chondrogenic, muscle, and neural markers. The depicted "molecular signature" of these early uncommitted progenitors, in the absence of differentiation stimuli, is consistent with their multipotentiality and plasticity as suggested by several in vitro and in vivo studies.

Poly(γ-Glutamic Acid) as an Exogenous Promoter of Chondrogenic Differentiation of Human Mesenchymal Stem/Stromal Cells.

PubMed

Antunes, Joana C; Tsaryk, Roman; Gonçalves, Raquel M; Pereira, Catarina Leite; Landes, Constantin; Brochhausen, Christoph; Ghanaati, Shahram; Barbosa, Mário A; Kirkpatrick, C James

2015-06-01

Cartilage damage and/or aging effects can cause constant pain, which limits the patient's quality of life. Although different strategies have been proposed to enhance the limited regenerative capacity of cartilage tissue, the full production of native and functional cartilaginous extracellular matrix (ECM) has not yet been achieved. Poly(γ-glutamic acid) (γ-PGA), a naturally occurring polyamino acid, biodegradable into glutamate residues, has been explored for tissue regeneration. In this work, γ-PGA's ability to support the production of cartilaginous ECM by human bone marrow mesenchymal stem/stromal cells (MSCs) and nasal chondrocytes (NCs) was investigated. MSC and NC pellets were cultured in basal medium (BM), chondrogenic medium (CM), and CM-γ-PGA-supplemented medium (CM+γ-PGA) over a period of 21 days. Pellet size/shape was monitored with time. At 14 and 21 days of culture, the presence of sulfated glycosaminoglycans (sGAGs), type II collagen (Col II), Sox-9, aggrecan, type XI collagen (Col XI), type X collagen (Col X), calcium deposits, and type I collagen (Col I) was analyzed. After excluding γ-PGA's cytotoxicity, earlier cell condensation, higher sGAG content, Col II, Sox-9 (day 14), aggrecan, and Col X (day 14) production was observed in γ-PGA-supplemented MSC cultures, with no signs of mineralization or Col I. These effects were not evident with NCs. However, Sox-9 (at day 14) and Col X (at days 14 and 21) were increased, decreased, or absent, respectively. Overall, γ-PGA improved chondrogenic differentiation of MSCs, increasing ECM production earlier in culture. It is proposed that γ-PGA incorporation in novel biomaterials has a beneficial impact on future approaches for cartilage regeneration.

Poly(γ-Glutamic Acid) as an Exogenous Promoter of Chondrogenic Differentiation of Human Mesenchymal Stem/Stromal Cells

PubMed Central

Antunes, Joana C.; Tsaryk, Roman; Gonçalves, Raquel M.; Pereira, Catarina Leite; Landes, Constantin; Brochhausen, Christoph; Ghanaati, Shahram

2015-01-01

Cartilage damage and/or aging effects can cause constant pain, which limits the patient's quality of life. Although different strategies have been proposed to enhance the limited regenerative capacity of cartilage tissue, the full production of native and functional cartilaginous extracellular matrix (ECM) has not yet been achieved. Poly(γ-glutamic acid) (γ-PGA), a naturally occurring polyamino acid, biodegradable into glutamate residues, has been explored for tissue regeneration. In this work, γ-PGA's ability to support the production of cartilaginous ECM by human bone marrow mesenchymal stem/stromal cells (MSCs) and nasal chondrocytes (NCs) was investigated. MSC and NC pellets were cultured in basal medium (BM), chondrogenic medium (CM), and CM-γ-PGA-supplemented medium (CM+γ-PGA) over a period of 21 days. Pellet size/shape was monitored with time. At 14 and 21 days of culture, the presence of sulfated glycosaminoglycans (sGAGs), type II collagen (Col II), Sox-9, aggrecan, type XI collagen (Col XI), type X collagen (Col X), calcium deposits, and type I collagen (Col I) was analyzed. After excluding γ-PGA's cytotoxicity, earlier cell condensation, higher sGAG content, Col II, Sox-9 (day 14), aggrecan, and Col X (day 14) production was observed in γ-PGA-supplemented MSC cultures, with no signs of mineralization or Col I. These effects were not evident with NCs. However, Sox-9 (at day 14) and Col X (at days 14 and 21) were increased, decreased, or absent, respectively. Overall, γ-PGA improved chondrogenic differentiation of MSCs, increasing ECM production earlier in culture. It is proposed that γ-PGA incorporation in novel biomaterials has a beneficial impact on future approaches for cartilage regeneration. PMID:25760236

In vitro analysis of equine, bone marrow-derived mesenchymal stem cells demonstrates differences within age- and gender-matched horses.

PubMed

Carter-Arnold, J L; Neilsen, N L; Amelse, L L; Odoi, A; Dhar, M S

2014-09-01

Stem cell therapies are used routinely in equine practice. Most published reports characterise stem cells derived from younger horses; however, middle-aged horses are often in athletic performance, and experience degenerative medical conditions. Thus, mesenchymal stem cells (MSCs) from this group should be investigated. To describe differences in in vitro adherence, proliferation and potential for differentiation of equine bone marrow-derived MSCs (equine BMMSCs) harvested from middle-aged (10-13 years old) female donors. Descriptive study of stem cell characteristics. Equine BMMSCs from 6 horses were cultured in vitro and evaluated for viability, proliferation, osteogenesis, chondrogenesis, adipogenesis, cluster-of-differentiation markers and gene expression. Equine BMMSCs from all 6 donors demonstrated fibroblastic, cellular morphology, adherence to plastic and expression of cluster-of-differentiation markers. They varied in their rate of proliferation and trilineage differentiation. The equine BMMSCs of one of 6 donors demonstrated a higher rate of proliferation, enhanced ability for cell passaging and a more robust in vitro differentiation. Comparatively, equine BMMSCs from 2 donors demonstrated a lower rate of proliferation and lack of osteogenic and chondrogenic differentiation. The results of this study confirm that donor-to-donor variation in equine BMMSCs exists and this variation can be documented using in vitro assays. Subjective assessment suggests that the rate of proliferation tends to correlate with differentiation potential. © 2013 EVJ Ltd.

Isolation and characterization of porcine adipose tissue-derived adult stem cells.

PubMed

Williams, Kellie J; Picou, Alicia A; Kish, Sharon L; Giraldo, Angelica M; Godke, Robert A; Bondioli, Kenneth R

2008-01-01

Stem cell characteristics such as self-renewal, differentiation and expression of CD34 and CD44 stem cell markers have not been identified in porcine adipose tissue-derived adult stem (ADAS) cells. The objective of this study was to develop a protocol for the isolation and culture of porcine adipose tissue-derived cells and to determine stem cell-like characteristics. Primary cultures were established and cell cultures were maintained. Cloning capacity was determined using a ring cloning procedure. Primary cultures and clones were differentiated and stained for multiple differentiated phenotypes. CD34 and CD44 messenger ribonucleic acid (mRNA) was isolated and reverse transcriptase polymerase chain reaction was used to compare expression profiles. An average of 2,700,000 nucleated cells/ml was isolated; 26% were adherent, and cells completed a cell cycle approximately every 3.3 days. Ring cloning identified 19 colonies. Primary cultures and clones were determined to differentiate along osteogenic, adipogenic and chondrogenic tissue lineages. The mRNA expression profiles showed CD34 expression was higher for undifferentiated ADAS cells versus differentiated cell types and the CD34 expression level was lower than that of CD44 among differentiated cells. Improved culture conditions and defined cellular characteristics of these porcine ADAS cells have been identified. Porcine ADAS can self-renew, can differentiate into multiple tissue lineages and they express CD34. Copyright 2008 S. Karger AG, Basel.

Hyaluronic acid effect on adipose-derived stem cells. Biological in vitro evaluation.

PubMed

Moreno, A; Martínez, A; Olmedillas, S; Bello, S; de Miguel, F

2015-01-01

To evaluate the in vitro effects of hyaluronic acid (HA) on adipose-derived stem cells (ASC) in order to consider the possibility of their combined used in the treatment of knee arthrosis. The ASC cells were grown both in the presence and absence of AH, and several studies were carried out: proliferation (WST8) and cell viability studies (Alamar Blue® and Trypan Blue), possible chondrogenic differentiation (collagen type 2 expression) by RT-PCR, AH receptor expression (CD44) by flow cytometry and RT-QPCR, and expression of inflammatory and anti-inflammatory factors (IL-6, TGFß, IL-10) by RT-QPCR. The number of ASC significantly increased after 7 days with HA (158±39%, p <0.05). Additionally, the cell viability of the ASC treated with HA after 1, 3, 5 and 7 days was similar to that of the control cells, being considered non-toxic. There were no changes observed in the expression of CD44 and chondrogenic differentiation. TGFß expression was not modified after AH treatment, but there was a 4-fold decrease in IL-6 expression and IL-10 expression increased up to 2-fold compared to control cells. Hyaluronic acid favours ASC proliferation without causing cellular toxicity, and inducing an anti-inflammatory profile in these cells. Hyaluronic acid appears to be a suitable vehicle for the intra-articular administration of mesenchymal stem cells. Copyright © 2014 SECOT. Published by Elsevier Espana. All rights reserved.

Fibroblast growth factor-2 stimulates adipogenic differentiation of human adipose-derived stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kakudo, Natsuko; Shimotsuma, Ayuko; Kusumoto, Kenji

2007-07-27

Adipose-derived stem cells (ASCs) have demonstrated a capacity for differentiating into a variety of lineages, including bone, cartilage, or fat, depending on the inducing stimuli and specific growth and factors. It is acknowledged that fibroblast growth factor-2 (FGF-2) promotes chondrogenic and inhibits osteogenic differentiation of ASCs, but thorough investigations of its effects on adipogenic differentiation are lacking. In this study, we demonstrate at the cellular and molecular levels the effect of FGF-2 on adipogenic differentiation of ASCs, as induced by an adipogenic hormonal cocktail consisting of 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, insulin, and indomethacin. FGF-2 significantly enhances the adipogenic differentiation of humanmore » ASCs. Furthermore, in cultures receiving FGF-2 before adipogenic induction, mRNA expression of peroxisome proliferator-activated receptor {gamma}2 (PPAR{gamma}2), a key transcription factor in adipogenesis, was upregulated. The results of FGF-2 supplementation suggest the potential applications of FGF-2 and ASCs in adipose tissue regeneration.« less

Generation of hyaline cartilaginous tissue from mouse adult dermal fibroblast culture by defined factors

PubMed Central

Hiramatsu, Kunihiko; Sasagawa, Satoru; Outani, Hidetatsu; Nakagawa, Kanako; Yoshikawa, Hideki; Tsumaki, Noriyuki

2011-01-01

Repair of cartilage injury with hyaline cartilage continues to be a challenging clinical problem. Because of the limited number of chondrocytes in vivo, coupled with in vitro de-differentiation of chondrocytes into fibrochondrocytes, which secrete type I collagen and have an altered matrix architecture and mechanical function, there is a need for a novel cell source that produces hyaline cartilage. The generation of induced pluripotent stem (iPS) cells has provided a tool for reprogramming dermal fibroblasts to an undifferentiated state by ectopic expression of reprogramming factors. Here, we show that retroviral expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) induces polygonal chondrogenic cells directly from adult dermal fibroblast cultures. Induced cells expressed marker genes for chondrocytes but not fibroblasts, i.e., the promoters of type I collagen genes were extensively methylated. Although some induced cell lines formed tumors when subcutaneously injected into nude mice, other induced cell lines generated stable homogenous hyaline cartilage–like tissue. Further, the doxycycline-inducible induction system demonstrated that induced cells are able to respond to chondrogenic medium by expressing endogenous Sox9 and maintain chondrogenic potential after substantial reduction of transgene expression. Thus, this approach could lead to the preparation of hyaline cartilage directly from skin, without generating iPS cells. PMID:21293062

Comparative analysis of rat mesenchymal stem cells derived from slow and fast skeletal muscle in vitro.

PubMed

Okumachi, Etsuko; Lee, Sang Yang; Niikura, Takahiro; Iwakura, Takashi; Dogaki, Yoshihiro; Waki, Takahiro; Takahara, Shunsuke; Ueha, Takeshi; Sakai, Yoshitada; Kuroda, Ryosuke; Kurosaka, Masahiro

2015-03-01

Skeletal muscle comprises different kinds of muscle fibres that can be classified as slow and fast fibres. The purpose of this study was to compare the yield, proliferation, and multi-potentiality of rat mesenchymal stem cells (MSCs) from the tibialis anterior (TA; fast muscle) and soleus (SO; slow muscle) in vitro. The TA and SO muscles were harvested, and isolated cells were plated. After two hours, the cells were washed extensively to remove any cell that did not adhere to the cell culture plate. The adherent cells, namely MSCs, were then cultured. Both types of MSCs were differentiated toward the osteogenic, chondrogenic and adipogenic lineages using lineage specific induction factors. The colony-forming unit fibroblast (CFU-F) assay revealed that the SO contained significantly higher quantities of MSCs than the TA. The self-renewal capacity of MSCs derived from the TA was significantly higher at later passages (passage 9-11). Both types of MSCs exhibited similar cell surface antigens to bone marrow (BM)-derived MSCs and were positive for CD29, CD44, and CD90 and negative for CD11b, CD34, and CD45. TA-derived MSCs were superior in terms of osteogenic differentiation capacity, but there was no significant difference in chondrogenic and adipogenic differentiation capacity. Our results demonstrated significant differences in the properties of muscle-derived MSCs from different muscle types (i.e. fast or slow muscles). The greater expandability and osteogenic differentiation ability of TA-derived MSCs suggests that fast muscle may be a better source for generating large numbers of MSCs for bone regeneration.

Thyroid Hormone-Induced Hypertrophy in Mesenchymal Stem Cell Chondrogenesis Is Mediated by Bone Morphogenetic Protein-4

PubMed Central

Karl, Alexandra; Olbrich, Norman; Pfeifer, Christian; Berner, Arne; Zellner, Johannes; Kujat, Richard; Angele, Peter; Nerlich, Michael

2014-01-01

Chondrogenic differentiating mesenchymal stem cells (MSCs) express markers of hypertrophic growth plate chondrocytes. As hypertrophic cartilage undergoes ossification, this is a concern for the application of MSCs in articular cartilage tissue engineering. To identify mechanisms that elicit this phenomenon, we used an in vitro hypertrophy model of chondrifying MSCs for differential gene expression analysis and functional experiments with the focus on bone morphogenetic protein (BMP) signaling. Hypertrophy was induced in chondrogenic MSC pellet cultures by transforming growth factor β (TGFβ) and dexamethasone withdrawal and addition of triiodothyronine. Differential gene expression analysis of BMPs and their receptors was performed. Based on these results, the in vitro hypertrophy model was used to investigate the effect of recombinant BMP4 and the BMP inhibitor Noggin. The enhancement of hypertrophy could be shown clearly by an increased cell size, alkaline phosphatase activity, and collagen type X deposition. Upon induction of hypertrophy, BMP4 and the BMP receptor 1B were upregulated. Addition of BMP4 further enhanced hypertrophy in the absence, but not in the presence of TGFβ and dexamethasone. Thyroid hormone induced hypertrophy by upregulation of BMP4 and this induced enhancement of hypertrophy could be blocked by the BMP antagonist Noggin. BMP signaling is an important modulator of the late differentiation stages in MSC chondrogenesis and the thyroid hormone induces this pathway. As cartilage tissue engineering constructs will be exposed to this factor in vivo, this study provides important insight into the biology of MSC-based cartilage. Furthermore, the possibility to engineer hypertrophic cartilage may be helpful for critical bone defect repair. PMID:23937304

Development of a new method to harvest chondroprogenitor cells from underneath cartilage defects in the knees.

PubMed

Elvenes, Jan; Knutsen, Gunnar; Johansen, Oddmund; Moe, Bjørn T; Martinez, Inigo

2009-07-01

Mesenchymal progenitor cells from bone marrow hold great potential as a cell source for cartilage repair. Aspiration from the iliac crest is the most widely used method to harvest bone marrow cells for cartilage repair. The objective of our study was to establish a new method to isolate mesenchymal progenitor cells by direct aspiration of bone marrow from the subchondral spongious bone underneath cartilage defects during microfracture treatment and to confirm the chondrogenic potential of the resulting cell cultures. Bone marrow was aspirated arthroscopically from patients treated for isolated cartilage defects. Adherent stromal cells were isolated, expanded in monolayer cultures, and characterized by flow cytometry. Chondrogenic induction of cells was achieved by combination of spheroid cultures in hanging drops and the concomitant use of transforming growth factor-beta (TGFbeta). Articular chondrocytes established in three-dimensional (3D) cultures were used as positive cartilage-forming units, and skin fibroblasts were used as negative controls. Three-dimensional constructs were stained for immunohistochemical and histological examination, and a real-time polymerase chain reaction (PCR) was performed to quantify the expression of aggrecan, collagen types 1 and 2, and Sox9. Mesenchymal stem cell-like progenitor cells (MSCs) displaying chondrogenic differentiation capacity were harvested arthroscopically from underneath cartilage lesions on distal femurs using the one-hole technique. Stem cell-related surface antigens analyzed by flow cytometry confirmed the nature of the isolated adherent cells. MSC spheroids stained positive for glycosaminoglycans and collagen type 2. Realtime PCR showed that MSCs in 3D spheroids significantly increased gene expression of collagen type 2, aggrecan, and Sox 9 and down-regulated expression of collagen type 1 when compared to the mRNA levels measured in MSCs monolayers. We describe a new technique that may be applied for harvesting bone marrow cells from cartilage defects during arthroscopic intervention of the knee. Cells harvested in this way hold full chondrogenic differentiation potential. Our data imply that MSC storage may be established by using marrow from this approach, bypassing the need for cell aspiration from the iliac crest.

Transfection of the IHH gene into rabbit BMSCs in a simulated microgravity environment promotes chondrogenic differentiation and inhibits cartilage aging.

PubMed

Liu, Peng-Cheng; Liu, Kuan; Liu, Jun-Feng; Xia, Kuo; Chen, Li-Yang; Wu, Xing

2016-09-27

The effect of overexpressing the Indian hedgehog (IHH) gene on the chondrogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells (BMSCs) was investigated in a simulated microgravity environment. An adenovirus plasmid encoding the rabbit IHH gene was constructed in vitro and transfected into rabbit BMSCs. Two large groups were used: conventional cell culture and induction model group and simulated microgravity environment group. Each large group was further divided into blank control group, GFP transfection group, and IHH transfection group. During differentiation induction, the expression levels of cartilage-related and cartilage hypertrophy-related genes and proteins in each group were determined. In the conventional model, the IHH transfection group expressed high levels of cartilage-related factors (Coll2 and ANCN) at the early stage of differentiation induction and expressed high levels of cartilage hypertrophy-related factors (Coll10, annexin 5, and ALP) at the late stage. Under the simulated microgravity environment, the IHH transfection group expressed high levels of cartilage-related factors and low levels of cartilage hypertrophy-related factors at all stages of differentiation induction. Under the simulated microgravity environment, transfection of the IHH gene into BMSCs effectively promoted the generation of cartilage and inhibited cartilage aging and osteogenesis. Therefore, this technique is suitable for cartilage tissue engineering.

Micro-Topographies Promote Late Chondrogenic Differentiation Markers in the ATDC5 Cell Line.

PubMed

Le, Bach Q; Vasilevich, Aliaksei; Vermeulen, Steven; Hulshof, Frits; Stamatialis, Dimitrios F; van Blitterswijk, Clemens A; de Boer, Jan

2017-05-01

Chemical and mechanical cues are well-established influencers of in vitro chondrogenic differentiation of ATDC5 cells. Here, we investigate the role of topographical cues in this differentiation process, a study not been explored before. Previously, using a library of surface micro-topographies we found some distinct patterns that induced alkaline phosphatase (ALP) production in human mesenchymal stromal cells. ALP is also a marker for hypertrophy, the end stage of chondrogenic differentiation preceding bone formation. Thus, we hypothesized that these patterns could influence end-stage chondrogenic differentiation of ATDC5 cells. In this study, we randomly selected seven topographies among the ALP influencing hits. Cells grown on these surfaces displayed varying nuclear shape and actin filament structure. When stimulated with insulin-transferrin-selenium (ITS) medium, nodule formation occurred and in some cases showed alignment to the topographical patterns. Gene expression analysis of cells growing on topographical surfaces in the presence of ITS medium revealed a downregulation of early markers and upregulation of late markers of chondrogenic differentiation compared to cells grown on a flat surface. In conclusion, we demonstrated that surface topography in addition to other cues can promote hypertrophic differentiation suitable for bone tissue engineering.

Chondrogenic Differentiation Processes in Human Bone Marrow Aspirates Seeded in Three-Dimensional Woven Poly(ε-Caprolactone) Scaffolds Enhanced by rAAV-Mediated SOX9 Gene Transfer.

PubMed

Venkatesan, Jagadeesh Kumar; Moutos, Franklin T; Rey-Rico, Ana; Estes, Bradley T; Frisch, Janina; Schmitt, Gertrud; Madry, Henning; Guilak, Farshid; Cucchiarini, Magali

2018-05-02

Combining gene therapy approaches with tissue engineering procedures is an active area of translational research for the effective treatment of articular cartilage lesions, especially to target chondrogenic progenitor cells such as those derived from the bone marrow. Here, we evaluated the effect of genetically modifying concentrated human mesenchymal stem cells from bone marrow to induce chondrogenesis by recombinant adeno-associated viral (rAAV) vector gene transfer of the sex-determining region Y-type high-mobility group box 9 (SOX9) factor upon seeding in three-dimensional (3D) woven poly(ε-caprolactone) (PCL) scaffolds that provide mechanical properties mimicking those of native articular cartilage. Prolonged, effective SOX9 expression was reported in the constructs for at least 21 days, the longest time point evaluated, leading to enhanced metabolic and chondrogenic activities relative to the control conditions (reporter lacZ gene transfer or absence of vector treatment) but without affecting the proliferative activities in the samples. The application of the rAAV SOX9 vector also prevented undesirable hypertrophic and terminal differentiation in the seeded concentrates. As bone marrow is readily accessible during surgery, such findings reveal the therapeutic potential of providing rAAV-modified marrow concentrates within 3D woven PCL scaffolds for repair of focal cartilage lesions.

Jellyfish collagen and alginate: Combined marine materials for superior chondrogenesis of hMSC.

PubMed

Pustlauk, W; Paul, B; Gelinsky, M; Bernhardt, A

2016-07-01

Marine, hybrid constructs of porous scaffolds from fibrillized jellyfish collagen and alginate hydrogel are mimicking both of the main tissue components of cartilage, thus being a promising approach for chondrogenic differentiation of human mesenchymal stem cells (hMSC). Investigating their potential for articular cartilage repair, the present study examined scaffolds being either infiltrated with an alginate-cell-suspension (ACS) or seeded with hMSC and embedded in alginate after cell adhesion (EAS). Hybrid constructs with 2×10(5) and 4.5×10(5)hMSC/scaffold were compared to hMSC encapsulated in pure alginate discs, both chondrogenically stimulated for 21days. Typical round, chondrocyte-like morphology was observed in pure alginate gels and ACS scaffolds, while cells in EAS were elongated and tightly attached to the collagen pores. Col 2 gene expression was comparable in all scaffold types examined. However, the Col 2/Col 1 ratio was higher for pure alginate discs and ACS scaffolds compared to EAS. In contrast, cells in EAS scaffolds displayed higher gene expression of Sox 9, Col 11 and ACAN compared to ACS and pure alginate. Secretion of sulfated glycosaminoglycans (sGAG) was comparable for ACS and EAS scaffolds. In conclusion hybrid constructs of jellyfish collagen and alginate support hMSC chondrogenic differentiation and provide more stable and constructs compared to pure hydrogels. Copyright © 2016 Elsevier B.V. All rights reserved.

A new fibrin sealant as a three-dimensional scaffold candidate for mesenchymal stem cells

PubMed Central

2014-01-01

Introduction The optimization of an organic scaffold for specific types of applications and cells is vital to successful tissue engineering. In this study, we investigated the effects of a new fibrin sealant derived from snake venom as a scaffold for mesenchymal stem cells, to demonstrate the ability of cells to affect and detect the biological microenvironment. Methods The characterization of CD34, CD44 and CD90 expression on mesenchymal stem cells was performed by flow cytometry. In vitro growth and cell viability were evaluated by light and electron microscopy. Differentiation into osteogenic, adipogenic and chondrogenic lineages was induced. Results The fibrin sealant did not affect cell adhesion, proliferation or differentiation and allowed the adherence and growth of mesenchymal stem cells on its surface. Hoechst 33342 and propidium iodide staining demonstrated the viability of mesenchymal stem cells in contact with the fibrin sealant and the ability of the biomaterial to maintain cell survival. Conclusions The new fibrin sealant is a three-dimensional scaffolding candidate that is capable of maintaining cell survival without interfering with differentiation, and might also be useful in drug delivery. Fibrin sealant has a low production cost, does not transmit infectious diseases from human blood and has properties of a suitable scaffold for stem cells because it permits the preparation of differentiated scaffolds that are suitable for every need. PMID:24916098

High-throughput bone and cartilage micropellet manufacture, followed by assembly of micropellets into biphasic osteochondral tissue.

PubMed

Babur, Betul Kul; Futrega, Kathryn; Lott, William B; Klein, Travis Jacob; Cooper-White, Justin; Doran, Michael Robert

2015-09-01

Engineered biphasic osteochondral tissues may have utility in cartilage defect repair. As bone-marrow-derived mesenchymal stem/stromal cells (MSC) have the capacity to make both bone-like and cartilage-like tissues, they are an ideal cell population for use in the manufacture of osteochondral tissues. Effective differentiation of MSC to bone-like and cartilage-like tissues requires two unique medium formulations and this presents a challenge both in achieving initial MSC differentiation and in maintaining tissue stability when the unified osteochondral tissue is subsequently cultured in a single medium formulation. In this proof-of-principle study, we used an in-house fabricated microwell platform to manufacture thousands of micropellets formed from 166 MSC each. We then characterized the development of bone-like and cartilage-like tissue formation in the micropellets maintained for 8-14 days in sequential combinations of osteogenic or chondrogenic induction medium. When bone-like or cartilage-like micropellets were induced for only 8 days, they displayed significant phenotypic changes when the osteogenic or chondrogenic induction medium, respectively, was swapped. Based on these data, we developed an extended 14-day protocol for the pre-culture of bone-like and cartilage-like micropellets in their respective induction medium. Unified osteochondral tissues were formed by layering 12,000 osteogenic micropellets and 12,000 chondrogenic micropellets into a biphasic structure and then further culture in chondrogenic induction medium. The assembled tissue was cultured for a further 8 days and characterized via histology. The micropellets had amalgamated into a continuous structure with distinctive bone-like and cartilage-like regions. This proof-of-concept study demonstrates the feasibility of micropellet assembly for the formation of osteochondral-like tissues for possible use in osteochondral defect repair.

PLGA-based microcarriers induce mesenchymal stem cell chondrogenesis and stimulate cartilage repair in osteoarthritis.

PubMed

Morille, Marie; Toupet, Karine; Montero-Menei, Claudia N; Jorgensen, Christian; Noël, Danièle

2016-05-01

In the present study, we aimed at evaluating the ability of novel PLGA-P188-PLGA-based microspheres to induce the differentiation of mesenchymal stem/stromal cells (MSC) into chondrocytes. To this aim, we tested microspheres releasing TGFβ3 (PAM-T) in vitro and in situ, in a pathological osteoarthritic (OA) environment. We first evaluated the chondrogenic differentiation of human MSCs seeded onto PAM-T in vitro and confirmed the up-regulation of chondrogenic markers while the secretome of the cells was not changed by the 3D environment. We then injected human MSC seeded onto PAM-T in the knee joints of mice with collagenase-induced OA. After 6 weeks, histological analysis revealed that formation of a cartilage-like tissue occurred at the vicinity of PAM-T that was not observed when MSCs were seeded onto PAM. We also noticed that the endogenous articular cartilage was less degraded. The extent of cartilage protection was further analysed by confocal laser microscopy. When MSCs seeded onto PAM-T were injected early after OA induction, protection of cartilage against degradation was evidenced and this effect was associated to a higher survival of MSCs in presence of TGFβ3. This study points to the interest of using MSCs seeded onto PAM for cartilage repair and stimulation of endogenous cartilage regeneration. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparison of Uncultured Marrow Mononuclear Cells and Culture-Expanded Mesenchymal Stem Cells in 3D Collagen-Chitosan Microbeads for Orthopedic Tissue Engineering

PubMed Central

Wise, Joel K.; Alford, Andrea I.; Goldstein, Steven A.

2014-01-01

Stem cell-based therapies have shown promise in enhancing repair of bone and cartilage. Marrow-derived mesenchymal stem cells (MSC) are typically expanded in vitro to increase cell number, but this process is lengthy, costly, and there is a risk of contamination and altered cellular properties. Potential advantages of using fresh uncultured bone marrow mononuclear cells (BMMC) include heterotypic cell and paracrine interactions between MSC and other marrow-derived cells including hematopoietic, endothelial, and other progenitor cells. In the present study, we compared the osteogenic and chondrogenic potential of freshly isolated BMMC to that of cultured-expanded MSC, when encapsulated in three-dimensional (3D) collagen-chitosan microbeads. The effect of low and high oxygen tension on cell function and differentiation into orthopedic lineages was also examined. Freshly isolated rat BMMC (25×106 cells/mL, containing an estimated 5×104 MSC/mL) or purified and culture-expanded rat bone marrow-derived MSC (2×105 cells/mL) were added to a 65–35 wt% collagen-chitosan hydrogel mixture and fabricated into 3D microbeads by emulsification and thermal gelation. Microbeads were cultured in control MSC growth media in either 20% O2 (normoxia) or 5% O2 (hypoxia) for an initial 3 days, and then in control, osteogenic, or chondrogenic media for an additional 21 days. Microbead preparations were evaluated for viability, total DNA content, calcium deposition, and osteocalcin and sulfated glycosaminoglycan expression, and they were examined histologically. Hypoxia enhanced initial progenitor cell survival in fresh BMMC-microbeads, but it did not enhance osteogenic potential. Fresh uncultured BMMC-microbeads showed a similar degree of osteogenesis as culture-expanded MSC-microbeads, even though they initially contained only 1/10th the number of MSC. Chondrogenic differentiation was not strongly supported in any of the microbead formulations. This study demonstrates the microbead-based approach to culturing and delivering cells for tissue regeneration, and suggests that fresh BMMC may be an alternative to using culture-expanded MSC for bone tissue engineering. PMID:23879621

Comparison of uncultured marrow mononuclear cells and culture-expanded mesenchymal stem cells in 3D collagen-chitosan microbeads for orthopedic tissue engineering.

PubMed

Wise, Joel K; Alford, Andrea I; Goldstein, Steven A; Stegemann, Jan P

2014-01-01

Stem cell-based therapies have shown promise in enhancing repair of bone and cartilage. Marrow-derived mesenchymal stem cells (MSC) are typically expanded in vitro to increase cell number, but this process is lengthy, costly, and there is a risk of contamination and altered cellular properties. Potential advantages of using fresh uncultured bone marrow mononuclear cells (BMMC) include heterotypic cell and paracrine interactions between MSC and other marrow-derived cells including hematopoietic, endothelial, and other progenitor cells. In the present study, we compared the osteogenic and chondrogenic potential of freshly isolated BMMC to that of cultured-expanded MSC, when encapsulated in three-dimensional (3D) collagen-chitosan microbeads. The effect of low and high oxygen tension on cell function and differentiation into orthopedic lineages was also examined. Freshly isolated rat BMMC (25 × 10(6) cells/mL, containing an estimated 5 × 10(4) MSC/mL) or purified and culture-expanded rat bone marrow-derived MSC (2 × 10(5) cells/mL) were added to a 65-35 wt% collagen-chitosan hydrogel mixture and fabricated into 3D microbeads by emulsification and thermal gelation. Microbeads were cultured in control MSC growth media in either 20% O2 (normoxia) or 5% O2 (hypoxia) for an initial 3 days, and then in control, osteogenic, or chondrogenic media for an additional 21 days. Microbead preparations were evaluated for viability, total DNA content, calcium deposition, and osteocalcin and sulfated glycosaminoglycan expression, and they were examined histologically. Hypoxia enhanced initial progenitor cell survival in fresh BMMC-microbeads, but it did not enhance osteogenic potential. Fresh uncultured BMMC-microbeads showed a similar degree of osteogenesis as culture-expanded MSC-microbeads, even though they initially contained only 1/10th the number of MSC. Chondrogenic differentiation was not strongly supported in any of the microbead formulations. This study demonstrates the microbead-based approach to culturing and delivering cells for tissue regeneration, and suggests that fresh BMMC may be an alternative to using culture-expanded MSC for bone tissue engineering.

Effect of Uniaxial Tensile Cyclic Loading Regimes on Matrix Organization and Tenogenic Differentiation of Adipose-Derived Stem Cells Encapsulated within 3D Collagen Scaffolds

PubMed Central

Stasuk, Alexander

2017-01-01

Adipose-derived mesenchymal stem cells have become a popular cell choice for tendon repair strategies due to their relative abundance, ease of isolation, and ability to differentiate into tenocytes. In this study, we investigated the solo effect of different uniaxial tensile strains and loading frequencies on the matrix directionality and tenogenic differentiation of adipose-derived stem cells encapsulated within three-dimensional collagen scaffolds. Samples loaded at 0%, 2%, 4%, and 6% strains and 0.1 Hz and 1 Hz frequencies for 2 hours/day over a 7-day period using a custom-built uniaxial tensile strain bioreactor were characterized in terms of matrix organization, cell viability, and musculoskeletal gene expression profiles. The results displayed that the collagen fibers of the loaded samples exhibited increased matrix directionality with an increase in strain values. Gene expression analyses demonstrated that ASC-encapsulated collagen scaffolds loaded at 2% strain and 0.1 Hz frequency showed significant increases in extracellular matrix genes and tenogenic differentiation markers. Importantly, no cross-differentiation potential to osteogenic, chondrogenic, and myogenic lineages was observed at 2% strain and 0.1 Hz frequency loading condition. Thus, 2% strain and 0.1 Hz frequency were identified as the appropriate mechanical loading regime to induce tenogenic differentiation of adipose-derived stem cells cultured in a three-dimensional environment. PMID:29375625

Complete human serum maintains viability and chondrogenic potential of human synovial stem cells: suitable conditions for transplantation.

PubMed

Mizuno, Mitsuru; Katano, Hisako; Otabe, Koji; Komori, Keiichiro; Kohno, Yuji; Fujii, Shizuka; Ozeki, Nobutake; Horie, Masafumi; Tsuji, Kunikazu; Koga, Hideyuki; Muneta, Takeshi; Sekiya, Ichiro

2017-06-13

In our clinical practice, we perform transplantations of autologous synovial mesenchymal stem cells (MSCs) for cartilage and meniscus regenerative medicine. One of the most important issues to ensuring clinical efficacy involves the transport of synovial MSCs from the processing facility to the clinic. Complete human serum (100% human serum) is an attractive candidate material in which to suspend synovial MSCs for their preservation during transport. The purpose of this study was to investigate whether complete human serum maintained MSC viability and chondrogenic potential and to examine the optimal temperature conditions for the preservation of human synovial MSCs. Human synovium was harvested from the knees of 14 donors with osteoarthritis during total knee arthroplasty. Passage 2 synovial MSCs were suspended at 2 million cells/100 μL in Ringer's solution or complete human serum at 4, 13, and 37 °C for 48 h. These cells were analyzed for live cell rates, cell surface marker expression, metabolic activity, proliferation, and adipogenic, calcification, and chondrogenic differentiation potentials before and after preservation. After preservation, synovial MSCs maintained higher live cell rates in human serum than in Ringer's solution at 4 and 13 °C. Synovial MSCs preserved in human serum at 4 and 13 °C also maintained high ratios of propidium iodide - and annexin V - cells. MSC surface marker expression was not altered in cells preserved at 4 and 13 °C. The metabolic activities of cells preserved in human serum at 4 and 13 °C was maintained, while significantly reduced in other conditions. Replated MSCs retained their proliferation ability when preserved in human serum at 4 and 13 °C. Adipogenesis and calcification potential could be observed in cells preserved in each condition, whereas chondrogenic potential was retained only in cells preserved in human serum at 4 and 13 °C. The viability and chondrogenic potential of synovial MSCs were maintained when the cells were suspended in human serum at 4 and 13 °C.

Effect of nanodiamond modification of siloxane surfaces on stem cell behaviour

NASA Astrophysics Data System (ADS)

Keremidarska, M.; Hikov, T.; Radeva, E.; Pramatarova, L.; Krasteva, N.

2014-12-01

Mesenchymal stem cells (MSCs) hold a great promise for use in many cell therapies and tissue engineering due to their remarkable potential to replicate indefinitely and differentiate into various cell types. Many efforts have been put to study the factors controlling stem cell differentiation. However, still little knowledge has been gained to what extent biomaterials properties influence stem cell adhesion, growth and differentiation. Research utilizing bone marrow-derived MSCs has concentrated on development of specific materials which can enhance specific differentiation of stem cells e.g. osteogenic and chondrogenic. In the present work we have modified an organosilane, hexamethyldisiloxane (HMDS) with detonation nanodiamond (DND) particles aiming to improve adhesion, growth and osteodifferentiation of rat mesenchymal stem cells. HMDS/DND films were deposited on cover glass using two approaches: premixing of both compounds, followed by plasma polymerization (PP) and PP of HMDS followed by plasma deposition of DND particles. We did not observe however an increase in rMSCs adhesion and growth on DND-modified PPHMDS surfaces compared to unmodified PPHMDS. When we studied alkaline phosphatase (ALP) activity, which is a major sign for early osteodifferentiation, we found the highest ALP activity on the PPHMDS/DND material, prepared by consequent deposition while on the other composite material ALP activity was the lowest. These results suggested that DND-modified materials were able to control osteodifferention in MSCs depending on the deposition approach. Modification of HMDS with DND particles by consequent plasma deposition seems to be a promising approach to produce biomaterials capable to guide stem cell differentiation toward osteoblasts and thus to be used in bone tissue engineering.

Immunochemical, ultrastructural and electrophysiological investigations of bone-derived stem cells in the course of neuronal differentiation.

PubMed

Wenisch, Sabine; Trinkaus, Katja; Hild, Anne; Hose, Dirk; Heiss, Christian; Alt, Volker; Klisch, Christopher; Meissl, Hilmar; Schnettler, Reinhard

2006-06-01

Numerous reports have highlighted the use of mesenchymal stem cells (MSC) for tissue engineering because of the capacity of the cells to differentiate along the osteogenic, chondrogenic or adipogenic pathway. As MSC also display neuronal morphologies under appropriate culture conditions, the differentiation capacity of stem cells seems to be more complex than initially thought, but it requires careful characterization of the cells. This is especially the case because recently it has been suggested that neuronal differentiation of stem cells is only an artifact. Here, we investigate the sequence of ultrastructural changes of bone-derived stem cells during neuronal induction and compare these data with immunocytochemical and electrophysiological properties of the cells. For further comparative analyses, stem cells were incubated with non-neurologically inducing stressors. The stem cells were harvested from human osseous debris and were characterized morphologically, immunocytochemically and by using FACS. After 6 h of neuronal induction, the cells had assumed neuronal morphologies and expressed neuron-specific enolase, beta-III-tubulin, neurofilament-H and HNK-1, while only a subpopulation expressed CD15 and synaptophysin. However, electrical signaling could not be detected, neither spontaneously nor after electrical stimulation. Nevertheless, transmission electron microscopy revealed cellular features of neuritogenesis and synaptogenesis in the course of neuronal induction and suggested that the cells have features similar to those observed in immature neurons. Based upon the results, it can be concluded that neuronal induction had initiated the early steps of neuronal differentiation, while exposure of the cells to non-neurological stressors had caused necrotic alterations.

Sonic hedgehog protein promotes proliferation and chondrogenic differentiation of bone marrow-derived mesenchymal stem cells in vitro.

PubMed

Warzecha, Jörg; Göttig, Stephan; Brüning, Christian; Lindhorst, Elmar; Arabmothlagh, Mohammad; Kurth, Andreas

2006-10-01

Sonic hedgehog (Shh) protein is known to be an important signaling protein in early embryonic development. Also, Shh is involved in the induction of early cartilaginous differentiation of mesenchymal cells in the limb and in the spine. The impact of Shh on adult stem cells, human bone marrow-derived mesenchymal stem cells (MSCs), was tested. The MSCs were treated either with recombinant Sonic hedgehog protein (r-Shh) or with transforming growth factor-beta 1 (TGF-beta(1)) as a positive control in vitro for 3 weeks. The effects on cartilaginous differentiation and proliferation were assayed. MSCs when treated with either Shh or TGF-beta(1) showed expression of cartilage markers aggrecan, Sox9, CEP-68, and collagen type II and X within 3 weeks. Only r-Shh-treated cells showed a very strong cell proliferation and much higher BrdU incorporation in cell assay systems. These are the first data that indicate an important role of Shh for the induction of cartilage production by MSCs in vitro.

Effects of non-steroidal anti-inflammatory drugs on proliferation, differentiation and migration in equine mesenchymal stem cells.

PubMed

Müller, Maike; Raabe, Oksana; Addicks, Klaus; Wenisch, Sabine; Arnhold, Stefan

2011-03-01

In equine medicine, stem cell therapies for orthopaedic diseases are routinely accompanied by application of NSAIDs (non-steroidal anti-inflammatory drugs). Thus, it has to be analysed how NSAIDs actually affect the growth and differentiation potential of MSCs (mesenchymal stem cells) in vitro in order to predict the influence of NSAIDs such as phenylbutazone, meloxicam, celecoxib and flunixin on MSCs after grafting in vivo. The effects of NSAIDs were evaluated regarding cell viability and proliferation. Additionally, the multilineage differentiation capacity and cell migration was analysed. NSAIDs at lower concentrations (0.1-1 μM for celecoxib and meloxicam and 10-50 μM for flunixin) exert a positive effect on cell proliferation and migration, while at higher concentrations (10-200 μM for celecoxib and meloxicam and 100-1000 μM for flunixin and phenylbutazone), there is rather a negative influence. While there is hardly any influence on the adipogenic as well as on the chondrogenic MSC differentiation, the osteogenic differentiation potential, as demonstrated with the von Kossa staining, is significantly disturbed. Thus, it can be concluded that the effects of NSAIDs on MSCs are largely dependent on the concentrations used. Additionally, for some differentiation lineages, also the choice of NSAID is critical.

Nontoxic Genetic Engineering of Mesenchymal Stem Cells Using Serum-Compatible Pullulan-Spermine/DNA Anioplexes

PubMed Central

Thakor, Devang K.; Obata, Hideaki; Nagane, Kentaro; Saito, Shigeru

2011-01-01

Genetic modification of stem cells could be applied to initiate/enhance their secretion of therapeutic molecules, alter their biological properties, or label them for in vivo tracking. We recently developed a negatively charged gene carrier (“anioplex”) based on pullulan-spermine, a conjugate prepared from a natural polysaccharide and polyamine. In rat mesenchymal stem cells (MSCs), anioplex-derived reporter gene activity was comparable to or exceeded that obtained using a commercial cationic lipid reagent. Transfection in the growth medium with 15% serum and antibiotics was approximately sevenfold more effective than in serum-free conditions. Cytotoxicity was essentially indiscernible after 24 h of anioplex transfection with 20 μg/mL DNA, in contrast to cationic lipid transfection that resulted in 40%–60% death of target MSCs. Anioplex-derived reporter gene activity persisted throughout the entire 3-week study, with post-transfection MSCs appearing to maintain osteogenic, adipogenic, and chondrogenic multipotency. In particular, chondrogenic pellet formation of differentiating human MSCs was significantly inhibited after lipofection but not after aniofection, which further indicates the biological inertness of pullulan-spermine/DNA anioplexes. Collectively, these data introduce a straightforward technology for genetic engineering of adult stem/progenitor cells under physiological niche-like conditions. Moreover, reporter gene activity was observed in rat spinal cords after minimally invasive intrathecal implantation, suggesting effective engraftment of donor MSCs. It is therefore plausible that anioplex-transfected MSCs or other stem/progenitor cells with autologous potential could be applied to disorders such as neurotrauma or neuropathic pain that involve the spinal cord and brain. PMID:20698746

Osteogenic and chondrogenic master genes expression is dependent on the Kir2.1 potassium channel through the bone morphogenetic protein pathway.

PubMed

Pini, Jonathan; Giuliano, Serena; Matonti, Julia; Simkin, Dina; Rouleau, Matthieu; Bendahhou, Saïd

2018-05-29

Andersen's syndrome is a rare disorder affecting muscle, heart, and bone, that is associated with mutations leading to a loss of function of the inwardly rectifying K + channel Kir2.1. While the Kir2.1 function can be anticipated in excitable cells by controlling the electrical activity, its role in non-excitable cells remains to be investigated. Using Andersen's syndrome induced Pluripotent Stem cells, we investigated the cellular and molecular events during the osteoblastic and chondrogenic differentiation that are affected by the loss of the Ik1 current. We show that loss of Kir2.1 channel function impairs both osteoblastic and chondrogenic processes through the down regulation master gene expression. This down regulation is due to an impairment of the bone morphogenetic proteins signaling pathway through de-phosphorylation of the Smad proteins. Restoring Kir2.1 channel function in Andersen's syndrome cells rescued master genes expression, and restored normal osteoblasts and chondrocytes behavior. Our results show that Kir2.1-mediated activity controls endochondral and intramembranous ossification signaling pathways. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Key Transcription Factors in the Differentiation of Mesenchymal Stem Cells

PubMed Central

Almalki, Sami G.; Agrawal, Devendra K.

2016-01-01

Mesenchymal stem cells (MSCs) are multipotent cells that represent a promising source for regenerative medicine. MSCs are capable of osteogenic, chondrogenic, adipogenic and myogenic differentiation. Efficacy of differentiated MSCs to regenerate cells in the injured tissues requires the ability to maintain the differentiation toward the desired cell fate. Since MSCs represent an attractive source for autologous transplantation, cellular and molecular signaling pathways and micro-environmental changes have been studied in order to understand the role of cytokines, chemokines, and transcription factors on the differentiation of MSCs. The differentiation of MSC into a mesenchymal lineage is genetically manipulated and promoted by specific transcription factors associated with a particular cell lineage. Recent studies have explored the integration of transcription factors, including Runx2, Sox9, PPARγ, MyoD, GATA4, and GATA6 in the differentiation of MSCs. Therefore, the overexpression of a single transcription factor in MSCs may promote trans-differentiation into specific cell lineage, which can be used for treatment of some diseases. In this review, we critically discussed and evaluated the role of transcription factors and related signaling pathways that affect the differentiation of MSCs toward adipocytes, chondrocytes, osteocytes, skeletal muscle cells, cardiomyocytes, and smooth muscle cells. PMID:27012163

How electromagnetic fields can influence adult stem cells: positive and negative impacts.

PubMed

Maziarz, Aleksandra; Kocan, Beata; Bester, Mariusz; Budzik, Sylwia; Cholewa, Marian; Ochiya, Takahiro; Banas, Agnieszka

2016-04-18

The electromagnetic field (EMF) has a great impact on our body. It has been successfully used in physiotherapy for the treatment of bone disorders and osteoarthritis, as well as for cartilage regeneration or pain reduction. Recently, EMFs have also been applied in in vitro experiments on cell/stem cell cultures. Stem cells reside in almost all tissues within the human body, where they exhibit various potential. These cells are of great importance because they control homeostasis, regeneration, and healing. Nevertheless, stem cells when become cancer stem cells, may influence the pathological condition. In this article we review the current knowledge on the effects of EMFs on human adult stem cell biology, such as proliferation, the cell cycle, or differentiation. We present the characteristics of the EMFs used in miscellaneous assays. Most research has so far been performed during osteogenic and chondrogenic differentiation of mesenchymal stem cells. It has been demonstrated that the effects of EMF stimulation depend on the intensity and frequency of the EMF and the time of exposure to it. However, other factors may affect these processes, such as growth factors, reactive oxygen species, and so forth. Exploration of this research area may enhance the development of EMF-based technologies used in medical applications and thereby improve stem cell-based therapy and tissue engineering.

Analyses of chondrogenic induction of adipose mesenchymal stem cells by combined co-stimulation mediated by adenoviral gene transfer

PubMed Central

2013-01-01

Introduction Adipose-derived stem cells (ASCs) have the potential to differentiate into cartilage under stimulation with some reported growth and transcriptional factors, which may constitute an alternative for cartilage replacement approaches. In this study, we analyzed the in vitro chondrogenesis of ASCs transduced with adenoviral vectors encoding insulin-like growth factor-1 (IGF-1), transforming growth factor beta-1 (TGF-β1), fibroblast growth factor-2 (FGF-2), and sex-determining region Y-box 9 (SOX9) either alone or in combinations. Methods Aggregate cultures of characterized ovine ASCs were transduced with 100 multiplicity of infections of Ad.IGF-1, Ad.TGF-β1, Ad.FGF-2, and Ad.SOX9 alone or in combination. These were harvested at various time points for detection of cartilage-specific genes expression by quantitative real-time PCR or after 14 and 28 days for histologic and biochemical analyses detecting proteoglycans, collagens (II, I and X), and total sulfated glycosaminoglycan and collagen content, respectively. Results Expression analyses showed that co-expression of IGF-1 and FGF-2 resulted in higher significant expression levels of aggrecan, biglycan, cartilage matrix, proteoglycan, and collagen II (all P ≤0.001 at 28 days). Aggregates co-transduced with Ad.IGF-1/Ad.FGF-2 showed a selective expression of proteoglycans and collagen II, with limited expression of collagens I and × demonstrated by histological analyses, and had significantly greater glycosaminoglycan and collagen production than the positive control (P ≤0.001). Western blot analyses for this combination also demonstrated increased expression of collagen II, while expression of collagens I and × was undetectable and limited, respectively. Conclusion Combined overexpression of IGF-1/FGF-2 within ASCs enhances their chondrogenic differentiation inducing the expression of chondrogenic markers, suggesting that this combination is more beneficial than the other factors tested for the development of cell-based therapies for cartilage repair. PMID:23899094

Canine Mesenchymal Stem Cell Potential and the Importance of Dog Breed: Implication for Cell-Based Therapies.

PubMed

Bertolo, Alessandro; Steffen, Frank; Malonzo-Marty, Cherry; Stoyanov, Jivko

2015-01-01

The study of canine bone marrow-derived mesenchymal stem cells (MSCs) has a prominent position in veterinary cell-based applications. Yet the plethora of breeds, their different life spans, and interbreed variations provide unclearness on what can be achieved specifically by such therapies. In this study, we compared a set of morphological, physiological, and genetic markers of MSCs derived from large dog breeds, namely, Border collie, German shepherd, Labrador, Malinois, Golden retriever, and Hovawart. We compared colony-forming units (CFUs) assay, population doubling time (PDT), senescence-associated β-galactosidase (SA-β-gal) activity, telomere length, and gene expression of MSCs, as well as the ability of cells to differentiate to osteogenic, adipogenic, and chondrogenic phenotypes. The influence of the culture media α-MEM, low-glucose DMEM, and high-glucose DMEM, used in cell isolation and expansion, was investigated in the presence and absence of basic fibroblast growth factor (bFGF). Initial cell yield was not affected by culturing medium, but MSCs expanded best in α-MEM supplemented with bFGF. After isolation, the number of MSCs was similar among breeds--as shown by equivalent CFUs--except in the Hovawart samples, which had fivefold less CFU. Telomere lengths were similar among breeds. MSCs divided actively only for 4 weeks in culture (PDT = ∼50 h/division), except Border collie cells divided for a longer time than cells from other groups. The percentage of senescent cells increased linearly in all breeds with time, with a faster rate in German shepherd, Labrador, and Golden retriever. Border collie cells underwent efficient osteogenic differentiation, Hovawart cells performed the best in chondrogenic differentiation, and Labrador cells in both, while German shepherd cells had the lower differentiation potential. MSCs from all breeds preserved the same adipogenic differentiation potential. In conclusion, despite variations, isolated MSCs can be expanded and differentiated in vitro, and all breeds are eligible for MSC-based therapies.

Biological characteristics of human-urine-derived stem cells: potential for cell-based therapy in neurology.

PubMed

Guan, Jun-Jie; Niu, Xin; Gong, Fei-Xiang; Hu, Bin; Guo, Shang-Chun; Lou, Yuan-Lei; Zhang, Chang-Qing; Deng, Zhi-Feng; Wang, Yang

2014-07-01

Stem cells in human urine have gained attention in recent years; however, urine-derived stem cells (USCs) are far from being well elucidated. In this study, we compared the biological characteristics of USCs with adipose-derived stem cells (ASCs) and investigated whether USCs could serve as a potential cell source for neural tissue engineering. USCs were isolated from voided urine with a modified culture medium. Through a series of experiments, we examined the growth rate, surface antigens, and differentiation potential of USCs, and compared them with ASCs. USCs showed robust proliferation ability. After serial propagation, USCs retained normal karyotypes. Cell surface antigen expression of USCs was similar to ASCs. With lineage-specific induction factors, USCs could differentiate toward the osteogenic, chondrogenic, adipogenic, and neurogenic lineages. To assess the ability of USCs to survive, differentiate, and migrate, they were seeded onto hydrogel scaffold and transplanted into rat brain. The results showed that USCs were able to survive in the lesion site, migrate to other areas, and express proteins that were associated with neural phenotypes. The results of our study demonstrate that USCs possess similar biological characteristics with ASCs and have multilineage differentiation potential. Moreover USCs can differentiate to neuron-like cells in rat brain. The present study shows that USCs are a promising cell source for tissue engineering and regenerative medicine.

Novel bio-synthetic hybrid materials and coculture systems for musculoskeletal tissue engineering

NASA Astrophysics Data System (ADS)

Lee, Hyeseung Janice

Tissue Engineering is a truly exciting field of this age, trying to regenerate and repair impaired tissues. Unlike the old artificial implants, tissue engineering aims at making a long-term functional biological replacement. One strategy for such tissue engineering requires the following three components: cells, scaffolds, and soluble factors. Cells are cultured in a three-dimensional (3D) scaffold with medium containing various soluble factors. Once a tissue is developed in vitro, then it is implanted in vivo. The overall goal of this thesis was to develop novel bio-synthetic hybrid scaffolds and coculture system for musculoskeletal tissue engineering. The most abundant cartilage extracellular matrix (ECM) components are collagen and glycosaminoglycan (GAG), which are the natural scaffold for chondrocytes. As two different peptides, collagen mimetic peptide (CMP) and hyaluronic acid binding peptide (HABPep) were previously shown to bind to collagen and hyaluronic acid (HA) of GAG, respectively, it was hypothesized that immobilizing CMP and HABP on 3D scaffold would results in an interaction between ECM components and synthetic scaffolds via peptide-ECM bindings. CMP or HABPep-conjugated photopolymerizable poly(ethylene oxide) diacrylate (PEODA) hydrogels were synthesized and shown to retain encapsulated collagen or HA, respectively. This result supported that conjugated CMP and HABPep can interact with collagen and HA, respectively, and can serve as biological linkers in 3D synthetic hydrogels. When chondrocytes or mesenchymal stem cells (MSCs) were seeded, cells in CMP-conjugated scaffolds produced significantly more amount of type II collagen and GAG, compared to those in control scaffolds. Moreover, MSCs cultured in CMP-conjugated scaffolds exhibited lower level of hypertrophic markers, cbfa-1 and type X collagen. These results demonstrated that enhanced interaction between collagen and scaffold via CMP improves chondrogenesis of chondrocytes and MSCs and further reduces hypertrophy of differentiating MSCs. On the other hand, although cells in HABPep-conjugated scaffolds produced less ECM components, they survived and proliferated significantly more than those in control, resulting in overall increase in ECM contents per scaffold. Once implanted in vivo, HABPep-conjugated constructs increased GAG and type II collagen contents further, compared to those of the control hydrogel. These results showed that enhanced interaction between HA and scaffold via HABPep improved the in vitro culture expansion of MSCs and further ECM production in vivo. Effects of cell-secreted bioactive factors via cell-cell communication on stem cell differentiation were also investigated in 3D bilayer system. First, when mesenchymal progenitor cells (MPCs) were cocultured with ES-derived cells (ESDC), morphogenetic factors secreted by ESDCs showed a potential to improve MPC chondrogenesis in both control and chondrogenic medium by increasing not only MPC's chondrogenic gene expression, but also ECM production. Moreover, the effect of ESDC cell-mediated chondrogenesis of MSC could not be mimicked by chondrogenic medium supplemented with TGF-beta1 and dexamethasone. Secondly, coculturing hepatic cells enhanced specific chondrogenic differentiation of ES cells in the 3D bilayer system. These studies demonstrated that cell-secreted soluble factors can be used to guide stem cell differentiation.

Dynamic culturing of cartilage tissue: the significance of hydrostatic pressure.

PubMed

Correia, Cristina; Pereira, Ana L; Duarte, Ana R C; Frias, Ana M; Pedro, Adriano J; Oliveira, João T; Sousa, Rui A; Reis, Rui L

2012-10-01

Human articular cartilage functions under a wide range of mechanical loads in synovial joints, where hydrostatic pressure (HP) is the prevalent actuating force. We hypothesized that the formation of engineered cartilage can be augmented by applying such physiologic stimuli to chondrogenic cells or stem cells, cultured in hydrogels, using custom-designed HP bioreactors. To test this hypothesis, we investigated the effects of distinct HP regimens on cartilage formation in vitro by either human nasal chondrocytes (HNCs) or human adipose stem cells (hASCs) encapsulated in gellan gum (GG) hydrogels. To this end, we varied the frequency of low HP, by applying pulsatile hydrostatic pressure or a steady hydrostatic pressure load to HNC-GG constructs over a period of 3 weeks, and evaluated their effects on cartilage tissue-engineering outcomes. HNCs (10×10(6) cells/mL) were encapsulated in GG hydrogels (1.5%) and cultured in a chondrogenic medium under three regimens for 3 weeks: (1) 0.4 MPa Pulsatile HP; (2) 0.4 MPa Steady HP; and (3) Static. Subsequently, we applied the pulsatile regimen to hASC-GG constructs and varied the amplitude of loading, by generating both low (0.4 MPa) and physiologic (5 MPa) HP levels. hASCs (10×10(6) cells/mL) were encapsulated in GG hydrogels (1.5%) and cultured in a chondrogenic medium under three regimens for 4 weeks: (1) 0.4 MPa Pulsatile HP; (2) 5 MPa Pulsatile HP; and (3) Static. In the HNC study, the best tissue development was achieved by the pulsatile HP regimen, whereas in the hASC study, greater chondrogenic differentiation and matrix deposition were obtained for physiologic loading, as evidenced by gene expression of aggrecan, collagen type II, and sox-9; metachromatic staining of cartilage extracellular matrix; and immunolocalization of collagens. We thus propose that both HNCs and hASCs detect and respond to physical forces, thus resembling joint loading, by enhancing cartilage tissue development in a frequency- and amplitude-dependant manner.

Advances and Prospects in Stem Cells for Cartilage Regeneration

PubMed Central

Wang, Mingjie; Yuan, Zhiguo; Ma, Ning; Hao, Chunxiang; Guo, Weimin; Zou, Gengyi; Zhang, Yu; Chen, Mingxue; Gao, Shuang; Wang, Aiyuan; Wang, Yu; Sui, Xiang; Xu, Wenjing; Lu, Shibi

2017-01-01

The histological features of cartilage call attention to the fact that cartilage has a little capacity to repair itself owing to the lack of a blood supply, nerves, or lymphangion. Stem cells have emerged as a promising option in the field of cartilage tissue engineering and regenerative medicine and could lead to cartilage repair. Much research has examined cartilage regeneration utilizing stem cells. However, both the potential and the limitations of this procedure remain controversial. This review presents a summary of emerging trends with regard to using stem cells in cartilage tissue engineering and regenerative medicine. In particular, it focuses on the characterization of cartilage stem cells, the chondrogenic differentiation of stem cells, and the various strategies and approaches involving stem cells that have been used in cartilage repair and clinical studies. Based on the research into chondrocyte and stem cell technologies, this review discusses the damage and repair of cartilage and the clinical application of stem cells, with a view to increasing our systematic understanding of the application of stem cells in cartilage regeneration; additionally, several advanced strategies for cartilage repair are discussed. PMID:28246531

Restoring the IL-1β/NF-κB-induced impaired chondrogenesis by diallyl disulfide in human adipose-derived mesenchymal stem cells via attenuation of reactive oxygen species and elevation of antioxidant enzymes.

PubMed

Bahrampour Juybari, Kobra; Kamarul, Tunku; Najafi, Mohammad; Jafari, Davood; Sharifi, Ali Mohammad

2018-03-26

Strategies based on mesenchymal stem cell (MSC) therapy for restoring injured articular cartilage are not effective enough in osteoarthritis (OA). Due to the enhanced inflammation and oxidative stress in OA microenvironment, differentiation of MSCs into chondrocytes would be impaired. This study aims to explore the effects of diallyl disulfide (DADS) on IL-1β-mediated inflammation and oxidative stress in human adipose derived mesenchymal stem cells (hADSCs) during chondrogenesis. MTT assay was employed to examine the effects of various concentrations of DADS on the viability of hADSCs at different time scales to obtain non-cytotoxic concentration range of DADS. The effects of DADS on IL-1β-induced intracellular ROS generation and lipid peroxidation were evaluated in hADSCs. Western blotting was used to analyze the protein expression levels of IκBα (np), IκBα (p), NF-κB (np) and NF-κB (p). Furthermore, the gene expression levels of antioxidant enzymes in hADSCs and chondrogenic markers at days 7, 14 and 21 of differentiation were measured using qRT-PCR. The results showed that addition of DADS significantly enhanced the mRNA expression levels of antioxidant enzymes as well as reduced ROS elevation, lipid peroxidation, IκBα activation and NF-κB nuclear translocation in hADSCs treated with IL-1β. In addition, DADS could significantly increase the expression levels of IL-1β-induced impaired chondrogenic marker genes in differentiated hADSCs. Treatment with DADS may provide an effective approach to prevent the pro-inflammatory cytokines and oxidative stress as catabolic causes of chondrocyte cell death and enhance the protective anabolic effects by promoting chondrogenesis associated gene expressions in hADSCs exposed to OA condition.

Cell therapy: a therapeutic alternative to treat focal cartilage lesions.

PubMed

Gimeno, M J; Maneiro, E; Rendal, E; Ramallal, M; Sanjurjo, L; Blanco, F J

2005-11-01

Human mesenchymal stem cells (MSCs) are present in most of the tissue matrix, taking part in their regeneration when injury or damage occurs. The aim of this study was to investigate the presence of cells with pluripotential characteristics in synovial membranes from osteoarthritic (OA) patients and the capacity of these cells to differentiate to chondrocytes. Synovial membranes (n = 8) from OA patients were digested with collagenase. Isolated cells were cultured with DMEM, 20% FBS, and FGFb10 ng/mL. Cells from second subculture were used to carry out phenotypic characterization experiments (flow cytometry analysis with 11 monoclonal antibodies) and chondrogenic differentiation experiments(micropellet cultured in chondrogenic medium). Chondrogenic differentiation of cells was assessment by quantification of cartilage extracellular matrix components by following techniques: Safranin O, Toluidine Blue, and Alcian Blue stains to detect proteoglycans and immunohistochemistry to detect type I and II collagen. Flow cytometry analyses showed that in our population more than 90% of cells were positive for MSC markers: CD29 (95%), CD44 (90%), CD73 (95%), CD90 (98%). Cells were negative for hematopoietic markers (CD11b, CD34, and CD45). Furthermore, cells showed positive stain to multipotent markers such as CD117 (c-kit) (98%), CD166 (74%), and STRO-1 (88%) and to quiescent satellite cells like PAX-7 (35%). The micropellet analyses showed that the culture of these cells with TGFbeta-3 for 2 and 3 weeks stimulates proteoglycan and collagen type II synthesis. Both molecules are characteristic of hyaline articular cartilage. In this work, we demonstrate the presence of a cellular population with MSC characteristics in synovial tissue from OA patients. As MSC takes part in reparative processes of adult tissues, these cells could play an important role in OA pathogenesis and treatment.

Identification of Multipotent Stem/Progenitor Cells in Murine Sclera

PubMed Central

Tsai, Chia-Ling; Wu, Pei-Chang; Fini, M. Elizabeth; Shi, Songtao

2011-01-01

Purpose. The sclera forms the fibrous outer coat of the eyeball and acts as a supportive framework. The purpose of this study was to examine whether the sclera contains mesenchymal stem/progenitor cells. Method. Scleral tissue from C57BL6/J mice was separated from the retina and choroid and subsequently enzyme digested to release single cells. Proliferation capacity, self-renewal capacity, and ability for multipotent differentiation were analyzed by BrdU labeling, flow cytometry, reverse transcriptase–polymerase chain reaction, immunocytochemistry, and in vivo transplantation. Results. The scleral stem/progenitor cells (SSPCs) possessed clonogenic and high doubling capacities. These cells were positive for the mesenchymal markers Sca-1, CD90.2, CD44, CD105, and CD73 and negative for the hematopoietic markers CD45, CD11b, Flk1, CD34, and CD117. In addition to expressing stem cell genes ABCG2, Six2, Notch1, and Pax6, SSPCs were able to differentiate to adipogenic, chondrogenic, and neurogenic lineages. Conclusions. This study indicates that the sclera contains multipotent mesenchymal stem cells. Further study of SSPCs may help elucidate the cellular and molecular mechanism of scleral diseases such as scleritis and myopia. PMID:21788434

Effects of intra-articular injection of mesenchymal stem cells associated with platelet-rich plasma in a rabbit model of osteoarthritis.

PubMed

Hermeto, L C; DeRossi, R; Oliveira, R J; Pesarini, J R; Antoniolli-Silva, A C M B; Jardim, P H A; Santana, A E; Deffune, E; Rinaldi, J C; Justulin, L A

2016-09-02

The current study aims to evaluate the macroscopic and histological effects of autologous mesenchymal stem cells (MSC) and platelet-rich plasma on knee articular cartilage regeneration in an experimental model of osteoarthritis. Twenty-four rabbits were randomly divided into four groups: control group, platelet-rich plasma group, autologous MSC undifferentiated group, and autologous MSC differentiated into chondrocyte group. Collagenase solution was used to induce osteoarthritis, and treatments were applied to each group at 6 weeks following osteoarthritis induction. After 60 days of therapy, the animals were euthanized and the articular surfaces were subjected to macroscopic and histological evaluations. The adipogenic, chondrogenic, and osteogenic differentiation potentials of MSCs were evaluated. Macroscopic and histological examinations revealed improved tissue repair in the MSC-treated groups. However, no difference was found between MSC-differentiated and undifferentiated chondrocytes. We found that MSCs derived from adipose tissue and platelet-rich plasma were associated with beneficial effects in articular cartilage regeneration during experimental osteoarthritis.

Adipose-derived stem cell: a better stem cell than BMSC.

PubMed

Zhu, Yanxia; Liu, Tianqing; Song, Kedong; Fan, Xiubo; Ma, Xuehu; Cui, Zhanfeng

2008-08-01

To further study the proliferation and multi-differentiation potentials of adipose-derived stem cells (ADSCs), the cells were isolated with improved methods and their growth curves were achieved with cck-8. Surface protein expression was analyzed by flow cytometry to characterize the cell phenotype. The multi-lineage potential of ADSCs was testified by differentiating cells with adipogenic, chondrogenic, osteogenic, and myogenic inducers. The results showed that about 5 x 10(5) stem cells could be obtained from 400 to 600 mg adipose tissue. The ADSCs can be continuously cultured in vitro for up to 1 month without passage and they have several logarithmic growth phases during the culture period. Also, the flow cytometry analysis showed that ADSCs expressed high levels of stem cell-related antigens (CD13, CD29, CD44, CD105, and CD166), while did not express hematopoiesis-related antigens CD34 and CD45, and human leukocyte antigen HLA-DR was also negative. Moreover, stem cell-related transcription factors, Nanog, Oct-4, Sox-2, and Rex-1 were positively expressed in ADSCs. The expression of alkaline phosphatase (ALP) was detected in the early osteogenic induction and the calcified nodules were observed by von Kossa staining. Intracellular lipid droplets could be observed by Oil Red staining. Differentiated cardiomyocytes were observed by connexin43 fluorescent staining. In order to obtain more stem cells, we can subculture ADSCs every 14 days instead of the normal 5 days. ADSCs still keep strong proliferation ability, maintain their phenotypes, and have stronger multi-differentiation potential after 25 passages. Copyright 2008 John Wiley & Sons, Ltd.

Mesenchymal stem cells reside in anterior cruciate ligament remnants in situ.

PubMed

Fu, Weili; Li, Qi; Tang, Xin; Chen, Gang; Zhang, Chenghao; Li, Jian

2016-07-01

It has been reported that the anterior cruciate ligament (ACL) has certain self-healing ability after acute injury or with primary suture repair. Many studies have confirmed that a remnant preservation technique with ACL reconstruction contributes to biological augmentation for ACL healing. However, it remains unclear whether mesenchymal stem cells (MSC) reside in ACL remnants in situ. The aim of this study was to investigate the methods of culture and identification of MSC derived from the remnants of ACL rupture patients and to analyse these MSC's properties. The cells of ACL remnants from the ACL rupture patients were isolated by the methods of enzymatic digestion and cultured in vitro to the third passage under the microscope to observe their morphology and growth status. The third passage of isolated cells was analysed for the identification of immunophenotype, osteogenic, adipogenic and chondrogenic differentiation. On the third to fifth days of in vitro culture, a few cells of long fusiform shape appeared and were adherent to the plastic walls. On the sixth to ninth days, cells clustered and colonies were observed. The third passage cells showed uniform cell morphology and good proliferation, with appearance of the typical surface markers of MSC, CD29, CD44, CD90 and CD105. The surface markers of CD34 and CD45 of haematopoietic stem cells were not expressed. Under appropriate conditions of in vitro culture, isolated cells could be differentiated into osteoblasts that deposit mineralised matrix and express early osteogenic markers, adipocytes that accumulate lipid droplets in cytoplasm and chondrocytes that secrete chondrogenic-specific matrix aggrecan and collagen II. Real-time polymerase chain reaction (PCR) analysis demonstrated that the specific mRNA expression of osteogenesis, adipogenesis and chondrogenesis increased significantly compared with the control groups at day zero. Stem cells derived in situ from the human ACL stump were successfully isolated and characterised. Those isolated cells were identified as MSC according to their adherent ability, morphology, surface markers and multilineage differentiation potential. MSC derived from ACL remnants could be a potential source of seeding cells for ligament regeneration.

Differential Properties of Human ALP+ Periodontal Ligament Stem Cells vs Their ALP- Counterparts

PubMed Central

Tran, Quynh T; El-Ayachi, Ikbale; Bhatti, Fazal-Ur-Rehman; Bahabri, Rayan; Al-Habib, Mey; Huang, George TJ

2015-01-01

Characterizing subpopulations of stem cells is important to understand stem cell properties. Tissue-nonspecific alkaline phosphatase (ALP) is associated with mineral tissue forming cells as well as stem cells. Information regarding ALP subpopulation of human periodontal ligament stem cells (hPDLSCs) is limited. In the present study, we examined ALP+ and ALP− hPDLSC subpopulations, their surface markers STRO-1 and CD146, and the expression of stemness genes at various cell passages. We found that ALP+ subpopulation had higher levels of STRO-1 (30.6 ± 5.6%) and CD146 (90.4 ± 3.3%) compared to ALP− (STRO-1: 0.5 ± 0.1%; CD146: 75.3 ± 7.2%). ALP+ cells expressed significantly higher levels of stemness associated genes, NANOG, OCT4 and SOX than ALP− cells at low cell passages of 2-3 (p<0.05). ALP+ and ALP− cells had similar osteogenic, chondrogenic and neurogenic potential while ALP−, not ALP+ cells, lacked adipogenic potential. Upon continuous culturing and passaging, ALP+ continued to express higher stemness genes and STRO-1 and CD146 than ALP− cells at ≥passage 19. Under conditions (over-confluence and vitamin C treatment) when ALP+ subpopulation was increased, the stemness gene levels of ALP+ was no longer significantly higher than those in ALP− cells. In conclusion, ALP+ hPDLSCs possess differential properties from their ALP− counterparts. PMID:26807329

Synovial fluid progenitors expressing CD90+ from normal but not osteoarthritic joints undergo chondrogenic differentiation without micro-mass culture.

PubMed

Krawetz, Roman J; Wu, Yiru Elizabeth; Martin, Liam; Rattner, Jerome B; Matyas, John R; Hart, David A

2012-01-01

Mesenchymal progenitor cells (MPCs) can differentiate into osteoblasts, adipocytes, and chondrocytes, and are in part responsible for maintaining tissue integrity. Recently, a progenitor cell population has been found within the synovial fluid that shares many similarities with bone marrow MPCs. These synovial fluid MPCs (sfMPCs) share the ability to differentiate into bone and fat, with a bias for cartilage differentiation. In this study, sfMPCs were isolated from human and canine synovial fluid collected from normal individuals and those with osteoarthritis (human: clinician-diagnosed, canine: experimental) to compare the differentiation potential of CD90+ vs. CD90- sfMPCs, and to determine if CD90 (Thy-1) is a predictive marker of synovial fluid progenitors with chondrogenic capacity in vitro. sfMPCs were derived from synovial fluid from normal and OA knee joints. These cells were induced to differentiate into chondrocytes and analyzed using quantitative PCR, immunofluorescence, and electron microscopy. The CD90+ subpopulation of sfMPCs had increased chondrogenic potential compared to the CD90- population. Furthermore, sfMPCs derived from healthy joints did not require a micro-mass step for efficient chondrogenesis. Whereas sfMPCs from OA synovial fluid retain the ability to undergo chondrogenic differentiation, they require micro-mass culture conditions. Overall, this study has demonstrated an increased chondrogenic potential within the CD90+ fraction of human and canine sfMPCs and that this population of cells derived from healthy normal joints do not require a micro-mass step for efficient chondrogenesis, while sfMPCs obtained from OA knee joints do not differentiate efficiently into chondrocytes without the micro-mass procedure. These results reveal a fundamental shift in the chondrogenic ability of cells isolated from arthritic joint fluids, and we speculate that the mechanism behind this change of cell behavior is exposure to the altered milieu of the OA joint fluid, which will be examined in further studies.

Advancing osteochondral tissue engineering: bone morphogenetic protein, transforming growth factor, and fibroblast growth factor signaling drive ordered differentiation of periosteal cells resulting in stable cartilage and bone formation in vivo.

PubMed

Mendes, L F; Katagiri, H; Tam, W L; Chai, Y C; Geris, L; Roberts, S J; Luyten, F P

2018-02-21

Chondrogenic mesenchymal stem cells (MSCs) have not yet been used to address the clinical demands of large osteochondral joint surface defects. In this study, self-assembling tissue intermediates (TIs) derived from human periosteum-derived stem/progenitor cells (hPDCs) were generated and validated for stable cartilage formation in vivo using two different animal models. hPDCs were aggregated and cultured in the presence of a novel growth factor (GF) cocktail comprising of transforming growth factor (TGF)-β1, bone morphogenetic protein (BMP)2, growth differentiation factor (GDF)5, BMP6, and fibroblast growth factor (FGF)2. Quantitative polymerase chain reaction (PCR) and immunohistochemistry were used to study in vitro differentiation. Aggregates were then implanted ectopically in nude mice and orthotopically in critical-size osteochondral defects in nude rats and evaluated by microcomputed tomography (µCT) and immunohistochemistry. Gene expression analysis after 28 days of in vitro culture revealed the expression of early and late chondrogenic markers and a significant upregulation of NOGGIN as compared to human articular chondrocytes (hACs). Histological examination revealed a bilayered structure comprising of chondrocytes at different stages of maturity. Ectopically, TIs generated both bone and mineralized cartilage at 8 weeks after implantation. Osteochondral defects treated with TIs displayed glycosaminoglycan (GAG) production, type-II collagen, and lubricin expression. Immunostaining for human nuclei protein suggested that hPDCs contributed to both subchondral bone and articular cartilage repair. Our data indicate that in vitro derived osteochondral-like tissues can be generated from hPDCs, which are capable of producing bone and cartilage ectopically and behave orthotopically as osteochondral units.

Decellularized Cartilage May Be a Chondroinductive Material for Osteochondral Tissue Engineering

PubMed Central

Sutherland, Amanda J.; Beck, Emily C.; Dennis, S. Connor; Converse, Gabriel L.; Hopkins, Richard A.; Berkland, Cory J.; Detamore, Michael S.

2015-01-01

Extracellular matrix (ECM)-based materials are attractive for regenerative medicine in their ability to potentially aid in stem cell recruitment, infiltration, and differentiation without added biological factors. In musculoskeletal tissue engineering, demineralized bone matrix is widely used, but recently cartilage matrix has been attracting attention as a potentially chondroinductive material. The aim of this study was thus to establish a chemical decellularization method for use with articular cartilage to quantify removal of cells and analyze the cartilage biochemical content at various stages during the decellularization process, which included a physically devitalization step. To study the cellular response to the cartilage matrix, rat bone marrow-derived mesenchymal stem cells (rBMSCs) were cultured in cell pellets containing cells only (control), chondrogenic differentiation medium (TGF-β), chemically decellularized cartilage particles (DCC), or physically devitalized cartilage particles (DVC). The chemical decellularization process removed the vast majority of DNA and about half of the glycosaminoglycans (GAG) within the matrix, but had no significant effect on the amount of hydroxyproline. Most notably, the DCC group significantly outperformed TGF-β in chondroinduction of rBMSCs, with collagen II gene expression an order of magnitude or more higher. While DVC did not exhibit a chondrogenic response to the extent that DCC did, DVC had a greater down regulation of collagen I, collagen X and Runx2. A new protocol has been introduced for cartilage devitalization and decellularization in the current study, with evidence of chondroinductivity. Such bioactivity along with providing the ‘raw material’ building blocks of regenerating cartilage may suggest a promising role for DCC in biomaterials that rely on recruiting endogenous cell recruitment and differentiation for cartilage regeneration. PMID:25965981

Impact of human platelet lysate on the expansion and chondrogenic capacity of cultured human chondrocytes for cartilage cell therapy.

PubMed

Sykes, J G; Kuiper, J H; Richardson, J B; Roberts, S; Wright, K T; Kuiper, N J

2018-05-01

High hopes have been pinned on regenerative medicine strategies in order to prevent the progression of cartilage damage to osteoarthritis, particularly by autologous chondrocyte implantation (ACI). The loss of chondrocyte phenotype during in vitro monolayer expansion, a necessary step to obtain sufficient cell numbers, may be a key limitation in ACI. In this study, it was determined whether a shorter monolayer expansion approach could improve chondrogenic differentiation. The effects of two supplement types, foetal bovine serum (FBS) and Stemulate™ (a commercial source of human platelet lysate), on the expansion and re-differentiation potential of human chondrocytes, isolated from five individuals, were compared. Chondrocytes were expanded with 10 % FBS or 10 % Stemulate™. Pellets were cultured for 28 d in chondrogenic differentiation medium and assessed for the presence of cartilage matrix molecules and genes associated with chondrogenicity. Stemulate™ significantly enhanced the proliferation rate [average population doubling times: FBS, 25.07 ± 6.98 d (standard error of the mean, SEM) vs. Stemulate™, 13.10 ± 2.57 d (SEM)]. Sulphated glycosaminoglycans (sGAG), total collagen and qRT-PCR analyses of cartilage genes showed that FBS-expanded chondrocytes demonstrated significantly better chondrogenic capacity than Stemulate™-expanded chondrocytes. Histologically, FBS-expanded chondrocyte pellets appeared to be more stable, with a more intense staining for toluidine blue, indicating a greater chondrogenic capacity. Although Stemulate™ positively influenced chondrocyte proliferation, it had a negative effect on chondrogenic differentiation potential. This suggested that, in the treatment of cartilage defects, Stemulate™ might not be the ideal supplement for expanding chondrocytes (which maintained a chondrocyte phenotype) and, hence, for cell therapies (including ACI).

Functional cartilage repair capacity of de-differentiated, chondrocyte- and mesenchymal stem cell-laden hydrogels in vitro

PubMed Central

Rackwitz, Lars; Djouad, Farida; Janjanin, Sasa; Nöth, Ulrich; Tuan, Rocky S.

2017-01-01

Objective The long-term performance of cell seeded matrix based cartilage constructs depends on (1) the development of sufficient biomechanical properties, and (2) lateral integration with host tissues, both of which require cartilage specific matrix deposition within the scaffold. In this study, we have examined the potential of tissue-engineered cartilage analogs developed using different cell types, i.e., MSCs versus chondrocytes and de-differentiated chondrocytes, in an established “construct in cartilage ring” model. Design Cell-laden constructs of differentiated chondrocytes, de-differentiated chondrocytes after 2, 5 or 8 population doublings, and MSCs were either implanted into a native cartilage ring immediately after fabrication (immature group) or pretreated for 21 days in a transforming growth factor-β3 (TGF-β3) containing medium prior to implantation. After additional culture for 28 days in a serum-free, chemically defined medium, the extent of lateral integration, and biochemical and biomechanical characteristics of the implants as hybrid constructs were assessed. Results The quality of integration, the amount of accumulated cartilage-specific matrix components and associated biomechanical properties were found to be highest when using differentiated chondrocytes. De-differentiation of chondrocytes negatively impacted the properties of the implants, as even two population doublings of the chondrocytes in culture significantly lowered cartilage repair capacity. In contrast, MSCs showed chondrogenic differentiation with TGF-β3 pre-treatment and superior integrational behavior. Conclusions Chondrocyte expansion and de-differentiation impaired the cell response, resulting in inferior cartilage repair in vitro. With TGF-β3 pre-treatment, MSCs were able to undergo sustained chondrogenic differentiation and exhibited superior matrix deposition and integration compared to de-differentiated chondrocytes. PMID:24887551

Mesenchymal Stem Cells for Cartilage Regeneration of TMJ Osteoarthritis

PubMed Central

Li, Hongyu; Xu, Xin; Ye, Ling; Zhou, Xuedong

2017-01-01

Temporomandibular joint osteoarthritis (TMJ OA) is a degenerative disease, characterized by progressive cartilage degradation, subchondral bone remodeling, synovitis, and chronic pain. Due to the limited self-healing capacity in condylar cartilage, traditional clinical treatments have limited symptom-modifying and structure-modifying effects to restore impaired cartilage as well as other TMJ tissues. In recent years, stem cell-based therapy has raised much attention as an alternative approach towards tissue repair and regeneration. Mesenchymal stem cells (MSCs), derived from the bone marrow, synovium, and even umbilical cord, play a role as seed cells for the cartilage regeneration of TMJ OA. MSCs possess multilineage differentiation potential, including chondrogenic differentiation as well as osteogenic differentiation. In addition, the trophic modulations of MSCs exert anti-inflammatory and immunomodulatory effects under aberrant conditions. Furthermore, MSCs combined with appropriate scaffolds can form cartilaginous or even osseous compartments to repair damaged tissue and impaired function of TMJ. In this review, we will briefly discuss the pathogenesis of cartilage degeneration in TMJ OA and emphasize the potential sources of MSCs and novel approaches for the cartilage regeneration of TMJ OA, particularly focusing on the MSC-based therapy and tissue engineering. PMID:29123550

Generation of chondrocytes from embryonic stem cells.

PubMed

Khillan, Jaspal Singh

2006-01-01

Pluripotent embryonic stem (ES) cells have complete potential for all the primary germ layers, such as ectoderm, mesoderm, and endoderm. However, the cellular and molecular mechanisms that control their lineage-restricted differentiation are not understood. Although embryoid bodies, which are formed because of the spontaneous differentiation of ES cells, have been used to study the differentiation into different cell types, including neurons, chondrocytes, insulin-producing cells, bone-forming cells, hematopoietic cells, and so on, this system has limitations for investigating the upstream events that lead to commitment of cells that occur during the inaccessible period of development. Recent developments in human ES cells have offered a challenge to develop strategies for understanding the basic mechanisms that play a key role in differentiation of stem cell into specific cell types for their applications in regenerative medicine and cell-based therapies. A micromass culture system was developed to induce the differentiation of ES cells into chondrocytes, the cartilage-producing cells, as a model to investigate the upstream events of stem cell differentiation. ES cells were co-cultured with limb bud progenitor cells. A high percentage of differentiated cells exhibit typical morphological characteristics of chondrocytes and express cartilage matrix genes such as collagen type II and proteoglycans, suggesting that signals from the progenitor cells are sufficient to induce ES cells into the chondrogenic lineage. Degeneration of cartilage in the joints is associated with osteoarthritis, which affects the quality of life of human patients. Therefore, the quantitative production of chondrocytes can be a powerful resource to alleviate the suffering of those patients.

Programmed Application of Transforming Growth Factor β3 and Rac1 Inhibitor NSC23766 Committed Hyaline Cartilage Differentiation of Adipose-Derived Stem Cells for Osteochondral Defect Repair.

PubMed

Zhu, Shouan; Chen, Pengfei; Wu, Yan; Xiong, Si; Sun, Heng; Xia, Qingqing; Shi, Libing; Liu, Huanhuan; Ouyang, Hong Wei

2014-10-01

Hyaline cartilage differentiation is always the challenge with application of stem cells for joint repair. Transforming growth factors (TGFs) and bone morphogenetic proteins can initiate cartilage differentiation but often lead to hypertrophy and calcification, related to abnormal Rac1 activity. In this study, we developed a strategy of programmed application of TGFβ3 and Rac1 inhibitor NSC23766 to commit the hyaline cartilage differentiation of adipose-derived stem cells (ADSCs) for joint cartilage repair. ADSCs were isolated and cultured in a micromass and pellet culture model to evaluate chondrogenic and hypertrophic differentiation. The function of Rac1 was investigated with constitutively active Rac1 mutant and dominant negative Rac1 mutant. The efficacy of ADSCs with programmed application of TGFβ3 and Rac1 inhibitor for cartilage repair was studied in a rat model of osteochondral defects. The results showed that TGFβ3 promoted ADSCs chondro-lineage differentiation and that NSC23766 prevented ADSC-derived chondrocytes from hypertrophy in vitro. The combination of ADSCs, TGFβ3, and NSC23766 promoted quality osteochondral defect repair in rats with much less chondrocytes hypertrophy and significantly higher International Cartilage Repair Society macroscopic and microscopic scores. The findings have illustrated that programmed application of TGFβ3 and Rac1 inhibitor NSC23766 can commit ADSCs to chondro-lineage differentiation and improve the efficacy of ADSCs for cartilage defect repair. These findings suggest a promising stem cell-based strategy for articular cartilage repair. ©AlphaMed Press.

Cartilage regeneration by selected chondrogenic clonal mesenchymal stem cells in the collagenase-induced monkey osteoarthritis model.

PubMed

Jiang, Li; Ma, Anlun; Song, Lijun; Hu, Yanxin; Dun, Hao; Daloze, Pierre; Yu, Yonglin; Jiang, Jianyuan; Zafarullah, Muhammad; Chen, Huifang

2014-11-01

Osteoarthritis (OA) is the most common form of arthritis, in which cartilage is irreversibly degraded, causing severe pain and disability. Current therapeutic strategies cannot repair damaged cartilage. We evaluated the repair potential of selected chondrogenic clonal MSCs (sC-MSCs) by delivering them into the injured cartilage site in a collagenase-induced OA model in Cynomolgus monkeys. In vitro characterization showed that the isolated monkey sC-MSCs and polyclonal MSCs (P-MSCs) expressed mesenchymal stem cell markers and could differentiate into chondrocytes. The articular cartilage lesions in animals were treated with normal saline (NS), autologous P-MSCs and sC-MSCs, respectively, by direct delivery. The clinical parameters, radiographic images, histological and immunohistochemical examinations at weeks 8, 16 and 24 post-treatment demonstrated that the abrasions of articular cartilage were significantly improved and repaired by MSC-based treatment, particularly in the sC-MSC-treated group, which displayed consistently higher histological scores than those of other groups. In summary, treatment with sC-MSCs can effectively improve the healing of cartilage lesions in the Cynomolgus monkey collagenase-induced OA model. Due to the genetic proximity of monkey and human, the therapeutic strategy presented in this study will have broad applications in clinical practice. Copyright © 2013 John Wiley & Sons, Ltd.

Devitalisation of human cartilage by high hydrostatic pressure treatment: Subsequent cultivation of chondrocytes and mesenchymal stem cells on the devitalised tissue

PubMed Central

Hiemer, B.; Genz, B.; Jonitz-Heincke, A.; Pasold, J.; Wree, A.; Dommerich, S.; Bader, R.

2016-01-01

The regeneration of cartilage lesions still represents a major challenge. Cartilage has a tissue-specific architecture, complicating recreation by synthetic biomaterials. A novel approach for reconstruction is the use of devitalised cartilage. Treatment with high hydrostatic pressure (HHP) achieves devitalisation while biomechanical properties are remained. Therefore, in the present study, cartilage was devitalised using HHP treatment and the potential for revitalisation with chondrocytes and mesenchymal stem cells (MSCs) was investigated. The devitalisation of cartilage was performed by application of 480 MPa over 10 minutes. Effective cellular inactivation was demonstrated by the trypan blue exclusion test and DNA quantification. Histology and electron microscopy examinations showed undamaged cartilage structure after HHP treatment. For revitalisation chondrocytes and MSCs were cultured on devitalised cartilage without supplementation of chondrogenic growth factors. Both chondrocytes and MSCs significantly increased expression of cartilage-specific genes. ECM stainings showed neocartilage-like structure with positive AZAN staining as well as collagen type II and aggrecan deposition after three weeks of cultivation. Our results showed that HHP treatment caused devitalisation of cartilage tissue. ECM proteins were not influenced, thus, providing a scaffold for chondrogenic differentiation of MSCs and chondrocytes. Therefore, using HHP-treated tissue might be a promising approach for cartilage repair. PMID:27671122

Yolk Sac Mesenchymal Progenitor Cells from New World Mice (Necromys lasiurus) with Multipotent Differential Potential

PubMed Central

Favaron, Phelipe Oliveira; Mess, Andrea; Will, Sônia Elisabete; Maiorka, Paulo César; de Oliveira, Moacir Franco; Miglino, Maria Angelica

2014-01-01

Fetal membranes are abundant, ethically acceptable and readily accessible sources of stem cells. In particular, the yolk sac is a source of cell lineages that do not express MHCs and are mainly free from immunological incompatibles when transferred to a recipient. Although data are available especially for hematopoietic stem cells in mice and human, whereas other cell types and species are dramatically underrepresented. Here we studied the nature and differentiation potential of yolk sac derived mesenchymal stem cells from a New World mouse, Necromys lasiurus. Explants from mid-gestation were cultured in DMEM-High glucose medium with 10% defined fetal bovine serum. The cells were characterized by standard methods including immunophenotyping by fluorescence and flow cytometry, growth and differentiation potential and tumorigenicity assays. The first adherent cells were observed after 7 days of cell culture and included small, elongated fibroblast-like cells (92.13%) and large, round epithelial-like cells with centrally located nuclei (6.5%). Only the fibroblast-like cells survived the first passages. They were positive to markers for mesenchymal stem cells (Stro-1, CD90, CD105, CD73) and pluripotency (Oct3/4, Nanog) as well as precursors of hematopoietic stem cells (CD117). In differentiation assays, they were classified as a multipotent lineage, because they differentiated into osteogenic, adipogenic, and chondrogenic lineages and, finally, they did not develop tumors. In conclusion, mesenchymal progenitor cells with multipotent differentiation potential and sufficient growth and proliferation abilities were able to be obtained from Necromys yolk sacs, therefore, we inferred that these cells may be promising for a wide range of applications in regenerative medicine. PMID:24918429

Using Polymeric Materials to Control Stem Cell Behavior for Tissue Regeneration

PubMed Central

Zhang, Nianli; Kohn, David H.

2017-01-01

Patients with organ failure often suffer from increased morbidity and decreased quality of life. Current strategies of treating organ failure have limitations, including shortage of donor organs, low efficiency of grafts, and immunological problems. Tissue engineering emerged about two decades ago as a strategy to restore organ function with a living, functional engineered substitute. However, the ability to engineer a functional organ substitute is limited by a limited understanding of the interactions between materials and cells that are required to yield functional tissue equivalents. Polymeric materials are one of the most promising classes of materials for use in tissue engineering due to their biodegradability, flexibility in processing and property design, and the potential to use polymer properties to control cell function. Stem cells offer potential in tissue engineering because of their unique capacity to self renew and differentiate into neurogenic, osteogenic, chondrogenic, myogenic lineages under appropriate stimuli from extracellular components. This review examines recent advances in stem cell-polymer interactions for tissue regeneration, specifically highlighting control of polymer properties to direct adhesion, proliferation, and differentiation of stem cells, and how biomaterials can be designed to provide some of the stimuli to cells that the natural extracellular matrix does. PMID:22457178

Natural Type II Collagen Hydrogel, Fibrin Sealant, and Adipose-Derived Stem Cells as a Promising Combination for Articular Cartilage Repair.

PubMed

Lazarini, Mariana; Bordeaux-Rego, Pedro; Giardini-Rosa, Renata; Duarte, Adriana S S; Baratti, Mariana Ozello; Zorzi, Alessandro Rozim; de Miranda, João Batista; Lenz Cesar, Carlos; Luzo, Ângela; Olalla Saad, Sara Teresinha

2017-10-01

Objective Articular cartilage is an avascular tissue with limited ability of self-regeneration and the current clinical treatments have restricted capacity to restore damages induced by trauma or diseases. Therefore, new techniques are being tested for cartilage repair, using scaffolds and/or stem cells. Although type II collagen hydrogel, fibrin sealant, and adipose-derived stem cells (ASCs) represent suitable alternatives for cartilage formation, their combination has not yet been investigated in vivo for focal articular cartilage defects. We performed a simple experimental procedure using the combination of these 3 compounds on cartilage lesions of rabbit knees. Design The hydrogel was developed in house and was first tested in vitro for chondrogenic differentiation. Next, implants were performed in chondral defects with or without ASCs and the degree of regeneration was macroscopically and microscopically evaluated. Results Production of proteoglycans and the increased expression of collagen type II (COL2α1), aggrecan (ACAN), and sex-determining region Y-box 9 (SOX9) confirmed the chondrogenic character of ASCs in the hydrogel in vitro. Importantly, the addition of ASC induced a higher overall repair of the chondral lesions and a better cellular organization and collagen fiber alignment compared with the same treatment without ASCs. This regenerating tissue also presented the expression of cartilage glycosaminoglycan and type II collagen. Conclusions Our results indicate that the combination of the 3 compounds is effective for articular cartilage repair and may be of future clinical interest.

Pleiotrophin Commits Human Bone Marrow Mesenchymal Stromal Cells towards Hypertrophy during Chondrogenesis

PubMed Central

Bouderlique, Thibault; Henault, Emilie; Lebouvier, Angelique; Frescaline, Guilhem; Bierling, Phillipe; Rouard, Helene; Courty, José

2014-01-01

Pleiotrophin (PTN) is a growth factor present in the extracellular matrix of the growth plate during bone development and in the callus during bone healing. Bone healing is a complicated process that recapitulates endochondral bone development and involves many cell types. Among those cells, mesenchymal stromal cells (MSC) are able to differentiate toward chondrogenic and osteoblastic lineages. We aimed to determine PTN effects on differentiation properties of human bone marrow stromal cells (hBMSC) under chondrogenic induction using histological analysis and quantitative reverse transcription polymerase chain reaction. PTN dramatically potentiated chondrogenic differentiation as indicated by a strong increase of collagen 2 protein, and cartilage-related gene expression. Moreover, PTN increased transcription of hypertrophic chondrocyte markers such as MMP13, collagen 10 and alkaline phosphatase and enhanced calcification and the content of collagen 10 protein. These effects are dependent on PTN receptors signaling and PI3 K pathway activation. These data suggest a new role of PTN in bone regeneration as an inducer of hypertrophy during chondrogenic differentiation of hBMSC. PMID:24516627

Mesenchymal stem cells derived from human exocrine pancreas express transcription factors implicated in beta-cell development.

PubMed

Baertschiger, Reto M; Bosco, Domenico; Morel, Philippe; Serre-Beinier, Veronique; Berney, Thierry; Buhler, Leo H; Gonelle-Gispert, Carmen

2008-07-01

Transplantation of in vitro generated islets or insulin-producing cells represents an attractive option to overcome organ shortage. The aim of this study was to isolate, expand, and characterize cells from human exocrine pancreas and analyze their potential to differentiate into beta cells. Fibroblast-like cells growing out of human exocrine tissue were characterized by flow cytometry and by their capacity to differentiate into mesenchymal cell lineages. During cell expansion and after differentiation toward beta cells, expression of transcription factors of endocrine pancreatic progenitors was analyzed by reverse transcription polymerase chain reaction. Cells emerged from 14/18 human pancreatic exocrine fractions and were expanded up to 40 population doublings. These cells displayed surface antigens similar to mesenchymal stem cells from bone marrow. A culture of these cells in adipogenic and chondrogenic differentiation media allowed differentiation into adipocyte- and chondrocyte-like cells. During expansion, cells expressed transcription factors implicated in islet development such as Isl1, Nkx2.2, Nkx6.1, nestin, Ngn3, Pdx1, and NeuroD. Activin A and hepatocyte growth factor induced an expression of insulin, glucagon, and glucokinase. Proliferating cells with characteristics of mesenchymal stem cells and endocrine progenitors were isolated from exocrine tissue. Under specific conditions, these cells expressed little insulin. Human pancreatic exocrine tissue might thus be a source of endocrine cell progenitors.

Human Mesenchymal Stem Cells Pretreated with Interleukin-1β and Stimulated with Bone Morphogenetic Growth Factor-3 Enhance Chondrogenesis.

PubMed

Hingert, Daphne; Barreto Henriksson, Helena; Brisby, Helena

2018-05-01

Low back pain is one of the most common ailments in western countries afflicting more than 80% of the population, and the main cause is considered to be degeneration of intervertebral discs. Interleukin-1β (IL-1β) is a vital inflammatory cytokine found in abundance in degenerated disc environment, whereas bone morphogenetic growth factor-3 (BMP-3) is believed to promote chondrogenesis through transforming growth factor-beta (TGF-β) pathway. The aim was to study the effects of BMP-3, IL-1β, and combination (pretreatment with IL-1β) on human mesenchymal stem cells (hMSCs) encapsulated in PuraMatrix™ hydrogel (Phg) especially in the absence of TGF-β in order to investigate the proliferation and differentiation ability of hMSCs over 28-day period. One hundred microliters of hMSCs' cell suspension was encapsulated between two layers of 100 μL hydrogels forming a sandwich-like structure. The encapsulated hMSCs were cultured in two sets of media, chondrogenic (C) and nonchondrogenic (nC) media, along with addition of BMP-3 (10 ng/mL) and IL-1β (10 ng/mL). To study the combined effects of BMP-3 and IL-1β, the encapsulated hMSCs were first pretreated with relevant media containing IL-1β for 24 h, and then the media was replaced by media containing BMP-3 for the remaining experimental time period. IL-1β pretreatment was carried out in both C and nC media. The samples were collected at day 7, 14, and 28. Proliferation and differentiation of hMSCs into chondrocyte-like cells were observed in all samples. Proteoglycan accumulation was observed in pretreatment samples in C media. The protein and gene expression of Sox-9 and COL2A1, respectively, showed the occurrence of chondrogenesis in all samples. High cell viability, proliferation, and differentiation were achieved in this in vitro model confirming that BMP-3 alone in the absence of TGF-β could drive hMSCs into chondrogenic lineage. Pretreatment with IL-1β followed by BMP-3 stimulation resulted in high proteoglycan accumulation compared to stimulation with growth factors or cytokine alone. This suggests that pretreatment with a pro-inflammatory cytokine before driving them into a chondrogenic lineage might be of importance also in vivo.

The influence of skeletal maturity on allogenic synovial mesenchymal stem cell-based repair of cartilage in a large animal model.

PubMed

Shimomura, Kazunori; Ando, Wataru; Tateishi, Kosuke; Nansai, Ryosuke; Fujie, Hiromichi; Hart, David A; Kohda, Hideyuki; Kita, Keisuke; Kanamoto, Takashi; Mae, Tatsuo; Nakata, Ken; Shino, Konsei; Yoshikawa, Hideki; Nakamura, Norimasa

2010-11-01

One of the potential factors that may affect the results of mesenchymal stem cell (MSC)-based therapy is the age of donors and recipients. However, there have been no controlled studies to investigate the influence of skeletal maturity on the MSC-based repair of cartilage. The purpose of this study was to compare the repair quality of damaged articular cartilage treated by a scaffold-free three-dimensional tissue-engineered construct (TEC) derived from synovial MSCs between immature and mature pigs. Synovial MSCs were isolated from immature and mature pigs and the proliferation and chondrogenic differentiation capacities were compared. The TEC derived from the synovial MSCs were then implanted into equivalent chondral defects in the medial femoral condyle of both immature and mature pigs, respectively. The implanted defects were morphologically and biomechanically evaluated at 6 months postoperatively. There was no skeletal maturity-dependent difference in proliferation or chondrogenic differentiation capacity of the porcine synovial MSCs. The TEC derived from synovial MSCs promoted the repair of chondral lesion in both immature and mature pigs without the evidence of immune reaction. The repaired tissue by the TEC also exhibited similar viscoelastic properties to normal cartilage regardless of the skeletal maturity. The results of the present study not only suggest the feasibility of allogenic MSC-based cartilage repair over generations but also may validate the use of immature porcine model as clinically relevant to test the feasibility of synovial MSC-based therapies in chondral lesions. Copyright 2010 Elsevier Ltd. All rights reserved.

RAC1 regulate tumor necrosis factor-α-mediated impaired osteogenic differentiation of dental pulp stem cells.

PubMed

Feng, Guijuan; Shen, Qijie; Lian, Min; Gu, Zhifeng; Xing, Jing; Lu, Xiaohui; Huang, Dan; Li, Liren; Huang, Shen; Wang, Yi; Zhang, Jinlong; Shi, Jiahai; Zhang, Dongmei; Feng, Xingmei

2015-09-01

Human dental pulp contains a rapidly proliferative subpopulation of precursor cells termed dental pulp stem cells (DPSCs) that show self-renewal and multilineage differentiation, including neurogenic, chondrogenic, osteogenic and adipogenic. We previously reported that tomuor necrosis factor-α (TNF-α) (10 ng/mL) triggered osteogenic differentiation of human DPSCs via the nuclear factor-κB (NF-κB) signaling pathway. While previous studies showed that cells treated with TNF-α at higher concentrations showed decreased osteogenic differentiation capability. In this study we analyze the function of TNF-α (100 ng/mL) on osteogenic differentiation of human DPSCs for the first time and identify the underlying molecule mechanisms. Our data revealed that TNF-α with higher concentration significantly reduced mineralization and the expression of bone morphogenetic protein 2 (BMP2), alkaline phosphatase (ALP) and runt-related transcription factor 2 (RUNX2). Further, we revealed that TNF-α could suppress the osteogenic differentiation of DPSCs via increasing the expression of RAC1, which could activate the Wnt/β-catenin signaling pathway and liberate β-catenin to translocate into the nucleus. Genetic silencing of RAC1 expression using siRNA restored osteogenic differentiation of DPSCs. Our findings may provide a potential approach to bone regeneration in inflammatory microenvironments. © 2015 Japanese Society of Developmental Biologists.

Directing the osteoblastic and chondrocytic differentiations of mesenchymal stem cells: matrix vs. induction media

PubMed Central

He, Jing; Guo, Jianglong; Jiang, Bo; Yao, Ruijuan; Wu, Yao

2017-01-01

Abstract While both induction culture media and matrix have been reported to regulate the stem cell fate, little is known about which factor plays a more decisive role in directing the MSC differentiation lineage as well as the underlying mechanisms. To this aim, we seeded MSCs on HA-collagen and HA-synthetic hydrogel matrixes, which had demonstrated highly different potentials toward osteoblastic and chondrocytic differentiation lineages, respectively, and cultured them with osteogenic, chondrogenic and normal culture media, respectively. A systematic comparison has been carried out on the effects of induction media and matrix on MSC adhesion, cytoskeleton organization, proliferation, and in particular differentiation into the osteoblastic and chondrocytic lineages. The results demonstrated that the matrix selection had a much more profound effect on directing the differentiation lineage than the induction media did. The strong modulation effect on the transcription activities might be the critical factor contributing to the above observations in our study, where canonical Wnt-β-Catenin signal pathway was directly involved in the matrix-driven osteoblastic differentiation. Such findings not only provide a critical insight on natural cellular events leading to the osteoblastic and chondrocytic differentiations, but also have important implications in biomaterial design for tissue engineering applications. PMID:29026640

Hydrostatic pressure enhances chondrogenic differentiation of human bone marrow stromal cells in osteochondrogenic medium.

PubMed

Wagner, Diane R; Lindsey, Derek P; Li, Kelvin W; Tummala, Padmaja; Chandran, Sheena E; Smith, R Lane; Longaker, Michael T; Carter, Dennis R; Beaupre, Gary S

2008-05-01

This study demonstrated the chondrogenic effect of hydrostatic pressure on human bone marrow stromal cells (MSCs) cultured in a mixed medium containing osteogenic and chondrogenic factors. MSCs seeded in type I collagen sponges were exposed to 1 MPa of intermittent hydrostatic pressure at a frequency of 1 Hz for 4 h per day for 10 days, or remained in identical culture conditions but without exposure to pressure. Afterwards, we compared the proteoglycan content of loaded and control cell/scaffold constructs with Alcian blue staining. We also used real-time PCR to evaluate the change in mRNA expression of selected genes associated with chondrogenic and osteogenic differentiation (aggrecan, type I collagen, type II collagen, Runx2 (Cbfa-1), Sox9, and TGF-beta1). With the hydrostatic pressure loading regime, proteoglycan staining increased markedly. Correspondingly, the mRNA expression of chondrogenic genes such as aggrecan, type II collagen, and Sox9 increased significantly. We also saw a significant increase in the mRNA expression of type I collagen, but no change in the expression of Runx2 or TGF-beta1 mRNA. This study demonstrated that hydrostatic pressure enhanced differentiation of MSCs in the presence of multipotent differentiation factors in vitro, and suggests the critical role that this loading regime may play during cartilage development and regeneration in vivo.

Dynamic Culturing of Cartilage Tissue: The Significance of Hydrostatic Pressure

PubMed Central

Pereira, Ana L.; Duarte, Ana R.C.; Frias, Ana M.; Pedro, Adriano J.; Oliveira, João T.; Sousa, Rui A.; Reis, Rui L.

2012-01-01

Human articular cartilage functions under a wide range of mechanical loads in synovial joints, where hydrostatic pressure (HP) is the prevalent actuating force. We hypothesized that the formation of engineered cartilage can be augmented by applying such physiologic stimuli to chondrogenic cells or stem cells, cultured in hydrogels, using custom-designed HP bioreactors. To test this hypothesis, we investigated the effects of distinct HP regimens on cartilage formation in vitro by either human nasal chondrocytes (HNCs) or human adipose stem cells (hASCs) encapsulated in gellan gum (GG) hydrogels. To this end, we varied the frequency of low HP, by applying pulsatile hydrostatic pressure or a steady hydrostatic pressure load to HNC-GG constructs over a period of 3 weeks, and evaluated their effects on cartilage tissue-engineering outcomes. HNCs (10×106 cells/mL) were encapsulated in GG hydrogels (1.5%) and cultured in a chondrogenic medium under three regimens for 3 weeks: (1) 0.4 MPa Pulsatile HP; (2) 0.4 MPa Steady HP; and (3) Static. Subsequently, we applied the pulsatile regimen to hASC-GG constructs and varied the amplitude of loading, by generating both low (0.4 MPa) and physiologic (5 MPa) HP levels. hASCs (10×106 cells/mL) were encapsulated in GG hydrogels (1.5%) and cultured in a chondrogenic medium under three regimens for 4 weeks: (1) 0.4 MPa Pulsatile HP; (2) 5 MPa Pulsatile HP; and (3) Static. In the HNC study, the best tissue development was achieved by the pulsatile HP regimen, whereas in the hASC study, greater chondrogenic differentiation and matrix deposition were obtained for physiologic loading, as evidenced by gene expression of aggrecan, collagen type II, and sox-9; metachromatic staining of cartilage extracellular matrix; and immunolocalization of collagens. We thus propose that both HNCs and hASCs detect and respond to physical forces, thus resembling joint loading, by enhancing cartilage tissue development in a frequency- and amplitude-dependant manner. PMID:22559784

Mesenchymal stem cell-derived extracellular matrix enhances chondrogenic phenotype of and cartilage formation by encapsulated chondrocytes in vitro and in vivo.

PubMed

Yang, Yuanheng; Lin, Hang; Shen, He; Wang, Bing; Lei, Guanghua; Tuan, Rocky S

2018-03-15

Mesenchymal stem cell derived extracellular matrix (MSC-ECM) is a natural biomaterial with robust bioactivity and good biocompatibility, and has been studied as a scaffold for tissue engineering. In this investigation, we tested the applicability of using decellularized human bone marrow derived MSC-ECM (hBMSC-ECM) as a culture substrate for chondrocyte expansion in vitro, as well as a scaffold for chondrocyte-based cartilage repair. hBMSC-ECM deposited by hBMSCs cultured on tissue culture plastic (TCP) was harvested, and then subjected to a decellularization process to remove hBMSCs. Compared with chondrocytes grown on TCP, chondrocytes seeded onto hBMSC-ECM exhibited significantly increased proliferation rate, and maintained better chondrocytic phenotype than TCP group. After being expanded to the same cell number and placed in high-density micromass cultures, chondrocytes from the ECM group showed better chondrogenic differentiation profile than those from the TCP group. To test cartilage formation ability, composites of hBMSC-ECM impregnated with chondrocytes were subjected to brief trypsin treatment to allow cell-mediated contraction, and folded to form 3-dimensional chondrocyte-impregnated hBMSC-ECM (Cell/ECM constructs). Upon culture in vitro in chondrogenic medium for 21 days, robust cartilage formation was observed in the Cell/ECM constructs. Similarly prepared Cell/ECM constructs were tested in vivo by subcutaneous implantation into SCID mice. Prominent cartilage formation was observed in the implanted Cell/ECM constructs 14 days post-implantation, with higher sGAG deposition compared to controls consisting of chondrocyte cell sheets. Taken together, these findings demonstrate that hBMSC-ECM is a superior culture substrate for chondrocyte expansion and a bioactive matrix potentially applicable for cartilage regeneration in vivo. Current cell-based treatments for focal cartilage defects face challenges, including chondrocyte dedifferentiation, need for xenogenic scaffolds, and suboptimal cartilage formation. We present here a novel technique that utilizes adult stem cell-derived extracellular matrix, as a culture substrate and/or encapsulation scaffold for human adult chondrocytes, for the repair of cartilage defects. Chondrocytes cultured in stem cell-derived matrix showed higher proliferation, better chondrocytic phenotype, and improved redifferentiation ability upon in vitro culture expansion. Most importantly, 3-dimensional constructs formed from chondrocytes folded within stem cell matrix manifested excellent cartilage formation both in vitro and in vivo. These findings demonstrate the suitability of stem cell-derived extracellular matrix as a culture substrate for chondrocyte expansion as well as a candidate bioactive matrix for cartilage regeneration. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Alginate Microcapsules Incorporating Hyaluronic Acid Recreate Closer in Vivo Environment for Mesenchymal Stem Cells.

PubMed

Cañibano-Hernández, Alberto; Saenz Del Burgo, Laura; Espona-Noguera, Albert; Orive, Gorka; Hernández, Rosa M; Ciriza, Jesús; Pedraz, Jose Luis

2017-07-03

The potential clinical application of alginate cell microencapsulation has advanced enormously during the past decade. However, the 3D environment created by alginate beads does not mimic the natural extracellular matrix surrounding cells in vivo, responsible of cell survival and functionality. As one of the most frequent macromolecules present in the extracellular matrix is hyaluronic acid, we have formed hybrid beads with alginate and hyaluronic acid recreating a closer in vivo cell environment. Our results show that 1% alginate-0.25% hyaluronic acid microcapsules retain 1.5% alginate physicochemical properties. Moreover, mesenchymal stem cells encapsulated in these hybrid beads show enhanced viability therapeutic protein release and mesenchymal stem cells' potential to differentiate into chondrogenic lineage. Although future studies with additional proteins need to be done in order to approach even more the extracellular matrix features, we have shown that hyaluronic acid protects alginate encapsulated mesenchymal stem cells by providing a niche-like environment and remaining them competent as a sustainable drug delivery system.

Skeletal stem cell isolation: A review on the state-of-the-art microfluidic label-free sorting techniques.

PubMed

Xavier, Miguel; Oreffo, Richard O C; Morgan, Hywel

2016-01-01

Skeletal stem cells (SSC) are a sub-population of bone marrow stromal cells that reside in postnatal bone marrow with osteogenic, chondrogenic and adipogenic differentiation potential. SSCs reside only in the bone marrow and have organisational and regulatory functions in the bone marrow microenvironment and give rise to the haematopoiesis-supportive stroma. Their differentiation capacity is restricted to skeletal lineages and therefore the term SSC should be clearly distinguished from mesenchymal stem cells which are reported to exist in extra-skeletal tissues and, critically, do not contribute to skeletal development. SSCs are responsible for the unique regeneration capacity of bone and offer unlimited potential for application in bone regenerative therapies. A current unmet challenge is the isolation of homogeneous populations of SSCs, in vitro, with homogeneous regeneration and differentiation capacities. Challenges that limit SSC isolation include a) the scarcity of SSCs in bone marrow aspirates, estimated at between 1 in 10-100,000 mononuclear cells; b) the absence of specific markers and thus the phenotypic ambiguity of the SSC and c) the complexity of bone marrow tissue. Microfluidics provides innovative approaches for cell separation based on bio-physical features of single cells. Here we review the physical principles underlying label-free microfluidic sorting techniques and review their capacity for stem cell selection/sorting from complex (heterogeneous) samples. Copyright © 2016 Elsevier Inc. All rights reserved.

Simultaneous isolation of vascular endothelial cells and mesenchymal stem cells from the human umbilical cord.

PubMed

Kadam, Sachin S; Tiwari, Shubha; Bhonde, Ramesh R

2009-01-01

The umbilical cord represents the link between mother and fetus during pregnancy. This cord is usually discarded as a biological waste after the child's birth; however, its importance as a "store house" of stem cells has been explored recently. We developed a method of simultaneous isolation of endothelial cells (ECs) from the vein and mesenchymal stem cells from umbilical cord Wharton's jelly of the same cord. The isolation protocol has been simplified, modified, and improvised with respect to choice of enzyme and enzyme mixture, digestion time, cell yield, cell growth, and culture medium. Isolated human umbilical vascular ECs (hUVECs) were positive for von-Willibrand factor, a classical endothelial marker, and could form capillary-like structures when seeded on Matrigel, thus proving their functionality. The isolated human umbilical cord mesenchymal stem cells (hUCMSCs) were found positive for CD44, CD90, CD 73, and CD117 and were found negative for CD33, CD34, CD45, and CD105 surface markers; they were also positive for cytoskeleton markers of smooth muscle actin and vimentin. The hUCMSCs showed multilineage differentiation potential and differentiated into adipogenic, chondrogenic, osteogenic, and neuronal lineages under influence of lineage specific differentiation medium. Thus, isolating endothelial cells as well as mesenchymal cells from the same umbilical cord could lead to complete utilization of the available tissue for the tissue engineering and cell therapy.

Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Varga, Nora; Vereb, Zoltan; Rajnavoelgyi, Eva

2011-10-28

Highlights: Black-Right-Pointing-Pointer MSC like cells were derived from hESC by a simple and reproducible method. Black-Right-Pointing-Pointer Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. Black-Right-Pointing-Pointer MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth ofmore » undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.« less

Expression of Pluripotency Genes in Chondrocyte-Like Cells Differentiated from Human Induced Pluripotent Stem Cells

PubMed Central

Stelcer, Ewelina; Kulcenty, Katarzyna; Rucinski, Marcin; Jopek, Karol; Trzeciak, Tomasz; Richter, Magdalena; Wroblewska, Joanna P.; Suchorska, Wiktoria M.

2018-01-01

Human induced pluripotent stem cells (hiPSCs) constitute an important breakthrough in regenerative medicine, particularly in orthopedics, where more effective treatments are urgently needed. Despite the promise of hiPSCs only limited data on in vitro chondrogenic differentiation of hiPSCs are available. Therefore, we compared the gene expression profile of pluripotent genes in hiPSC-derived chondrocytes (ChiPS) to that of an hiPSC cell line created by our group (GPCCi001-A). The results are shown on heatmaps and plots and confirmed by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) analysis. Unlike the ChiPS, our GPCCi001-A cells maintained their pluripotency state during long-term culture, thus demonstrating that this cell line was comprised of stable, fully pluripotent hiPSCs. Moreover, these chondrocyte-like cells not only presented features that are characteristic of chondrocytes, but they also lost their pluripotency, which is an important advantage in favor of using this cell line in future clinical studies. PMID:29439516

Delta-like 1/Fetal Antigen-1 (Dlk1/FA1) Is a Novel Regulator of Chondrogenic Cell Differentiation via Inhibition of the Akt Kinase-dependent Pathway*

PubMed Central

Chen, Li; Qanie, Diyako; Jafari, Abbas; Taipaleenmaki, Hanna; Jensen, Charlotte H.; Säämänen, Anna-Marja; Sanz, Maria Luisa Nueda; Laborda, Jorge; Abdallah, Basem M.; Kassem, Moustapha

2011-01-01

Delta-like 1 (Dlk1, also known as fetal antigen-1, FA1) is a member of Notch/Delta family that inhibits adipocyte and osteoblast differentiation; however, its role in chondrogenesis is still not clear. Thus, we overexpressed Dlk1/FA1 in mouse embryonic ATDC5 cells and tested its effects on chondrogenic differentiation. Dlk1/FA1 inhibited insulin-induced chondrogenic differentiation as evidenced by reduction of cartilage nodule formation and gene expression of aggrecan, collagen Type II and X. Similar effects were obtained either by using Dlk1/FA1-conditioned medium or by addition of a purified, secreted, form of Dlk1 (FA1) directly to the induction medium. The inhibitory effects of Dlk1/FA1 were dose-dependent and occurred irrespective of the chondrogenic differentiation stage: proliferation, differentiation, maturation, or hypertrophic conversion. Overexpression or addition of the Dlk1/FA1 protein to the medium strongly inhibited the activation of Akt, but not the ERK1/2, or p38 MAPK pathways, and the inhibition of Akt by Dlk1/FA1 was mediated through PI3K activation. Interestingly, inhibition of fibronectin expression by siRNA rescued the Dlk1/FA1-mediated inhibition of Akt, suggesting interaction of Dlk1/FA1 and fibronectin in chondrogenic cells. Our results identify Dlk1/FA1 as a novel regulator of chondrogenesis and suggest Dlk1/FA1 acts as an inhibitor of the PI3K/Akt pathways that leads to its inhibitory effects on chondrogenesis. PMID:21724852

Effect of silver nanoparticles on human mesenchymal stem cell differentiation

PubMed Central

Diendorf, Jörg; Epple, Matthias; Schildhauer, Thomas A; Köller, Manfred

2014-01-01

Summary Background: Silver nanoparticles (Ag-NP) are one of the fastest growing products in nano-medicine due to their enhanced antibacterial activity at the nanoscale level. In biomedicine, hundreds of products have been coated with Ag-NP. For example, various medical devices include silver, such as surgical instruments, bone implants and wound dressings. After the degradation of these materials, or depending on the coating technique, silver in nanoparticle or ion form can be released and may come into close contact with tissues and cells. Despite incorporation of Ag-NP as an antibacterial agent in different products, the toxicological and biological effects of silver in the human body after long-term and low-concentration exposure are not well understood. In the current study, we investigated the effects of both ionic and nanoparticulate silver on the differentiation of human mesenchymal stem cells (hMSCs) into adipogenic, osteogenic and chondrogenic lineages and on the secretion of the respective differentiation markers adiponectin, osteocalcin and aggrecan. Results: As shown through laser scanning microscopy, Ag-NP with a size of 80 nm (hydrodynamic diameter) were taken up into hMSCs as nanoparticulate material. After 24 h of incubation, these Ag-NP were mainly found in the endo-lysosomal cell compartment as agglomerated material. Cytotoxicity was observed for differentiated or undifferentiated hMSCs treated with high silver concentrations (≥20 µg·mL−1 Ag-NP; ≥1.5 µg·mL−1 Ag+ ions) but not with low-concentration treatments (≤10 µg·mL−1 Ag-NP; ≤1.0 µg·mL−1 Ag+ ions). Subtoxic concentrations of Ag-NP and Ag+ ions impaired the adipogenic and osteogenic differentiation of hMSCs in a concentration-dependent manner, whereas chondrogenic differentiation was unaffected after 21 d of incubation. In contrast to aggrecan, the inhibitory effect of adipogenic and osteogenic differentiation was confirmed by a decrease in the secretion of specific biomarkers, including adiponectin (adipocytes) and osteocalcin (osteoblasts). Conclusion: Aside from the well-studied antibacterial effect of silver, little is known about the influence of nano-silver on cell differentiation processes. Our results demonstrate that ionic or nanoparticulate silver attenuates the adipogenic and osteogenic differentiation of hMSCs even at non-toxic concentrations. Therefore, more studies are needed to investigate the effects of silver species on cells at low concentrations during long-term treatment. PMID:25551033

The osteogenic response of mesenchymal stem cells to an injectable PLGA bone regeneration system.

PubMed

Curran, Judith M; Fawcett, Sandra; Hamilton, Lloyd; Rhodes, Nicholas P; Rahman, Cheryl V; Alexander, Morgan; Shakesheff, Kevin; Hunt, John A

2013-12-01

The enrichment of substrates/surfaces with selected functional groups, methyl (-CH3), allyl amine (-NH2), allyl alcohol (-OH) and acrylic acid (-COOH), can be used to trigger mesenchymal stem (MSC) cell differentiation into specified lineages, minimising the need for exogenous biological supplementation. We present the successful translation of this research phenomenon to an injectable two phase injectable PLGA system, utilising plasma techniques, for the repair of bone defects. Modified microspheres were characterised using water contact angel (WCA), X-ray Photon Spectroscopy (XPS) and scanning electron microscopy (SEM). When cultured in contact with MSCs in vitro, the ability of the modified particles, within the 2 phase system, to induce differentiation was characterised using quantitative assays for cell viability and histological analysis for key markers of differentiation throughout the entirety of the three dimensional scaffold. Biological analysis proved that selected modified microspheres have the ability to induce MSC osteogenic (-NH2 modified scaffolds) and chondrogenic (-OH modified scaffolds) differentiation throughout the entirety of the formed scaffold. Therefore optimised plasma modification of microspheres is an effective tool for the production of injectable systems for the repair of bone and cartilage defects. Copyright © 2013 Elsevier Ltd. All rights reserved.

TrxR2 deficiencies promote chondrogenic differentiation and induce apoptosis of chondrocytes through mitochondrial reactive oxygen species

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, Jidong; Xu, Jing; Fei, Yao

Thioredoxin reductase 2 (TrxR2) is a selenium (Se) containing protein. Se deficiency is associated with an endemic osteoarthropathy characterized by impaired cartilage formation. It is unclear whether TrxR2 have roles in cartilage function. We examined the effects of TrxR2 on chondrogenic ATDC5 cells through shRNA-mediated gene silencing of TrxR2. We demonstrated TrxR2 deficiencies could enhance chondrogenic differentiation and apoptosis of ATDC5 cells. TrxR2 deficiencies increased accumulation of cartilage glycosaminoglycans (GAGs) and mineralization. TrxR2 deficiencies also stimulated expression of extracellular (ECM) gene including Collagen II and Aggrecan. The enhanced chondrogenic properties were further confirmed by activation of Akt signaling which aremore » required for chondrogenesis. In addition, TrxR2 deficiencies promoted chondrocyte proliferation through acceleration of cell cycle progression by increase in both S and G2/M phase cell distribution accompanied with induction of parathyroid hormone-related protein (PTHrP). Moreover, TrxR2 deficiencies induced chondrocyte death via apoptosis and increased cell sensitivity to exogenous oxidative stress. Furthermore, TrxR2 deficiencies induced emission of mitochondrial reactive oxygen species (ROS) without alteration of mitochondrial membrane potential and intracellular ATP content. Finally, treatment of TrxR2 deficiency cells with N-acetylcysteine (NAC) inhibited mitochondrial ROS production and chondrocyte apoptosis. NAC also prevented chondrogenic differentiation of TrxR2 deficiency cells by suppression of ECM gene expression, GAGs accumulation and mineralization, as well as attenuation of Akt signaling. Thus, TrxR2-mediated mitochondrial integrity is indispensable for chondrogenic differentiation of ATDC5 cells. TrxR2 deficiency-induced impaired proliferation and death of chondrocytes may be the pathological mechanism of the osteoarthropathy due to Se deficiency. Notably, this study also uncover the roles of mitochondrial ROS which could stimulate cartilage ECM synthesis that offer novel insights for development of therapeutic agent to prevent cartilage degeneration in human disease. - Highlights: • TrxR2 deficiencies enhance chondrogenic differentiation. • TrxR2 deficiencies stimulate chondrocyte proliferation. • TrxR2 deficiencies induce chondrocyte apoptosis. • TrxR2 deficiencies increase emission of mitochondrial ROS. • Mitochondrial ROS regulate chondrocyte proliferation, differentiation, and apoptosis.« less

Differentiation of mesenchymal stem cells for cartilage tissue engineering: Individual and synergetic effects of three-dimensional environment and mechanical loading.

PubMed

Panadero, J A; Lanceros-Mendez, S; Ribelles, J L Gomez

2016-03-01

Chondrogenesis of dedifferentiated chondrocytes and mesenchymal stem cells is influenced not only by soluble molecules like growth factors, but also by the cell environment itself. The latter is achieved through both mechanical cues - which act as stimulation factor and influences nutrient transport - and adhesion to extracellular matrix cues - which determine cell shape. Although the effects of soluble molecules and cell environment have been intensively addressed, few observations and conclusions about the interaction between the two have been achieved. In this work, we review the state of the art on the single effects between mechanical and biochemical cues, as well as on the combination of the two. Furthermore, we provide a discussion on the techniques currently used to determine the mechanical properties of materials and tissues generated in vitro, their limitations and the future research needs to properly address the identified problems. The importance of biomechanical cues in chondrogenesis is well known. This paper reviews the existing literature on the effect of mechanical stimulation on chondrogenic differentiation of mesenchymal stem cells in order to regenerate hyaline cartilage. Contradictory results found with respect to the effect of different modes of external loading can be explained by the different properties of the scaffolding system that holds the cells, which determine cell adhesion and morphology and spatial distribution of cells, as well as the stress transmission to the cells. Thus, this review seeks to provide an insight into the interplay between external loading program and scaffold properties during chondrogenic differentiation. The review of the literature reveals an important gap in the knowledge in this field and encourages new experimental studies. The main issue is that in each of the few cases in which the interplay is investigated, just two groups of scaffolds are compared, leaving intermediate adhesion conditions out of study. The authors propose broader studies implementing new high-throughput techniques for mechanical characterization of tissue engineering constructs and the inclusion of fatigue analysis as support methodology to more exhaustive mechanical characterization. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Electric stimulation at 448 kHz promotes proliferation of human mesenchymal stem cells.

PubMed

Hernández-Bule, María Luisa; Paíno, Carlos Luis; Trillo, María Ángeles; Úbeda, Alejandro

2014-01-01

Capacitive-resistive electric transfer (CRET) is a non invasive electrothermal therapy that applies electric currents within the 400 kHz - 450 kHz frequency range to the treatment of musculoskeletal lesions. Evidence exists that electric currents and electric or magnetic fields can influence proliferative and/or differentiating processes involved in tissue regeneration. This work investigates proliferative responses potentially underlying CRET effects on tissue repair. XTT assay, flow cytometry, immunofluorescence and Western Blot analyses were conducted to asses viability, proliferation and differentiation of adipose-derived stem cells (ADSC) from healthy donors, after short, repeated (5 m On/4 h Off) in vitro stimulation with a 448-kHz electric signal currently used in CRET therapy, applied at a subthermal dose of 50 μA/mm(2) RESULTS: The treatment induced PCNA and ERK1/2 upregulation, together with significant increases in the fractions of ADSC undergoing cycle phases S, G2 and M, and enhanced cell proliferation rate. This proliferative effect did not compromise the multipotential ability of ADSC for subsequent adipogenic, chondrogenic or osteogenic differentiation. These data identify cellular and molecular phenomena potentially underlying the response to CRET and indicate that CRET-induced lesion repair could be mediated by stimulation of the proliferation of stem cells present in the injured tissues. © 2014 S. Karger AG, Basel.

High molecular weight hyaluronic acid increases the differentiation potential of the murine chondrocytic ATDC5 cell line.

PubMed

Sato, Eiichi; Ando, Takashi; Ichikawa, Jiro; Okita, Genki; Sato, Nobutaka; Wako, Masanori; Ohba, Tetsuro; Ochiai, Satoshi; Hagino, Tetsuo; Jacobson, Richard; Haro, Hirotaka

2014-12-01

Osteoarthritis (OA) is a group of common, chronic, and painful inflammatory joint diseases. One important finding in OA patients is a remarkable decrease in the molecular weight of hyaluronic acid (HA) in the synovial fluid of affected joints. Therapeutic HA is available to patients in most parts of the world as a viscosupplementation product for the treatment of OA. Previous clinical reports show that high molecular weight HA (HMWHA) more effectively relieves pain than low molecular weight HA (LMWHA). However, the mechanism behind this finding remains unclear. In this study, we investigated whether a LMWHA (Low-0.9 MDa) and two types of HMWHA (High-1.9 MDa and 6 MDa) differentially affected chondroregulatory action. We tested this using ATDC5 cell, a murine chondrocytic cell line widely used in culture systems to study chondrogenic differentiation. We found that HMWHA, especially hylan G-F 20 (High-6 MDa), significantly induced aggrecan and proteoglycan accumulation, nodule formation, and mRNA expression of chondrogenic differentiation markers in a time- and dose-dependent manner. In addition, we showed that HMWHA prevented TNF-α induced inhibition of chondrogenic differentiation, with no effect on cell proliferation or viability. These results reveal that HMWHA significantly promotes chondrogenic differentiation of ATDC5 cells in vitro, and suggest that HMWHA plays a significant chondroregulatory role in vivo. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Effects of natural cartilaginous extracellular matrix on chondrogenic potential for cartilage cell transplantation.

PubMed

Yang, W; Lee, S; Jo, Y H; Lee, K M; Nemeno, J G; Nam, B M; Kim, B Y; Jang, I J; Kim, H N; Takebe, T; Lee, J I

2014-05-01

Autologous chondrocyte transplantation (ACT) has been established to contribute cartilage regeneration over the past years; however, many obstacles need to be overcome. Recently, newer ACT technique involves cotransplantation of chondrocytes and biomaterial. Although various proposed intelligent biomaterials exist, many of them remain insufficient and controversial. In this study, we aimed to examine the effects of natural extracellular matrix (ECM) to the proliferation rate and differentiation on the chondrocytes. We first derived a natural ECM sheet from 10-μm-thick frozen sections of porcine knee cartilages. We then cultured the chondrocytes derived from a rabbit's knee on a dish precoated with the natural ECM. Then we assessed differentiation and chondrogenic potential of the cells compared with those grown in untreated culture dishes. We characterized the gene expression of chondrogenic markers, such as collagen type II, SOX-9, and aggrecan, as well as the level of ECM protein with the use of reverse-transcription polymerase chain reaction analysis. The cells cultured with the ECM sheet showed highest chondrogenic potential and differentiation. Therefore, we can induce good chondrogenesis by with the use of a natural ECM sheet on the culture dish. The readily available and easy-to-handle thin ECM sheets create an environment that promotes efficient cartilage regeneration. Our data suggest that this natural ECM scaffold improved the chondrogenic differentiation of the cells in vitro by providing a favorable microenvironment. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.

Generating Chondromimetic Mesenchymal Stem Cell Spheroids by Regulating Media Composition and Surface Coating.

PubMed

Sridharan, BanuPriya; Laflin, Amy D; Detamore, Michael S

2018-04-01

Spheroids of mesenchymal stem cells (MSCs) in cartilage tissue engineering have been shown to enhance regenerative potential owing to their 3D structure. In this study, we explored the possibility of priming spheroids under different media to replace the use of inductive surface coatings for chondrogenic differentiation. Rat bone marrow-derived MSCs were organized into cell spheroids by the hanging drop technique and subsequently cultured on hyaluronic acid (HA) coated or non-coated well plates under different cell media conditions. Endpoint analysis included cell viability, DNA and Glycosaminoglycan (GAG) and collagen content, gene expression and immunohistochemistry. For chondrogenic applications, MSC spheroids derived on non-coated surfaces outperformed the spheroids derived from HA-coated surfaces in matrix synthesis and collagen II gene expression. Spheroids on non-coated surfaces gave rise to the highest collagen and GAG when primed with medium containing insulin-like growth factor (IGF) for 1 week during spheroid formation. Spheroids that were grown in chondroinductive raw material-inclusive media such as aggrecan or chondroitin sulfate exhibited the highest Collagen II gene expression in the non-coated surface at 1 week. Media priming by growth factors and raw materials might be a more predictive influencer of chondrogenesis compared to inductive-surfaces. Such tailored bioactivity of the stem cell spheroids in the stage of the spheroid formation may give rise to a platform technology that may eventually produce spheroids capable of chondrogenesis achieved by mere media manipulation, skipping the need for additional culture on a modified surface, that paves the way for cost-effective technologies.

Stem Cells for Cartilage Repair: Preclinical Studies and Insights in Translational Animal Models and Outcome Measures.

PubMed

Lo Monaco, Melissa; Merckx, Greet; Ratajczak, Jessica; Gervois, Pascal; Hilkens, Petra; Clegg, Peter; Bronckaers, Annelies; Vandeweerd, Jean-Michel; Lambrichts, Ivo

2018-01-01

Due to the restricted intrinsic capacity of resident chondrocytes to regenerate the lost cartilage postinjury, stem cell-based therapies have been proposed as a novel therapeutic approach for cartilage repair. Moreover, stem cell-based therapies using mesenchymal stem cells (MSCs) or induced pluripotent stem cells (iPSCs) have been used successfully in preclinical and clinical settings. Despite these promising reports, the exact mechanisms underlying stem cell-mediated cartilage repair remain uncertain. Stem cells can contribute to cartilage repair via chondrogenic differentiation, via immunomodulation, or by the production of paracrine factors and extracellular vesicles. But before novel cell-based therapies for cartilage repair can be introduced into the clinic, rigorous testing in preclinical animal models is required. Preclinical models used in regenerative cartilage studies include murine, lapine, caprine, ovine, porcine, canine, and equine models, each associated with its specific advantages and limitations. This review presents a summary of recent in vitro data and from in vivo preclinical studies justifying the use of MSCs and iPSCs in cartilage tissue engineering. Moreover, the advantages and disadvantages of utilizing small and large animals will be discussed, while also describing suitable outcome measures for evaluating cartilage repair.

Stem Cells for Cartilage Repair: Preclinical Studies and Insights in Translational Animal Models and Outcome Measures

PubMed Central

Ratajczak, Jessica; Gervois, Pascal; Clegg, Peter; Bronckaers, Annelies; Vandeweerd, Jean-Michel; Lambrichts, Ivo

2018-01-01

Due to the restricted intrinsic capacity of resident chondrocytes to regenerate the lost cartilage postinjury, stem cell-based therapies have been proposed as a novel therapeutic approach for cartilage repair. Moreover, stem cell-based therapies using mesenchymal stem cells (MSCs) or induced pluripotent stem cells (iPSCs) have been used successfully in preclinical and clinical settings. Despite these promising reports, the exact mechanisms underlying stem cell-mediated cartilage repair remain uncertain. Stem cells can contribute to cartilage repair via chondrogenic differentiation, via immunomodulation, or by the production of paracrine factors and extracellular vesicles. But before novel cell-based therapies for cartilage repair can be introduced into the clinic, rigorous testing in preclinical animal models is required. Preclinical models used in regenerative cartilage studies include murine, lapine, caprine, ovine, porcine, canine, and equine models, each associated with its specific advantages and limitations. This review presents a summary of recent in vitro data and from in vivo preclinical studies justifying the use of MSCs and iPSCs in cartilage tissue engineering. Moreover, the advantages and disadvantages of utilizing small and large animals will be discussed, while also describing suitable outcome measures for evaluating cartilage repair. PMID:29535784

Effects of in vitro low oxygen tension preconditioning of adipose stromal cells on their in vivo chondrogenic potential: application in cartilage tissue repair.

PubMed

Portron, Sophie; Merceron, Christophe; Gauthier, Olivier; Lesoeur, Julie; Sourice, Sophie; Masson, Martial; Fellah, Borhane Hakim; Geffroy, Olivier; Lallemand, Elodie; Weiss, Pierre; Guicheux, Jérôme; Vinatier, Claire

2013-01-01

Multipotent stromal cell (MSC)-based regenerative strategy has shown promise for the repair of cartilage, an avascular tissue in which cells experience hypoxia. Hypoxia is known to promote the early chondrogenic differentiation of MSC. The aim of our study was therefore to determine whether low oxygen tension could be used to enhance the regenerative potential of MSC for cartilage repair. MSC from rabbit or human adipose stromal cells (ASC) were preconditioned in vitro in control or chondrogenic (ITS and TGF-β) medium and in 21 or 5% O2. Chondrogenic commitment was monitored by measuring COL2A1 and ACAN expression (real-time PCR). Preconditioned rabbit and human ASC were then incorporated into an Si-HPMC hydrogel and injected (i) into rabbit articular cartilage defects for 18 weeks or (ii) subcutaneously into nude mice for five weeks. The newly formed tissue was qualitatively and quantitatively evaluated by cartilage-specific immunohistological staining and scoring. The phenotype of ASC cultured in a monolayer or within Si-HPMC in control or chondrogenic medium and in 21 or 5% O2 was finally evaluated using real-time PCR. 5% O2 increased the in vitro expression of chondrogenic markers in ASC cultured in induction medium. Cells implanted within Si-HPMC hydrogel and preconditioned in chondrogenic medium formed a cartilaginous tissue, regardless of the level of oxygen. In addition, the 3D in vitro culture of ASC within Si-HPMC hydrogel was found to reinforce the pro-chondrogenic effects of the induction medium and 5% O2. These data together indicate that although 5% O2 enhances the in vitro chondrogenic differentiation of ASC, it does not enhance their in vivo chondrogenesis. These results also highlight the in vivo chondrogenic potential of ASC and their potential value in cartilage repair.

How do culture media influence in vitro perivascular cell behavior?

PubMed

Huber, Birgit; Volz, Ann-Cathrin; Kluger, Petra Juliane

2015-12-01

Perivascular cells are multilineage cells located around the vessel wall and important for wall stabilization. In this study, we evaluated a stem cell media and a perivascular cell-specific media for the culture of primary perivascular cells regarding their cell morphology, doubling time, stem cell properties, and expression of cell type-specific markers. When the two cell culture media were compared to each other, perivascular cells cultured in the stem cell medium had a more elongated morphology and a faster doubling rate and cells cultured in the pericyte medium had a more typical morphology, with several filopodia, and a slower doubling rate. To evaluate stem cell properties, perivascular cells, CD146(-) cells, and mesenchymal stem cells (MSCs) were differentiated into the adipogenic, osteogenic, and chondrogenic lineages. It was seen that perivascular cells, as well as CD146(-) cells and MSCs, cultured in stem cell medium showed greater differentiation than cells cultured in pericyte-specific medium. The expression of pericyte-specific markers CD146, neural/glial antigen 2 (NG2), platelet-derived growth factor receptor-β (PDGFR-β), myosin, and α-smooth muscle actin (α-SMA) could be found in both pericyte cultures, as well as to varying amounts in CD146(-) cells, MSCs, and endothelial cells. The here presented work shows that perivascular cells can adapt to their in vitro environment and cell culture conditions influence cell functionality, such as doubling rate or differentiation behavior. Pericyte-specific markers were shown to be expressed also from cells other than perivascular cells. We can further conclude that CD146(+) perivascular cells are inhomogeneous cell population probably containing stem cell subpopulations, which are located perivascular around capillaries. © 2015 International Federation for Cell Biology.

Chemically defined serum-free conditions for cartilage regeneration from human embryonic stem cells.

PubMed

Yang, Dandan; Chen, Shubin; Gao, Changzhao; Liu, Xiaobo; Zhou, Yulai; Liu, Pengfei; Cai, Jinglei

2016-11-01

The aim of this study was to improve a method that induce cartilage differentiation of human embryoid stem cells (hESCs) in vitro, and test the effect of in vivo environments on the further maturation of hESCs derived cells. Embryoid bodies (EBs) formed from hESCs, with serum-free KSR-based medium and mesodermal specification related factors, CHIR, and Noggin for first 8days. Then cells were digested and cultured as micropellets in serum-free KSR-based chondrogenic medium that was supplemented with PDGF-BB, TGF β3, BMP4 in sequence for 24days. The morphology, FACS, histological staining as well as the expression of chondrogenic specific genes were detected in each stage, and further in vivo experiments, cell injections and tissue transplantations, further verified the formation of chondrocytes. We were able to obtain chondrocyte/cartilage from hESCs using serum-free KSR-based conditioned medium. qPCR analysis showed that expression of the chondroprogenitor genes and the chondrocyte/cartilage matrix genes. Morphology analysis demonstrated we got PG+COL2+COL1-particles. It indicated we obtained hyaline cartilage-like particles. 32-Day differential cells were injected subcutaneous. Staining results showed grafts developed further mature in vivo. But when transplanted in subrenal capsule, their effect was not good as in subcutaneous. Microenvironment might affect the cartilage formation. The results of this study provide an absolute serum-free and efficient approach for generation of hESC-derived chondrocytes, and cells will become further maturation in vivo. It provides evidence and technology for the hypothesis that hESCs may be a promising therapy for the treatment of cartilage disease. Copyright © 2016 Elsevier Inc. All rights reserved.

Suppressing Mesenchymal Stem Cell Hypertrophy and Endochondral Ossification in 3D Cartilage Regeneration with Nanofibrous Poly(l-Lactic Acid) Scaffold and Matrilin-3.

PubMed

Liu, Qihai; Wang, Jun; Chen, Yupeng; Zhang, Zhanpeng; Saunders, Laura; Schipani, Ernestina; Chen, Qian; Ma, Peter X

2018-06-22

Articular cartilage has a very limited ability to self-heal after injury or degeneration due to its low cellularity, poor proliferative activity, and avascular nature. Current clinical options are able to alleviate patient suffering, but cannot sufficiently regenerate the lost tissue. Biomimetic scaffolds that recapitulate the important features of the extracellular matrix (ECM) of cartilage are hypothesized to be advantageous in supporting cell growth, chondrogenic differentiation, and integration of regenerated cartilage with native cartilage, ultimately restoring the injured tissue to its normal function. It's a challenge to support and maintain articular cartilage regenerated by bone marrow-derived mesenchymal stem cells (BMSCs), which are prone to hypertrophy and endochondral ossification after implanted in vivo. In the present work, a nanofibrous poly(l-lactic acid) (NF PLLA) scaffold developed by our group was utilized because of the desired highly porous structure, high interconnectivity, collagen-like NF architecture to support rabbit BMSCs for articular cartilage regeneration. We further hypothesized that Matrilin-3 (MATN3), a non-collagenous, cartilage-specific ECM protein, would enhance the microenvironment of the NF PLLA scaffold for cartilage regeneration and maintaining its property. To test this hypothesis, we seeded BMSCs on the NF PLLA scaffold with or without MATN3. We found that MATN3 suppresses hypertrophy in this 3D culture system in vitro. Subcutaneous implantation of the chondrogenic cell/scaffold constructs in a nude mouse model showed that pretreatment with MATN3 was able to maintain chondrogenesis and prevent hypertrophy and endochondral ossification in vivo. These results demonstrate that the porous NF PLLA scaffold treated with MATN3 represents an advantageous 3D microenvironment for cartilage regeneration and phenotype maintenance, and is a promising strategy for articular cartilage repair. Articular cartilage defects, caused by trauma, inflammation, or joint instability, may ultimately lead to debilitating pain and disability. Bone marrow-derived mesenchymal stem cells (BMSCs) are an attractive cell source for articular cartilage tissue engineering. However, chondrogenic induction of BMSCs is often accompanied by undesired hypertrophy, which can lead to calcification and ultimately damage the cartilage. Therefore, a therapy to prevent hypertrophy and endochondral ossification is of paramount importance to adequately regenerate articular cartilage. We hypothesized that MATN3 (a non-collagenous ECM protein expressed exclusively in cartilage) may improve regeneration of articular cartilage with BMSCs by maintaining chondrogenesis and preventing hypertrophic transition in an ECM mimicking nanofibrous scaffold. Our results showed that the administration of MATN3 to the cell/nanofibrous scaffold constructs favorably maintained chondrogenesis and prevented hypertrophy/endochondral ossification in the chondrogenic constructs in vitro and in vivo. The combination of nanofibrous PLLA scaffolds and MATN3 treatment provides a very promising strategy to generate chondrogenic grafts with phenotypic stability for articular cartilage repair. Copyright © 2018. Published by Elsevier Ltd.

Ex vivo identification and characterization of a population of CD13(high) CD105(+) CD45(-) mesenchymal stem cells in human bone marrow.

PubMed

Muñiz, Carmen; Teodosio, Cristina; Mayado, Andrea; Amaral, Ana Teresa; Matarraz, Sergio; Bárcena, Paloma; Sanchez, Maria Luz; Alvarez-Twose, Iván; Diez-Campelo, María; García-Montero, Andrés C; Blanco, Juan F; Del Cañizo, Maria Consuelo; del Pino Montes, Javier; Orfao, Alberto

2015-09-07

Mesenchymal stem cells (MSCs) are multipotent cells capable of self-renewal and multilineage differentiation. Their multipotential capacity and immunomodulatory properties have led to an increasing interest in their biological properties and therapeutic applications. Currently, the definition of MSCs relies on a combination of phenotypic, morphological and functional characteristics which are typically evaluated upon in vitro expansion, a process that may ultimately lead to modulation of the immunophenotypic, functional and/or genetic features of these cells. Therefore, at present there is great interest in providing markers and phenotypes for direct in vivo and ex vivo identification and isolation of MSCs. Multiparameter flow cytometry immunophenotypic studies were performed on 65 bone marrow (BM) samples for characterization of CD13(high) CD105(+) CD45(-) cells. Isolation and expansion of these cells was performed in a subset of samples in parallel to the expansion of MSCs from mononuclear cells following currently established procedures. The protein expression profile of these cells was further assessed on (paired) primary and in vitro expanded BM MSCs, and their adipogenic, chondrogenic and osteogenic differentiation potential was also determined. Our results show that the CD13(high) CD105(+) CD45(-) immunophenotype defines a minor subset of cells that are systematically present ex vivo in normal/reactive BM (n = 65) and that display immunophenotypic features, plastic adherence ability, and osteogenic, adipogenic and chondrogenic differentiation capacities fully compatible with those of MSCs. In addition, we also show that in vitro expansion of these cells modulates their immunophenotypic characteristics, including changes in the expression of markers currently used for the definition of MSCs, such as CD105, CD146 and HLA-DR. BM MSCs can be identified ex vivo in normal/reactive BM, based on a robust CD13(high) CD105(+) and CD45(-) immunophenotypic profile. Furthermore, in vitro expansion of these cells is associated with significant changes in the immunophenotypic profile of MSCs.

The influence of hyaluronic acid hydrogel crosslinking density and macromolecular diffusivity on human MSC chondrogenesis and hypertrophy.

PubMed

Bian, Liming; Hou, Chieh; Tous, Elena; Rai, Reena; Mauck, Robert L; Burdick, Jason A

2013-01-01

Hyaluronic acid (HA) hydrogels formed via photocrosslinking provide stable 3D hydrogel environments that support the chondrogenesis of mesenchymal stem cells (MSCs). Crosslinking density has a significant impact on the physical properties of hydrogels, including their mechanical stiffness and macromolecular diffusivity. Variations in the HA hydrogel crosslinking density can be obtained by either changes in the HA macromer concentration (1, 3, or 5% w/v at 15 min exposure) or the extent of reaction through light exposure time (5% w/v at 5, 10, or 15 min). In this work, increased crosslinking by either method resulted in an overall decrease in cartilage matrix content and more restricted matrix distribution. Increased crosslinking also promoted hypertrophic differentiation of the chondrogenically induced MSCs, resulting in more matrix calcification in vitro. For example, type X collagen expression in the high crosslinking density 5% 15 min group was ~156 and 285% higher when compared to the low crosslinking density 1% 15 min and 5% 5 min groups on day 42, respectively. Supplementation with inhibitors of the small GTPase pathway involved in cytoskeletal tension or myosin II had no effect on hypertrophic differentiation and matrix calcification, indicating that the differential response is unlikely to be related to force-sensing mechanotransduction mechanisms. When implanted subcutaneously in nude mice, higher crosslinking density again resulted in reduced cartilage matrix content, restricted matrix distribution, and increased matrix calcification. This study demonstrates that hydrogel properties mediated through alterations in crosslinking density must be considered in the context of the hypertrophic differentiation of chondrogenically induced MSCs. Copyright © 2012 Elsevier Ltd. All rights reserved.

Adipose-derived mesenchymal stem cells for cartilage tissue engineering: state-of-the-art in in vivo studies.

PubMed

Veronesi, Francesca; Maglio, Melania; Tschon, Matilde; Aldini, Nicolò Nicoli; Fini, Milena

2014-07-01

Several therapeutic approaches have been developed to address hyaline cartilage regeneration, but to date, there is no universal procedure to promote the restoration of mechanical and functional properties of native cartilage, which is one of the most important challenges in orthopedic surgery. For cartilage tissue engineering, adult mesenchymal stem cells (MSCs) are considered as an alternative cell source to chondrocytes. Since little is known about adipose-derived mesenchymal stem cell (ADSC) cartilage regeneration potential, the aim of this review was to give an overview of in vivo studies about the chondrogenic potential and regeneration ability of culture-expanded ADSCs when implanted in heterotopic sites or in osteoarthritic and osteochondral defects. The review compares the different studies in terms of number of implanted cells and animals, cell harvesting sites, in vitro expansion and chondrogenic induction conditions, length of experimental time, defect dimensions, used scaffolds and post-explant analyses of the cartilage regeneration. Despite variability of the in vivo protocols, it seems that good cartilage formation and regeneration were obtained with chondrogenically predifferentiated ADSCs (1 × 10(7) cells for heterotopic cartilage formation and 1 × 10(6) cells/scaffold for cartilage defect regeneration) and polymeric scaffolds, even if many other aspects need to be clarified in future studies. © 2013 Wiley Periodicals, Inc.

Comparison of potentials between stem cells isolated from human anterior cruciate ligament and bone marrow for ligament tissue engineering.

PubMed

Cheng, Ming-Te; Liu, Chien-Lin; Chen, Tain-Hsiung; Lee, Oscar K

2010-07-01

We have previously isolated and identified stem cells from human anterior cruciate ligament (ACL). The purpose of this study was to evaluate the differences in proliferation, differentiation, and extracellular matrix (ECM) formation abilities between bone marrow stem cells (BMSCs) and ACL-derived stem cells (LSCs) from the same donors when cultured with different growth factors, including basic fibroblast growth factor (bFGF), epidermal growth factor, and transforming growth factor-beta 1 (TGF-beta1). Ligament tissues and bone marrow aspirate were obtained from patients undergoing total knee arthroplasty and ACL reconstruction surgeries. Proliferation, colony formation, and population doubling capacity as well as multilineage differentiation potentials of LSCs and BMSCs were compared. Gene expression and ECM production for ligament engineering were also evaluated. It was found that BMSCs possessed better osteogenic differentiation potential than LSCs, while similar adipogenic and chondrogenic differentiation abilities were observed. Proliferation rates of both LSCs and BMSCs were enhanced by bFGF and TGF-beta1. TGF-beta1 treatment significantly increased the expression of type I collagen, type III collagen, fibronectin, and alpha-smooth muscle actin in LSCs, but TGF-beta1 only upregulated type I collagen and tenascin-c in BMSCs. Protein quantification further confirmed the results of differential gene expression and suggested that LSCs and BMSCs increase ECM production upon TGF-beta1 treatment. In summary, in comparison with BMSCs, LSCs proliferate faster and maintain an undifferentiated state with bFGF treatment, whereas under TGF-beta1 treatment, LSCs upregulate major tendinous gene expression and produce a robust amount of ligament ECM protein, making LSCs a potential cell source in future applications of ACL tissue engineering.

Isolation of canine mesenchymal stem cells from amniotic fluid and differentiation into hepatocyte-like cells.

PubMed

Choi, Seon-A; Choi, Hoon-Sung; Kim, Keun Jung; Lee, Dong-Soo; Lee, Ji Hey; Park, Jie Yeun; Kim, Eun Young; Li, Xiaoxia; Oh, Hyun-Yang; Lee, Dong-Seok; Kim, Min Kyu

2013-01-01

Recent findings have demonstrated that amniotic fluid cells are an interesting and potential source of mesenchymal stem cells (MSCs). In this study, we isolated MSCs from canine amniotic fluid and then characterized their multilineage differentiation ability. Canine amniotic fluid stem (cAFS) cells at passage 5 had a fibroblast-like morphology instead of forming colonies and were positive for pluripotent stem cell markers such as OCT4, NANOG, and SOX2. Flow cytometry analysis showed the expression of MSC surface markers CD44, CD29, and CD90 on the cAFS cells. In addition, these cells were cultured under conditions favorable for adipogenic, chondrogenic, and osteogenic induction. The results of this experiment confirmed the mesenchymal nature of cAFS cells and their multipotent potential. Interestingly, although the cells exhibited a fibroblast-like morphology after hepatogenic induction, reverse transcription-polymerase chain reaction revealed that the expression of several hepatic genes, such as albumin, tyrosine aminotransferase, and alpha-1 antiproteinase, increased following maturation and differentiation. These findings indicated that cAFS cells have functional properties similar to those of hepatocytes. Taken together, the results of our study demonstrated that cAFS cells with mesenchymal characteristics can be successfully isolated from canine amniotic fluid and possess functional properties characteristic of hepatocytes. The findings of our work suggest that cAFS cells have the potential to be a resource for cell-based therapies in a canine model of hepatic disease.

Potential role of herbal remedies in stem cell therapy: proliferation and differentiation of human mesenchymal stromal cells.

PubMed

Udalamaththa, Vindya Lankika; Jayasinghe, Chanika Dilumi; Udagama, Preethi Vidya

2016-08-11

Stem cell therapy has revolutionized modern clinical therapy with the potential of stem cells to differentiate into many different cell types which may help to replace different cell lines of an organism. Innumerous trials are carried out to merge new scientific knowledge and techniques with traditional herbal extracts that may result in less toxic, affordable, and highly available natural alternative therapeutics. Currently, mesenchyamal stromal cell (MSC) lines are treated with individual and mixtures of crude herbal extracts, as well as with purified compounds from herbal extracts, to investigate the mechanisms and effects of these on stem cell growth and differentiation. Human MSCs (hMSCs) possess multilineage, i.e., osteogenic, neurogenic, adipogenic, chondrogenic, and myogenic, differentiation abilities. The proliferative and differentiation properties of hMSCs treated with herbal extracts have shown promise in diseases such as osteoporosis, neurodegenerative disorders, and other tissue degenerative disorders. Well characterized herbal extracts that result in increased rates of tissue regeneration may be used in both stem cell therapy and tissue engineering for replacement therapy, where the use of scaffolds and vesicles with enhanced attaching and proliferative properties could be highly advantageous in the latter. Although the clinical application of herbal extracts is still in progress due to the variability and complexity of bioactive constituents, standardized herbal preparations will strengthen their application in the clinical context. We have critically reviewed the proliferative and differentiation effects of individual herbal extracts on hMSCs mainly derived from bone marrow and elaborated on the plausible underlying mechanisms of action. To be fruitfully used in reparative and regenerative therapy, future directions in this area of study should (i) make use of hMSCs derived from different non-traditional sources, including medical waste material (umbilical cord, Wharton's jelly, and placenta), (ii) take account of the vast numbers of herbal extracts used in traditional medicine globally, and (iii) investigate the mechanisms and pathways of their effects on hMSCs.

In Vivo Exposure to Inorganic Arsenic Alters Differentiation-Specific Gene Expression of Adipose-Derived Mesenchymal Stem/Stromal Cells in C57BL/6J Mouse Model

PubMed Central

Shearer, Joseph J.; Figueiredo Neto, Manoel; Umbaugh, C. Samuel; Figueiredo, Marxa L.

2017-01-01

Abstract The number of mesenchymal stem cell (MSC) therapeutic modalities has grown in recent years. Adipose-derived mesenchymal stem/stromal cells (ASCs) can be isolated and expanded relatively easily as compared with their bone-marrow counterparts, making them a particularly promising source of MSCs. And although the biological mechanisms surrounding ASCs are actively being investigated, little is known about the effects that in vivo environmental exposures might have on their ability to properly differentiate. Therefore, we hypothesized that ASCs isolated from mice exposed to inorganic arsenic (iAs) would have an altered response towards adipogenic, osteogenic, and/or chondrogenic differentiation. To test this hypothesis, C57BL/6J male mice were provided drinking water containing 0, 300, or 1000 ppb iAs. ASCs were then isolated and differentiated, which was assessed by immunocytochemistry and real-time quantitative PCR (RT-qPCR). Our results showed that total urinary arsenic equilibrated within 1 week of exposure to iAs and was maintained throughout the study. ASCs isolated from each exposure group maintained differentiation capabilities for each lineage. The magnitude of differentiation-specific gene expression, however, appeared to be concentration dependent. For osteogenesis and chondrogenesis, differentiation-specific gene expression decreased, whereas adipogenesis showed a biphasic response with an initial decrease followed by an increase in adipogenic-related gene expression following iAs exposure. These results suggest that the level in which differentiation-specific genes are induced within these stromal cells might be sensitive to environmental contaminants. These findings highlight the need to take into account potential environmental exposures prior to selecting stromal cell donors, so ASCs can achieve optimal efficiency in regenerative therapy applications. PMID:28206643

Purinergic Signaling Regulates the Transforming Growth Factor-β3-Induced Chondrogenic Response of Mesenchymal Stem Cells to Hydrostatic Pressure.

PubMed

Steward, Andrew J; Kelly, Daniel J; Wagner, Diane R

2016-06-01

Although hydrostatic pressure (HP) is known to regulate chondrogenic differentiation of mesenchymal stromal/stem cells (MSCs), improved insight into the mechanotransduction of HP may form the basis for novel tissue engineering strategies. Previously, we demonstrated that matrix stiffness and calcium ion (Ca(++)) mobility regulate the mechanotransduction of HP; however, the mechanisms, by which these Ca(++) signaling pathways are initiated, are currently unknown. The purinergic pathway, in which adenosine triphosphate (ATP) is released and activates P-receptors to initiate Ca(++) signaling, plays a key role in the mechanotransduction of compression, but has yet to be investigated with regard to HP. Therefore, the objective of this study was to investigate the interplay between purinergic signaling, matrix stiffness, and the chondrogenic response of MSCs to HP. Porcine bone marrow-derived MSCs were seeded into soft or stiff agarose hydrogels and subjected to HP (10 MPa at 1 Hz for 4 h/d for 21 days) or kept in free swelling conditions. Stiff constructs were incubated with pharmacological inhibitors of extracellular ATP, P2 receptors, or hemichannels, or without any inhibitors as a control. As with other loading modalities, HP significantly increased ATP release in the control group; however, inhibition of hemichannels completely abrogated this response. The increase in sulfated glycosaminoglycan (sGAG) synthesis and vimentin reorganization observed in the control group in response to HP was suppressed in the presence of all three inhibitors, suggesting that purinergic signaling is involved in the mechanoresponse of MSCs to HP. Interestingly, ATP was released from both soft and stiff hydrogels in response to HP, but HP only enhanced chondrogenesis in the stiff hydrogels, indicating that matrix stiffness may act downstream of purinergic signaling to regulate the mechanoresponse of MSCs to HP. Addition of exogenous ATP did not replicate the effects of HP on chondrogenesis, suggesting that mechanisms other than purinergic signaling also regulate the response of MSCs to HP.

Macromolecular crowding meets oxygen tension in human mesenchymal stem cell culture - A step closer to physiologically relevant in vitro organogenesis

NASA Astrophysics Data System (ADS)

Cigognini, Daniela; Gaspar, Diana; Kumar, Pramod; Satyam, Abhigyan; Alagesan, Senthilkumar; Sanz-Nogués, Clara; Griffin, Matthew; O'Brien, Timothy; Pandit, Abhay; Zeugolis, Dimitrios I.

2016-08-01

Modular tissue engineering is based on the cells’ innate ability to create bottom-up supramolecular assemblies with efficiency and efficacy still unmatched by man-made devices. Although the regenerative potential of such tissue substitutes has been documented in preclinical and clinical setting, the prolonged culture time required to develop an implantable device is associated with phenotypic drift and/or cell senescence. Herein, we demonstrate that macromolecular crowding significantly enhances extracellular matrix deposition in human bone marrow mesenchymal stem cell culture at both 20% and 2% oxygen tension. Although hypoxia inducible factor - 1α was activated at 2% oxygen tension, increased extracellular matrix synthesis was not observed. The expression of surface markers and transcription factors was not affected as a function of oxygen tension and macromolecular crowding. The multilineage potential was also maintained, albeit adipogenic differentiation was significantly reduced in low oxygen tension cultures, chondrogenic differentiation was significantly increased in macromolecularly crowded cultures and osteogenic differentiation was not affected as a function of oxygen tension and macromolecular crowding. Collectively, these data pave the way for the development of bottom-up tissue equivalents based on physiologically relevant developmental processes.

Macromolecular crowding meets oxygen tension in human mesenchymal stem cell culture - A step closer to physiologically relevant in vitro organogenesis

PubMed Central

Cigognini, Daniela; Gaspar, Diana; Kumar, Pramod; Satyam, Abhigyan; Alagesan, Senthilkumar; Sanz-Nogués, Clara; Griffin, Matthew; O’Brien, Timothy; Pandit, Abhay; Zeugolis, Dimitrios I.

2016-01-01

Modular tissue engineering is based on the cells’ innate ability to create bottom-up supramolecular assemblies with efficiency and efficacy still unmatched by man-made devices. Although the regenerative potential of such tissue substitutes has been documented in preclinical and clinical setting, the prolonged culture time required to develop an implantable device is associated with phenotypic drift and/or cell senescence. Herein, we demonstrate that macromolecular crowding significantly enhances extracellular matrix deposition in human bone marrow mesenchymal stem cell culture at both 20% and 2% oxygen tension. Although hypoxia inducible factor - 1α was activated at 2% oxygen tension, increased extracellular matrix synthesis was not observed. The expression of surface markers and transcription factors was not affected as a function of oxygen tension and macromolecular crowding. The multilineage potential was also maintained, albeit adipogenic differentiation was significantly reduced in low oxygen tension cultures, chondrogenic differentiation was significantly increased in macromolecularly crowded cultures and osteogenic differentiation was not affected as a function of oxygen tension and macromolecular crowding. Collectively, these data pave the way for the development of bottom-up tissue equivalents based on physiologically relevant developmental processes. PMID:27478033

Interactions of Human Endothelial and Multipotent Mesenchymal Stem Cells in Cocultures

PubMed Central

Ern, Christina; Krump-Konvalinkova, Vera; Docheva, Denitsa; Schindler, Stefanie; Rossmann, Oliver; Böcker, Wolfgang; Mutschler, Wolf; Schieker, Matthias

2010-01-01

Current strategies for tissue engineering of bone rely on the implantation of scaffolds, colonized with human mesenchymal stem cells (hMSC), into a recipient. A major limitation is the lack of blood vessels. One approach to enhance the scaffold vascularisation is to supply the scaffolds with endothelial cells (EC). The main goal of this study was to establish a coculture system of hMSC and EC for the purposes of bone tissue engineering. Therefore, the cell behaviour, proliferation and differentiation capacity in various cell culture media as well as cell interactions in the cocultures were evaluated. The differentiation capacity of hMSC along osteogenic, chondrogenic, and adipogenic lineage was impaired in EC medium while in a mixed EC and hMSC media, hMSC maintained osteogenic differentiation. In order to identify and trace EC in the cocultures, EC were transduced with eGFP. Using time-lapse imaging, we observed that hMSC and EC actively migrated towards cells of their own type and formed separate clusters in long term cocultures. The scarcity of hMSC and EC contacts in the cocultures suggest the influence of growth factor-mediated cell interactions and points to the necessity of further optimization of the coculture conditions. PMID:21625373

Chondrogenic Differentiation of Mesenchymal Stem Cells: Challenges and Unfulfilled Expectations

PubMed Central

Somoza, Rodrigo A.; Welter, Jean F.; Correa, Diego

2014-01-01

Articular cartilage repair and regeneration provides a substantial challenge in Regenerative Medicine because of the high degree of morphological and mechanical complexity intrinsic to hyaline cartilage due, in part, to its extracellular matrix. Cartilage remains one of the most difficult tissues to heal; even state-of-the-art regenerative medicine technology cannot yet provide authentic cartilage resurfacing. Mesenchymal stem cells (MSCs) were once believed to be the panacea for cartilage repair and regeneration, but despite years of research, they have not fulfilled these expectations. It has been observed that MSCs have an intrinsic differentiation program reminiscent of endochondral bone formation, which they follow after exposure to specific reagents as a part of current differentiation protocols. Efforts have been made to avoid the resulting hypertrophic fate of MSCs; however, so far, none of these has recreated a fully functional articular hyaline cartilage without chondrocytes exhibiting a hypertrophic phenotype. We reviewed the current literature in an attempt to understand why MSCs have failed to regenerate articular cartilage. The challenges that must be overcome before MSC-based tissue engineering can become a front-line technology for successful articular cartilage regeneration are highlighted. PMID:24749845

Reduced Differentiation Efficiency of Murine Embryonic Stem Cells in Stirred Suspension Bioreactors

PubMed Central

Taiani, Jaymi T.; Krawetz, Roman J.; zur Nieden, Nicole I.; Wu, Yiru Elizabeth; Kallos, Michael S.; Matyas, John R.

2010-01-01

The use of embryonic stem cells (ESCs) for regenerative medicine has generated increased attention due to the favorable attributes of these cells; namely, they are pluripotent and possess long-term self-renewal capacity. The initial aims of the present study were: (i) to use stirred suspension bioreactors to expand and differentiate ESCs into osteogenic and chondrogenic cell types and (ii) to explore if these ESC-derived cells influenced skeletal healing in an in vivo fracture model. We show that differentiation protocols used in static culture are insufficient when applied directly to suspension culture bioreactors. Moreover, when bioreactor-differentiated cells are transplanted into a burr-hole defect in bone, severe disruption of the bone architecture was noted at the fracture site, as determined by microcomputed tomography (microCT) imaging and histopathology. Further characterization of the bioreactor-differentiated cultures revealed that a subpopulation of cells in the resulting aggregates expressed the pluripotency marker Oct-4 in the nucleus. Nuclear Oct-4 expression persisted even after 30 days of culture in the absence of leukemia inhibitory factor (LIF). Remarkably, and unlike ESCs differentiated into skeletal cell types in static cultures, bioreactor-differentiated aggregates implanted subcutaneously into SCID mice formed teratomas. The development of effective ESC differentiation protocols for suspension bioreactors will require a more complete understanding of the environmental conditions within these culture systems and the influence that these conditions have on the regulation of pluripotency and differentiation in ESCs. PMID:19775198

Animal serum-free expansion and differentiation of human mesenchymal stromal cells.

PubMed

Felka, Tino; Schäfer, Richard; De Zwart, Peter; Aicher, Wilhelm K

2010-04-01

Mesenchymal stromal cells (MSC) are attracting increasing interest for possible application in cell therapies. Fetal calf serum (FCS) is widely utilized for cell culture, but its use in the context of clinical applications is associated with too many risks. Therefore we tested FCS-free media for the expansion and differentiation of MSC in compliance with the European good manufacturing practice (GMP) regulations for medicinal products. MSC expansion medium was modified by replacing FCS with human plasma and platelet extract. Cells were characterized according to the defined minimal criteria for multipotent MSC. For chondrogenic differentiation, serum-free micromass cultures were employed. For adipogenic and osteogenic differentiation, the FCS was replaced by human plasma. After 28 days of incubation in differentiation media, cells were analyzed by cytochemical and immunohistochemical staining. Furthermore, mRNA expression of chondrogenic, adipogenic and osteogenic markers was investigated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Expansion and differentiation of MSC under FCS-free conditions yielded cells with chondrogenic, adipogenic and osteogenic phenotypes and a characteristic gene expression. Chondrocytes in micromass pellets revealed an accumulation of proteoglycans and type II collagen as well as a significantly increased mRNA expression of chondrogenic marker genes. The adipocytes displayed Oil red O staining and expressed peroxisome proliferator-activated receptor gamma(2) (ppARgamma2) and lipoprotein lipase (LPL) mRNA. The osteoblasts were positive for von Kossa staining and expressed mRNA of osteogenic marker genes. The results did not indicate any spontaneous differentiation. Human plasma is a suitable FCS replacement for the expansion and differentiation of MSC, providing a feasible alternative for tissue engineering with GMP-compatible protocols.

Early induction of a prechondrogenic population allows efficient generation of stable chondrocytes from human induced pluripotent stem cells.

PubMed

Lee, Jieun; Taylor, Sarah E B; Smeriglio, Piera; Lai, Janice; Maloney, William J; Yang, Fan; Bhutani, Nidhi

2015-08-01

Regeneration of human cartilage is inherently inefficient; an abundant autologous source, such as human induced pluripotent stem cells (hiPSCs), is therefore attractive for engineering cartilage. We report a growth factor-based protocol for differentiating hiPSCs into articular-like chondrocytes (hiChondrocytes) within 2 weeks, with an overall efficiency >90%. The hiChondrocytes are stable and comparable to adult articular chondrocytes in global gene expression, extracellular matrix production, and ability to generate cartilage tissue in vitro and in immune-deficient mice. Molecular characterization identified an early SRY (sex-determining region Y) box (Sox)9(low) cluster of differentiation (CD)44(low)CD140(low) prechondrogenic population during hiPSC differentiation. In addition, 2 distinct Sox9-regulated gene networks were identified in the Sox9(low) and Sox9(high) populations providing novel molecular insights into chondrogenic fate commitment and differentiation. Our findings present a favorable method for generating hiPSC-derived articular-like chondrocytes. The hiChondrocytes are an attractive cell source for cartilage engineering because of their abundance, autologous nature, and potential to generate articular-like cartilage rather than fibrocartilage. In addition, hiChondrocytes can be excellent tools for modeling human musculoskeletal diseases in a dish and for rapid drug screening. © FASEB.

Characterization of Mesenchymal Stem Cell-Like Cells Derived From Human iPSCs via Neural Crest Development and Their Application for Osteochondral Repair

PubMed Central

Ikeya, Makoto; Yasui, Yukihiko; Ikeda, Yasutoshi; Ebina, Kosuke; Moriguchi, Yu; Shimomura, Kazunori; Hideki, Yoshikawa

2017-01-01

Mesenchymal stem cells (MSCs) derived from induced pluripotent stem cells (iPSCs) are a promising cell source for the repair of skeletal disorders. Recently, neural crest cells (NCCs) were reported to be effective for inducing mesenchymal progenitors, which have potential to differentiate into osteochondral lineages. Our aim was to investigate the feasibility of MSC-like cells originated from iPSCs via NCCs for osteochondral repair. Initially, MSC-like cells derived from iPSC-NCCs (iNCCs) were generated and characterized in vitro. These iNCC-derived MSC-like cells (iNCMSCs) exhibited a homogenous population and potential for osteochondral differentiation. No upregulation of pluripotent markers was detected during culture. Second, we implanted iNCMSC-derived tissue-engineered constructs into rat osteochondral defects without any preinduction for specific differentiation lineages. The implanted cells remained alive at the implanted site, whereas they failed to repair the defects, with only scarce development of osteochondral tissue in vivo. With regard to tumorigenesis, the implanted cells gradually disappeared and no malignant cells were detected throughout the 2-month follow-up. While this study did not show that iNCMSCs have efficacy for repair of osteochondral defects when implanted under undifferentiated conditions, iNCMSCs exhibited good chondrogenic potential in vitro under appropriate conditions. With further optimization, iNCMSCs may be a new source for tissue engineering of cartilage. PMID:28607560

Microenvironment is involved in cellular response to hydrostatic pressures during chondrogenesis of mesenchymal stem cells.

PubMed

Ye, Rui; Hao, Jin; Song, Jinlin; Zhao, Zhihe; Fang, Shanbao; Wang, Yating; Li, Juan

2014-06-01

Chondrocytes integrate numerous microenvironmental cues to mount physiologically relevant differentiation responses, and the regulation of mechanical signaling in chondrogenic differentiation is now coming into intensive focus. To facilitate tissue-engineered chondrogenesis by mechanical strategy, a thorough understanding about the interactional roles of chemical factors under mechanical stimuli in regulating chondrogenesis is in great need. Therefore, this study attempts to investigate the interaction of rat MSCs with their microenvironment by imposing dynamic and static hydrostatic pressure through modulating gaseous tension above the culture medium. Under dynamic pressure, chemical parameters (pH, pO2, and pCO2) were kept in homeostasis. In contrast, pH was remarkably reduced due to increased pCO2 under static pressure. MSCs under the dynamically pressured microenvironment exhibited a strong accumulation of GAG within and outside the alginate beads, while cells under the statically pressured environment lost newly synthesized GAG into the medium with a speed higher than its production. In addition, the synergic influence on expression of chondrogenic genes was more persistent under dynamic pressure than that under static pressure. This temporal contrast was similar to that of activation of endogenous TGF-β1. Taken altogether, it indicates that a loading strategy which can keep a homeostatic chemical microenvironment is preferred, since it might sustain the stimulatory effects of mechanical stimuli on chondrogenesis via activation of endogenous TGF-β1. © 2013 Wiley Periodicals, Inc.

Matrilin-3 codelivery with adipose-derived mesenchymal stem cells promotes articular cartilage regeneration in a rat osteochondral defect model.

PubMed

Muttigi, Manjunatha S; Kim, Byoung Ju; Choi, Bogyu; Yoshie, Arai; Kumar, Hemant; Han, Inbo; Park, Hansoo; Lee, Soo-Hong

2018-03-01

Matrilin-3 is an essential extracellular matrix component present only in cartilaginous tissues. Matrilin-3 exerts chondroprotective effects by regulating an anti-inflammatory function and extracellular matrix components. We hypothesized that the codelivery of matrilin-3 with infrapatellar adipose-tissue-derived mesenchymal stem cells (Ad-MSCs) may enhance articular cartilage regeneration. Matrilin-3 treatment of Ad-MSCs in serum-free media induced collagen II and aggrecan expression, and matrilin-3 in chondrogenic media also enhanced in vitro chondrogenic differentiation. Next, the in vivo effect of matrilin-3 codelivery with Ad-MSCs on cartilage regeneration was assessed in an osteochondral defect model in Sprague Dawley rats: Ad-MSCs and hyaluronic acid were implanted at the defect site with or without matrilin-3 (140, 280, and 700 ng). Safranin O staining revealed that matrilin-3 (140 and 280 ng) treatment significantly improved cartilage regeneration and glycosaminoglycan accumulation. In the animals treated with 140-ng matrilin-3, in particular, the defect site exhibited complete integration with surrounding tissue and a smooth glistening surface. The International Cartilage Repair Society macroscopic and O'Driscoll microscopic scores for regenerated cartilage were furthermore shown to be considerably higher for this group (matrilin-3; 140 ng) compared with the other groups. Furthermore, the defects treated with 140-ng matrilin-3 revealed significant hyaline-like cartilage regeneration in the osteochondral defect model; in contrast, the defects treated with 700-ng matrilin-3 exhibited drastically reduced cartilage regeneration with mixed hyaline-fibrocartilage morphology. Codelivery of matrilin-3 with Ad-MSCs significantly influenced articular cartilage regeneration, supporting the potential use of this tissue-specific protein for a cartilage-targeted stem cell therapy. Copyright © 2017 John Wiley & Sons, Ltd.

Chondrogenesis of Human Bone Marrow Mesenchymal Stem Cells in 3-Dimensional, Photocrosslinked Hydrogel Constructs: Effect of Cell Seeding Density and Material Stiffness

PubMed Central

Sun, Aaron X.; Lin, Hang; Fritch, Madalyn R.; Shen, He; Alexander, Pete G.; DeHart, Michael; Tuan, Rocky S.

2018-01-01

Three-dimensional hydrogel constructs incorporated with live stem cells that support chondrogenic differentiation and maintenance offer a promising regenerative route towards addressing the limited self-repair capabilities of articular cartilage. In particular, hydrogel scaffolds that augment chondrogenesis and recapitulate the native physical properties of cartilage, such as compressive strength, can potentially be applied in point-of-care procedures. We report here the synthesis of two new materials, [poly-L-lactic acid/polyethylene glycol/poly-L-lactic acid] (PLLA-PEG 1000) and [poly-D,L-lactic acid/polyethylene glycol/poly-D,L-lactic acid] (PDLLA-PEG 1000), that are biodegradable, biocompatible (>80% viability post fabrication), and possess high, physiologically relevant mechanical strength (~1,500 to 1,800 kPa). This study examined the effects of physiologically relevant cell densities (4, 8, 20, and 50 × 106/mL) and hydrogel stiffnesses (~150kPa to ~1,500 kPa Young’s moduli) on chondrogenesis of human bone marrow stem cells incorporated in hydrogel constructs fabricated with these materials and a previously characterized PDLLA-PEG 4000. Results showed that 20 × 106 cells/mL, under a static culture condition, was the most efficient cell seeding density for extracellular matrix (ECM) production on the basis of hydroxyproline and glycosaminoglycan content. Interestingly, material stiffness did not significantly affect chondrogenesis, but rather material concentration was correlated to chondrogenesis with increasing levels at lower concentrations based on ECM production, chondrogenic gene expression, and histological analysis. These findings establish optimal cell densities for chondrogenesis within three-dimensional cell-incorporated hydrogels, inform hydrogel material development for cartilage tissue engineering, and demonstrate the efficacy and potential utility of PDLLA-PEG 1000 for point-of-care treatment of cartilage defects. PMID:28611002

Pretreatment with IL-1β enhances proliferation and chondrogenic potential of synovium-derived mesenchymal stem cells.

PubMed

Matsumura, Etsuko; Tsuji, Kunikazu; Komori, Keiichiro; Koga, Hideyuki; Sekiya, Ichiro; Muneta, Takeshi

2017-02-01

Synovial mesenchymal stem cells (MSCs) are an attractive cell source for cartilage regeneration because of their high proliferative ability and chondrogenic potential. We have performed clinical trials using synovial MSCs to regenerate articular cartilage. To achieve good clinical outcomes for cell transplantation therapy, it is important to control both quantity (cell number) and quality (pluripotency or chondrogenic potential) of the cells for transplantation. Interleukin (IL)-1β is a pro-inflammatory cytokine with significant pro-proliferative potential for mesenchymal cells. However, the effects of IL-1β on synovial MSCs remain unknown. We investigated the effects of pretreatment with IL-1β on synovial MSCs. Human synovial tissue was harvested during total knee arthroplasty. Nucleated cells were plated and cultured in the absence or presence of IL-1β at 10 -13 , 10 -12 , 10 -11 , 10 -10 , 10 -9 or 10 -8  g/mL for 14 days. The number of synovial MSCs increased in a concentration-dependent manner. When cultured for 21 days in chondrogenic medium after pretreatment with 10 -8   g/mL IL-1β, pellet aggregation was observed, whereas pretreatment with 10 -12 , 10 -11 or 10 -10  g/mL IL-1β significantly increased the weight of cartilage pellets (P <0.01). Surface markers for adhesion ability and pluripotency were reduced with high concentrations of IL-1β. IL-6 and IL-8 expression increased, but no changes in the expression level of growth factors were indicated by cytokine array. We have demonstrated that pretreatment of IL-1β increased the proliferation and chondrogenic potential of synovial MSCs, which may promote the regenerative potential of synovial MSCs. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Articular cartilage-derived cells hold a strong osteogenic differentiation potential in comparison to mesenchymal stem cells in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Salamon, Achim, E-mail: achim.salamon@med.uni-rostock.de; Jonitz-Heincke, Anika, E-mail: anika.jonitz@med.uni-rostock.de; Adam, Stefanie, E-mail: stefanie.adam@med.uni-rostock.de

Cartilaginous matrix-degenerative diseases like osteoarthritis (OA) are characterized by gradual cartilage erosion, and also by increased presence of cells with mesenchymal stem cell (MSC) character within the affected tissues. Moreover, primary chondrocytes long since are known to de-differentiate in vitro and to be chondrogenically re-differentiable. Since both findings appear to conflict with each other, we quantitatively assessed the mesenchymal differentiation potential of OA patient cartilage-derived cells (CDC) towards the osteogenic and adipogenic lineage in vitro and compared it to that of MSC isolated from adipose tissue (adMSC) of healthy donors. We analyzed expression of MSC markers CD29, CD44, CD105, andmore » CD166, and, following osteogenic and adipogenic induction in vitro, quantified their expression of osteogenic and adipogenic differentiation markers. Furthermore, CDC phenotype and proliferation were monitored. We found that CDC exhibit an MSC CD marker expression pattern similar to adMSC and a similar increase in proliferation rate during osteogenic differentiation. In contrast, the marked reduction of proliferation observed during adipogenic differentiation of adMSC was absent in CDC. Quantification of differentiation markers revealed a strong osteogenic differentiation potential for CDC, however almost no capacity for adipogenic differentiation. Since in the pathogenesis of OA, cartilage degeneration coincides with high bone turnover rates, the high osteogenic differentiation potential of OA patient-derived CDC may affect clinical therapeutic regimens aiming at autologous cartilage regeneration in these patients. - Highlights: • We analyze the mesenchymal differentiation capacity of cartilage-derived cells (CDC). • CDC express mesenchymal stem cell (MSC) markers CD29, CD44, CD105, and CD166. • CDC and MSC proliferation is reduced in adipogenesis and increased in osteogenesis. • Adipogenic differentiation is virtually absent in CDC, but strong in MSC. • Osteogenic differentiation is significantly stronger for CDC than for MSC.« less

PRMT5 is essential for the maintenance of chondrogenic progenitor cells in the limb bud

PubMed Central

Norrie, Jacqueline L.; Li, Qiang; Co, Swanie; Huang, Bau-Lin; Ding, Ding; Uy, Jann C.; Ji, Zhicheng; Mackem, Susan; Bedford, Mark T.; Galli, Antonella; Ji, Hongkai

2016-01-01

During embryonic development, undifferentiated progenitor cells balance the generation of additional progenitor cells with differentiation. Within the developing limb, cartilage cells differentiate from mesodermal progenitors in an ordered process that results in the specification of the correct number of appropriately sized skeletal elements. The internal pathways by which these cells maintain an undifferentiated state while preserving their capacity to differentiate is unknown. Here, we report that the arginine methyltransferase PRMT5 has a crucial role in maintaining progenitor cells. Mouse embryonic buds lacking PRMT5 have severely truncated bones with wispy digits lacking joints. This novel phenotype is caused by widespread cell death that includes mesodermal progenitor cells that have begun to precociously differentiate into cartilage cells. We propose that PRMT5 maintains progenitor cells through its regulation of Bmp4. Intriguingly, adult and embryonic stem cells also require PRMT5 for maintaining pluripotency, suggesting that similar mechanisms might regulate lineage-restricted progenitor cells during organogenesis. PMID:27827819

PRMT5 is essential for the maintenance of chondrogenic progenitor cells in the limb bud.

PubMed

Norrie, Jacqueline L; Li, Qiang; Co, Swanie; Huang, Bau-Lin; Ding, Ding; Uy, Jann C; Ji, Zhicheng; Mackem, Susan; Bedford, Mark T; Galli, Antonella; Ji, Hongkai; Vokes, Steven A

2016-12-15

During embryonic development, undifferentiated progenitor cells balance the generation of additional progenitor cells with differentiation. Within the developing limb, cartilage cells differentiate from mesodermal progenitors in an ordered process that results in the specification of the correct number of appropriately sized skeletal elements. The internal pathways by which these cells maintain an undifferentiated state while preserving their capacity to differentiate is unknown. Here, we report that the arginine methyltransferase PRMT5 has a crucial role in maintaining progenitor cells. Mouse embryonic buds lacking PRMT5 have severely truncated bones with wispy digits lacking joints. This novel phenotype is caused by widespread cell death that includes mesodermal progenitor cells that have begun to precociously differentiate into cartilage cells. We propose that PRMT5 maintains progenitor cells through its regulation of Bmp4 Intriguingly, adult and embryonic stem cells also require PRMT5 for maintaining pluripotency, suggesting that similar mechanisms might regulate lineage-restricted progenitor cells during organogenesis. © 2016. Published by The Company of Biologists Ltd.

Combating Osteoarthritis through Stem Cell Therapies by Rejuvenating Cartilage: A Review

PubMed Central

Dubey, Navneet Kumar; Mishra, Viraj Krishna; Dubey, Rajni; Syed-Abdul, Shabbir; Wang, Joseph R.; Wang, Peter D.

2018-01-01

Knee osteoarthritis (OA) is a chronic degenerative disorder which could be distinguished by erosion of articular cartilage, pain, stiffness, and crepitus. Not only aging-associated alterations but also the metabolic factors such as hyperglycemia, dyslipidemia, and obesity affect articular tissues and may initiate or exacerbate the OA. The poor self-healing ability of articular cartilage due to limited regeneration in chondrocytes further adversely affects the osteoarthritic microenvironment. Traditional and current surgical treatment procedures for OA are limited and incapable to reverse the damage of articular cartilage. To overcome these limitations, cell-based therapies are currently being employed to repair and regenerate the structure and function of articular tissues. These therapies not only depend upon source and type of stem cells but also on environmental conditions, growth factors, and chemical and mechanical stimuli. Recently, the pluripotent and various multipotent mesenchymal stem cells have been employed for OA therapy, due to their differentiation potential towards chondrogenic lineage. Additionally, the stem cells have also been supplemented with growth factors to achieve higher healing response in osteoarthritic cartilage. In this review, we summarized the current status of stem cell therapies in OA pathophysiology and also highlighted the potential areas of further research needed in regenerative medicine. PMID:29765416

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration.

PubMed

Van de Walle, Aurore; Wilhelm, Claire; Luciani, Nathalie

2017-04-27

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

[Current status of bone/cartilage tissue engineering towards clinical applications].

PubMed

Ohgushi, Hajime

2014-10-01

Osteo/chondrogenic differentiation capabilities are seen after in vivo implantation of mesenchymal stem cells (MSCs), which are currently used for the patients having bone/cartilage defects. Importantly, the differentiation capabilities are induced by culturing technology, resulting in in vitro bone/cartilage formation. Especially, the in vitro bone tissue is useful for bone tissue regeneration. For cartilage regeneration, culture expanded chondrocytes derived from patient's normal cartilage are also used for the patients having cartilage damages. Recently, the cultured chondrocytes embedded in atelocollagen gel are obtainable as tissue engineered products distributed by Japan Tissue Engineering Co. Ltd. The products are available in the well-regulated hospitals by qualified orthopedic surgeons. The criteria for these hospitals/surgeons have been established. This review paper focuses on current status of bone/cartilage tissue engineering towards clinical applications in Japan.

Chondrogenesis of Embryonic Stem Cell-Derived Mesenchymal Stem Cells Induced by TGFβ1 and BMP7 Through Increased TGFβ Receptor Expression and Endogenous TGFβ1 Production.

PubMed

Lee, Patrick T; Li, Wan-Ju

2017-01-01

For decades stem cells have proven to be invaluable to the study of tissue development. More recently, mesenchymal stem cells (MSCs) derived from embryonic stem cells (ESCs) (ESC-MSCs) have emerged as a cell source with great potential for the future of biomedical research due to their enhanced proliferative capability compared to adult tissue-derived MSCs and effectiveness of musculoskeletal lineage-specific cell differentiation compared to ESCs. We have previously compared the properties and differentiation potential of ESC-MSCs to bone marrow-derived MSCs. In this study, we evaluated the potential of TGFβ1 and BMP7 to induce chondrogenic differentiation of ESC-MSCs compared to that of TGFβ1 alone and further investigated the cellular phenotype and intracellular signaling in response to these induction conditions. Our results showed that the expression of cartilage-associated markers in ESC-MSCs induced by the TGFβ1 and BMP7 combination was increased compared to induction with TGFβ1 alone. The TGFβ1 and BMP7 combination upregulated the expression of TGFβ receptor and the production of endogenous TGFβs compared to TGFβ1 induction. The growth factor combination also increasingly activated both of the TGF and BMP signaling pathways, and inhibition of the signaling pathways led to reduced chondrogenesis of ESC-MSCs. Our findings suggest that by adding BMP7 to TGFβ1-supplemented induction medium, ESC-MSC chondrogenesis is upregulated through increased production of endogenous TGFβ and activities of TGFβ and BMP signaling. J. Cell. Biochem. 118: 172-181, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Investigating the mincing method for isolation of adipose-derived stem cells from pregnant women fat.

PubMed

Li, Yuan-Sheng; Chen, Pao-Jen; Wu, Li-Wei; Chou, Pei-Wen; Sun, Li-Yi; Chiou, Tzyy-Wen

2018-02-01

The success of stem cell application in regenerative medicine, usually require a stable source of stem or progenitor cells. Fat tissue represents a good source of stem cells because it is rich in stem cells and there are fewer ethical issues related to the use of such stem cells, unlike embryonic stem cells. Therefore, there has been increased interest in adipose-derived stem cells (ADSCs) for tissue engineering applications. Here, we aim to provide an easy processing method for isolating adult stem cells from human adipose tissue harvested from the subcutaneous fat of the abdominal wall during gynecologic surgery. We used a homogenizer to mince fat and compared the results with those obtained from the traditional cut method involving a sterile scalpel and forceps. Our results showed that our method provides another stable and quality source of stem cells that could be used in cases with a large quantity of fat. Furthermore, we found that pregnancy adipose-derived stem cells (P-ADSCs) could be maintained in vitro for extended periods with a stable population doubling and low senescence levels. P-ADSCs could also differentiate in vitro into adipogenic, osteogenic, chondrogenic, and insulin-producing cells in the presence of lineage-specific induction factors. In conclusion, like human lipoaspirates, adipose tissues obtained from pregnant women contain multipotent cells with better proliferation and showed great promise for use in both stem cell banking studies as well as in stem cell therapy.

Mitogenic and chondrogenic effects of fibroblast growth factor-2 in adipose-derived mesenchymal cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chiou, Michael; Xu Yue; Longaker, Michael T.

2006-05-05

Adipose-derived mesenchymal cells (AMCs) have demonstrated a great capacity for differentiating into bone, cartilage, and fat. Studies using bone marrow-derived mesenchymal cells (BMSCs) have shown that fibroblast growth factor (FGF)-2, a potent mitogenic factor, plays an important role in tissue engineering due to its effects in proliferation and differentiation for mesenchymal cells. The aim of this study was to investigate the function of FGF-2 in AMC chondrogenic differentiation and its possible contributions to cell-based therapeutics in skeletal tissue regeneration. Data demonstrated that FGF-2 significantly promoted the proliferation of AMCs and enhanced chondrogenesis in three-dimensional micromass culture. Moreover, priming AMCs withmore » treatment of FGF-2 at 10 ng/ml demonstrated that cells underwent chondrogenic phenotypic differentiation, possibly by inducing N-Cadherin, FGF-receptor 2, and transcription factor Sox9. Our results indicated that FGF-2 potentiates chondrogenesis in AMCs, similar to its functions in BMSCs, suggesting the versatile potential applications of FGF-2 in skeletal regeneration and cartilage repair.« less

Equine mesenchymal stem cells from bone marrow, adipose tissue and umbilical cord: immunophenotypic characterization and differentiation potential

PubMed Central

2014-01-01

Introduction Studies with mesenchymal stem cells (MSCs) are increasing due to their immunomodulatory, anti-inflammatory and tissue regenerative properties. However, there is still no agreement about the best source of equine MSCs for a bank for allogeneic therapy. The aim of this study was to evaluate the cell culture and immunophenotypic characteristics and differentiation potential of equine MSCs from bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and umbilical cord (UC-MSCs) under identical in vitro conditions, to compare these sources for research or an allogeneic therapy cell bank. Methods The BM-MSCs, AT-MSCs and UC-MSCs were cultured and evaluated in vitro for their osteogenic, adipogenic and chondrogenic differentiation potential. Additionally, MSCs were assessed for CD105, CD44, CD34, CD90 and MHC-II markers by flow cytometry, and MHC-II was also assessed by immunocytochemistry. To interpret the flow cytometry results, statistical analysis was performed using ANOVA. Results The harvesting and culturing procedures of BM-MSCs, AT-MSCs and UC-MSCs were feasible, with an average cell growth until the third passage of 25 days for BM-MSCs, 15 days for AT-MSCs and 26 days for UC-MSCs. MSCs from all sources were able to differentiate into osteogenic (after 10 days for BM-MSCs and AT-MSCs and 15 days for UC-MSCs), adipogenic (after 8 days for BM-MSCs and AT-MSCs and 15 days for UC-MSCs) and chondrogenic (after 21 days for BM-MSCs, AT-MSCs and UC-MSCs) lineages. MSCs showed high expression of CD105, CD44 and CD90 and low or negative expression of CD34 and MHC-II. The MHC-II was not detected by immunocytochemistry techniques in any of the MSCs studied. Conclusions The BM, AT and UC are feasible sources for harvesting equine MSCs, and their immunophenotypic and multipotency characteristics attained minimal criteria for defining MSCs. Due to the low expression of MHC-II by MSCs, all of the sources could be used in clinical trials involving allogeneic therapy in horses. However, the BM-MSCs and AT-MSCs showed fastest ‘‘in vitro’’ differentiation and AT-MSCs showed highest cell growth until third passage. These findings suggest that BM and AT may be preferable for cell banking purposes. PMID:24559797

Notochordal cell conditioned medium stimulates mesenchymal stem cell differentiation toward a young nucleus pulposus phenotype

PubMed Central

2010-01-01

Introduction Mesenchymal stem cells (MSCs) offer promise for intervertebral disc (IVD) repair and regeneration because they are easily isolated and expanded, and can differentiate into several mesenchymal tissues. Notochordal (NC) cells contribute to IVD development, incorporate into the nucleus pulposus (NP), and stimulate mature disc cells. However, there have been no studies investigating the effects of NC cells on adult stem cell differentiation. The premise of this study is that IVD regeneration is more similar to IVD development than to IVD maintenance, and we hypothesize that soluble factors from NC cells differentiate MSCs to a phenotype characteristic of nucleus pulposus (NP) cells during development. The eventual clinical goal would be to isolate or chemically/recombinantly produce the active agent to induce the therapeutic effects, and to use it as either an injectable therapy for early intervention on disc disease, or in developing appropriately pre-differentiated MSC cells in a tissue engineered NP construct. Methods Human MSCs from bone marrow were expanded and pelleted to form high-density cultures. MSC pellets were exposed to either control medium (CM), chondrogenic medium (CM with dexamethasone and transforming growth factor, (TGF)-β3) or notochordal cell conditioned medium (NCCM). NCCM was prepared from NC cells maintained in serum free medium for four days. After seven days culture, MSC pellets were analyzed for appearance, biochemical composition (glycosaminoglycans and DNA), and gene expression profile (sox-9, collagen types-II and III, laminin-β1 and TIMP1(tissue inhibitor of metalloproteinases-1)). Results Significantly higher glycosaminoglycan accumulation was seen in NCCM treated pellets than in CM or TGFβ groups. With NCCM treatment, increased gene expression of collagen III, and a trend of increasing expression of laminin-β1 and decreased expression of sox-9 and collagen II relative to the TGFβ group was observed. Conclusions Together, results suggest NCCM stimulates mesenchymal stem cell differentiation toward a potentially NP-like phenotype with some characteristics of the developing IVD. PMID:20565707

Different culture media affect growth characteristics, surface marker distribution and chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells.

PubMed

Hagmann, Sebastien; Moradi, Babak; Frank, Sebastian; Dreher, Thomas; Kämmerer, Peer Wolfgang; Richter, Wiltrud; Gotterbarm, Tobias

2013-07-30

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) play an important role in modern tissue engineering, while distinct variations of culture media compositions and supplements have been reported. Because MSCs are heterogeneous regarding their regenerative potential and their surface markers, these parameters were compared in four widely used culture media compositions. MSCs were isolated from bone marrow and expanded in four established cell culture media. MSC yield/1000 MNCs, passage time and growth index were observed. In P4, typical MSC surface markers were analysed by fluorescence cytometry. Additionally, chondrogenic, adipogenic and osteogenic differentiation potential were evaluated. Growth index and P0 cell yield varied importantly between the media. The different expansion media had a significant influence on the expression of CD10, CD90, CD105, CD140b CD146 and STRO-1. While no significant differences were observed regarding osteogenic and adipogenic differentiation, chondrogenic differentiation was superior in medium A as reflected by GAG/DNA content. The choice of expansion medium can have a significant influence on growth, differentiation potential and surface marker expression of mesenchymal stromal cells, which is of fundamental importance for tissue engineering procedures.

The role of calcium signalling in the chondrogenic response of mesenchymal stem cells to hydrostatic pressure.

PubMed

Steward, A J; Kelly, D J; Wagner, D R

2014-10-28

The object of this study was to elucidate the role of Ca++ signalling in the chondrogenic response of mesenchymal stem cells (MSCs) to hydrostatic pressure (HP). MSCs were seeded into agarose hydrogels, subjected to HP or kept in free swelling conditions, and were cultured either with or without pharmacological inhibitors of Ca++ mobility and downstream targets. Chelating free Ca++, inhibiting voltage-gated calcium channels, and depleting intracellular calcium storessuppressed the beneficial effect of HP on chondrogenesis, indicating that Ca++ mobility may play an important role in the mechanotransduction of HP. However, inhibition of stretch-activated calcium channels in the current experiment yielded similar results to the control group, suggesting that mechanotransduction of HP is distinct from loads that generate cell deformations. Inhibition of the downstream targets calmodulin, calmodulin kinase II, and calcineurin all knocked down the effect of HP on chondrogenesis, implicating these targets in MSCs response to HP. All of the pharmacological inhibitors that abolished the chondrogenic response to HP also maintained a punctate vimentin organisation in the presence of HP, as opposed to the mechanoresponsive groups where the vimentin structure became more diffuse. These results suggest that Ca++ signalling may transduce HP via vimentin adaptation to loading.

Expansion in microcarrier-spinner cultures improves the chondrogenic potential of human early mesenchymal stromal cells.

PubMed

Lin, Youshan Melissa; Lim, Jessica Fang Yan; Lee, Jialing; Choolani, Mahesh; Chan, Jerry Kok Yen; Reuveny, Shaul; Oh, Steve Kah Weng

2016-06-01

Cartilage tissue engineering with human mesenchymal stromal cells (hMSC) is promising for allogeneic cell therapy. To achieve large-scale hMSC propagation, scalable microcarrier-based cultures are preferred over conventional static cultures on tissue culture plastic. Yet it remains unclear how microcarrier cultures affect hMSC chondrogenic potential, and how this potential is distinguished from that of tissue culture plastic. Hence, our study aims to compare the chondrogenic potential of human early MSC (heMSC) between microcarrier-spinner and tissue culture plastic cultures. heMSC expanded on either collagen-coated Cytodex 3 microcarriers in spinner cultures or tissue culture plastic were harvested for chondrogenic pellet differentiation with empirically determined chondrogenic inducer bone morphogenetic protein 2 (BMP2). Pellet diameter, DNA content, glycosaminoglycan (GAG) and collagen II production, histological staining and gene expression of chondrogenic markers including SOX9, S100β, MMP13 and ALPL, were investigated and compared in both conditions. BMP2 was the most effective chondrogenic inducer for heMSC. Chondrogenic pellets generated from microcarrier cultures developed larger pellet diameters, and produced more DNA, GAG and collagen II per pellet with greater GAG/DNA and collagen II/DNA ratios compared with that of tissue culture plastic. Moreover, they induced higher expression of chondrogenic genes (e.g., S100β) but not of hypertrophic genes (e.g., MMP13 and ALPL). A similar trend showing enhanced chondrogenic potential was achieved with another microcarrier type, suggesting that the mechanism is due to the agitated nature of microcarrier cultures. This is the first study demonstrating that scalable microcarrier-spinner cultures enhance the chondrogenic potential of heMSC, supporting their use for large-scale cell expansion in cartilage cell therapy. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Mesenchymal cells condensation-inducible mesh scaffolds for cartilage tissue engineering.

PubMed

Kim, In Gul; Ko, Jaehoon; Lee, Hye Rim; Do, Sun Hee; Park, Kwideok

2016-04-01

Mesenchymal cells condensation is crucial in chondrogenic development. However current tissue-engineered scaffolds for chondrogenesis pay little attention to this phenomenon. In this study, we fabricate poly(l-lactide-co-glycolide) (PLGA)/poly(l-lactide) (PLLA) microfiber scaffolds and coat them with human fibroblast-derived matrix (hFDM) that is a decellularized extracellular matrix (ECM) obtained from in vitro cultured human lung fibroblasts (WI-38). Those scaffolds were then conjugated with heparin via EDC chemistry and subsequently immobilized with transforming growth factor (TGF)-β1. The amount of TGF-β1 was quantitatively measured and the release profile showed a continuous release of TGF-β1 for 4 weeks. Human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) were seeded in four different scaffolds; control, fibronectin (FN)-coated, hFDM-coated, hFDM/TGF-β1 and subjected to chondrogenic differentiation in vitro for up to 28 days. Both hFDM and hFDM/TGF-β1 groups exhibited significantly more synthesis of glycosaminoglycan (GAG) and much better upregulation of chondrogenic markers expression. Interestingly, MSCs condensation that led to cell aggregates was clearly observed with time in the two hFDM-coated groups and the quantitative difference was obvious compared to the control and FN group. A mechanistic study in gene and protein level indicated that focal adhesion kinase (FAK) was involved at the early stage of cell adhesion and cell-cell contact-related markers, N-cadherin and neural cell adhesion molecule (NCAM), were highly up-regulated at later time point. In addition histological analysis proved that hFDM/TGF-β1 group was the most effective in forming neocartilage tissue in a rabbit articular cartilage defect model. Taken together, this study demonstrates not only the positive effect of hFDM on chondrogenesis of MSCs and cartilage repair but also provides an important insight toward the significance of in vitro mesenchymal condensation on chondrogenic development of MSCs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Successful immortalization of mesenchymal progenitor cells derived from human placenta and the differentiation abilities of immortalized cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang Xiaohong; Soda, Yasushi; Takahashi, Kenji

2006-12-29

We reported previously that mesenchymal progenitor cells derived from chorionic villi of the human placenta could differentiate into osteoblasts, adipocytes, and chondrocytes under proper induction conditions and that these cells should be useful for allogeneic regenerative medicine, including cartilage tissue engineering. However, similar to human mesenchymal stem cells (hMSCs), though these placental cells can be isolated easily, they are difficult to study in detail because of their limited life span in vitro. To overcome this problem, we attempted to prolong the life span of human placenta-derived mesenchymal cells (hPDMCs) by modifying hTERT and Bmi-1, and investigated whether these modified hPDMCsmore » retained their differentiation capability and multipotency. Our results indicated that the combination of hTERT and Bmi-1 was highly efficient in prolonging the life span of hPDMCs with differentiation capability to osteogenic, adipogenic, and chondrogenic cells in vitro. Clonal cell lines with directional differentiation ability were established from the immortalized parental hPDMC/hTERT + Bmi-1. Interestingly, hPDMC/Bmi-1 showed extended proliferation after long-term growth arrest and telomerase was activated in the immortal hPDMC/Bmi-1 cells. However, the differentiation potential was lost in these cells. This study reports a method to extend the life span of hPDMCs with hTERT and Bmi-1 that should become a useful tool for the study of mesenchymal stem cells.« less

The Effect of Quercetin on the Osteogenesic Differentiation and Angiogenic Factor Expression of Bone Marrow-Derived Mesenchymal Stem Cells

PubMed Central

Zhou, Yuning; Wu, Yuqiong; Jiang, Xinquan; Zhang, Xiuli; Xia, Lunguo; Lin, Kaili; Xu, Yuanjin

2015-01-01

Bone marrow-derived mesenchymal stem cells (BMSCs) are widely used in regenerative medicine in light of their ability to differentiate along the chondrogenic and osteogenic lineages. As a type of traditional Chinese medicine, quercetin has been preliminarily reported to promote osteogenic differentiation in osteoblasts. In the present study, the effects of quercetin on the proliferation, viability, cellular morphology, osteogenic differentiation and angiogenic factor secretion of rat BMSCs (rBMSCs) were examined by MTT assay, fluorescence activated cell sorter (FACS) analysis, real-time quantitative PCR (RT-PCR) analysis, alkaline phosphatase (ALP) activity and calcium deposition assays, and Enzyme-linked immunosorbent assay (ELISA). Moreover, whether mitogen-activated protein kinase (MAPK) signaling pathways were involved in these processes was also explored. The results showed that quercetin significantly enhanced the cell proliferation, osteogenic differentiation and angiogenic factor secretion of rBMSCs in a dose-dependent manner, with a concentration of 2 μM achieving the greatest stimulatory effect. Moreover, the activation of the extracellular signal-regulated protein kinases (ERK) and p38 pathways was observed in quercetin-treated rBMSCs. Furthermore, these induction effects could be repressed by either the ERK inhibitor PD98059 or the p38 inhibitor SB202190, respectively. These data indicated that quercetin could promote the proliferation, osteogenic differentiation and angiogenic factor secretion of rBMSCs in vitro, partially through the ERK and p38 signaling pathways. PMID:26053266

Engineering anisotropic biomimetic fibrocartilage microenvironment by bioprinting mesenchymal stem cells in nanoliter gel droplets.

PubMed

Gurkan, Umut A; El Assal, Rami; Yildiz, Simin E; Sung, Yuree; Trachtenberg, Alexander J; Kuo, Winston P; Demirci, Utkan

2014-07-07

Over the past decade, bioprinting has emerged as a promising patterning strategy to organize cells and extracellular components both in two and three dimensions (2D and 3D) to engineer functional tissue mimicking constructs. So far, tissue printing has neither been used for 3D patterning of mesenchymal stem cells (MSCs) in multiphase growth factor embedded 3D hydrogels nor been investigated phenotypically in terms of simultaneous differentiation into different cell types within the same micropatterned 3D tissue constructs. Accordingly, we demonstrated a biochemical gradient by bioprinting nanoliter droplets encapsulating human MSCs, bone morphogenetic protein 2 (BMP-2), and transforming growth factor β1 (TGF- β1), engineering an anisotropic biomimetic fibrocartilage microenvironment. Assessment of the model tissue construct displayed multiphasic anisotropy of the incorporated biochemical factors after patterning. Quantitative real time polymerase chain reaction (qRT-PCR) results suggested genomic expression patterns leading to simultaneous differentiation of MSC populations into osteogenic and chondrogenic phenotype within the multiphasic construct, evidenced by upregulation of osteogenesis and condrogenesis related genes during in vitro culture. Comprehensive phenotypic network and pathway analysis results, which were based on genomic expression data, indicated activation of differentiation related mechanisms, via signaling pathways, including TGF, BMP, and vascular endothelial growth factor.

Engineering Anisotropic Biomimetic Fibrocartilage Microenvironment by Bioprinting Mesenchymal Stem Cells in Nanoliter Gel Droplets

PubMed Central

2015-01-01

Over the past decade, bioprinting has emerged as a promising patterning strategy to organize cells and extracellular components both in two and three dimensions (2D and 3D) to engineer functional tissue mimicking constructs. So far, tissue printing has neither been used for 3D patterning of mesenchymal stem cells (MSCs) in multiphase growth factor embedded 3D hydrogels nor been investigated phenotypically in terms of simultaneous differentiation into different cell types within the same micropatterned 3D tissue constructs. Accordingly, we demonstrated a biochemical gradient by bioprinting nanoliter droplets encapsulating human MSCs, bone morphogenetic protein 2 (BMP-2), and transforming growth factor β1 (TGF- β1), engineering an anisotropic biomimetic fibrocartilage microenvironment. Assessment of the model tissue construct displayed multiphasic anisotropy of the incorporated biochemical factors after patterning. Quantitative real time polymerase chain reaction (qRT-PCR) results suggested genomic expression patterns leading to simultaneous differentiation of MSC populations into osteogenic and chondrogenic phenotype within the multiphasic construct, evidenced by upregulation of osteogenesis and condrogenesis related genes during in vitro culture. Comprehensive phenotypic network and pathway analysis results, which were based on genomic expression data, indicated activation of differentiation related mechanisms, via signaling pathways, including TGF, BMP, and vascular endothelial growth factor. PMID:24495169

[Simulated uterus microenvironment induced human placental mesenchymal stem cells differentiation to uterus smooth muscle cells in vitro].

PubMed

Li, Chang-dong; Zhang, Wei-yuan; Yuan, Chun-li; Han, Li-ying

2008-12-09

To develop a new method to promote the differentiation of mesenchymal stem cells derived from human placenta (pMSC) to uterus smooth muscle cells (uSMC) in simulated uterus microenvironment. MSCs were isolated from human placenta, cultivated, and analyzed for their phenotype by flow cytometry. The multipotential differentiation of the pMSC was examined by chondrogenic, adipogenic, and osteogenetic induction. uSMC were isolated from uteri resected during operation and co-cultivated with the pMSC in a Transwell chamber simulating Two, 4, and 8 days later RT-PCR and Western blotting were used to detect the mRNA and protein expression of alpha-actin, calmodulin, and myosin heavy chains (MHC), the markers of smooth muscle differentiation at the early, middle, and late stages. On day 8 RT-PCR was used to detect the expression of estrogen receptor in these 2 groups of cells, then estrogen was used to stimulate these cells and the protein kinase C (PKC) activity was examined. The pMSC could be induced into adipocytes, osteocytes, and chondrocytes respectively. After co-culture with uSMC, the morphology of the pMSC changed closely into that of the uSMC, and MHC was expressed in the pMSC. Estrogen receptor was positive in both groups of cells. The PKC activity increased, especially in the cell membrane, after stimulation of estrogen. The postpartum human placenta can be used as an important and novel source of multipotent stem cells for tissue engineering and genetic engineering. Placental MSC have the potential to differentiate into smooth muscle cells under the simulated uterus microenvironment in vitro.

Transfection of gene regulation nanoparticles complexed with pDNA and shRNA controls multilineage differentiation of hMSCs.

PubMed

Kim, Hye Jin; Yi, Se Won; Oh, Hyun Jyung; Lee, Jung Sun; Park, Ji Sun; Park, Keun-Hong

2018-05-29

Overexpression and knockdown of specific proteins can control stem cell differentiation for therapeutic purposes. In this study, we fabricated RUNX2, SOX9, and C/EBPα plasmid DNAs (pDNAs) and ATF4-targeting shRNA (shATF4) to induce osteogenesis, chondrogenesis, and adipogenesis of human mesenchymal stem cells (hMSCs). The pDNAs and shATF4 were complexed with TRITC-gene regulation nanoparticles (GRN). Osteogenesis-related gene expression was reduced at early (12 h) and late (36 h) time points after co-delivery of shATF4 and SOX9 or C/EBPα pDNA, respectively, and osteogenesis was inhibited in these hMSCs. By contrast, osteogenesis-related genes were highly expressed upon co-delivery of RUNX2 and ATF4 pDNAs. DEX in GRN enhanced chondrogenic differentiation. Expression of osteogenesis-, chondrogenesis-, and adipogenesis-related genes was higher in hMSCs transfected with NPs complexed with RUNX2 and ATF4 pDNAs, shATF4 and SOX9 pDNA, and shATF4 and C/EBPα pDNA for 72 h than in control hMSCs, respectively. Moreover, delivery of these NPs also increased expression of osteogenesis-, chondrogenesis-, and adipogenesis-related proteins. These alterations in expression led to morphological changes, indicating that hMSCs differentiated into osteoblasts, chondrocytes, and adipose cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

One-step generation of murine embryonic stem cell-derived mesoderm progenitors and chondrocytes in a serum-free monolayer differentiation system.

PubMed

Waese, Elaine Y L; Stanford, William L

2011-01-01

Cartilage defects have limited capacity for repair and are often replaced by fibrocartilage with inferior mechanical properties. To overcome the limitations of artificial joint replacement, high-throughput screens (HTS) could be developed to identify molecules that stimulate differentiation and/or proliferation of articular cartilage for drug therapy or tissue engineering. Currently embryonic stem cells (ESCs) can differentiate into articular cartilage by forming aggregates (embryoid body (EB), pellet, micromass), which are difficult to image. We present a novel, single-step method of generating murine ESC-derived chondrocytes in monolayer cultures under chemically defined conditions. Mesoderm induction was achieved in cultures supplemented with BMP4, activin A, or Wnt3a. Prolonged culture with sustained activin A, TGFβ3, or BMP4 supplementation led to robust chondrogenic induction. A short pulse of activin A or BMP4 also induced chondrogenesis efficiently while Wnt3a acted as a later inducer. Long-term supplementation with activin A or with activin A followed by TGFβ3 promoted articular cartilage formation. Thus, we devised a serum-free (SF) culture system to generate ESC-derived chondrocytes without the establishment of 3D cultures or the aid of cell sorting. Cultures were governed by the same signaling pathways as 3D ESC differentiation systems and limb bud mesenchyme or articular cartilage explant cultures. Copyright © 2010 Elsevier B.V. All rights reserved.

Quality Evaluation of Human Bone Marrow Mesenchymal Stem Cells for Cartilage Repair

PubMed Central

Shiraishi, Katsunori; Takeuchi, Shunsuke; Yanada, Shinobu; Mera, Hisashi; Wakitani, Shigeyuki; Adachi, Nobuo

2017-01-01

Quality evaluation of mesenchymal stem cells (MSCs) based on efficacy would be helpful for their clinical application. In this study, we aimed to find the factors of human bone marrow MSCs relating to cartilage repair. The expression profiles of humoral factors, messenger RNAs (mRNAs), and microRNAs (miRNAs) were analyzed in human bone marrow MSCs from five different donors. We investigated the correlations of these expression profiles with the capacity of the MSCs for proliferation, chondrogenic differentiation, and cartilage repair in vivo. The mRNA expression of MYBL1 was positively correlated with proliferation and cartilage differentiation. By contrast, the mRNA expression of RCAN2 and the protein expression of TIMP-1 and VEGF were negatively correlated with proliferation and cartilage differentiation. However, MSCs from all five donors had the capacity to promote cartilage repair in vivo regardless of their capacity for proliferation and cartilage differentiation. The mRNA expression of HLA-DRB1 was positively correlated with cartilage repair in vivo. Meanwhile, the mRNA expression of TMEM155 and expression of miR-486-3p, miR-148b, miR-93, and miR-320B were negatively correlated with cartilage repair. The expression analysis of these factors might help to predict the ability of bone marrow MSCs to promote cartilage repair. PMID:28835756

Effect of Bone Marrow Aspirate Concentrate-Platelet-Rich Plasma on Tendon-Derived Stem Cells and Rotator Cuff Tendon Tear

PubMed Central

Kim, Sun Jeong; Song, Da Hyun; Park, Jong Wook; Park, Silvia; Kim, Sang Jun

2017-01-01

Bone marrow aspirate concentrates (BMACs) and platelet-rich plasma (PRP) are good sources to control the differentiation of tendon-derived stem cells (TDSCs), but there has been no study about the effect of the BMAC–PRP complex on TDSCs and tendinopathy. The aim of this study was to investigate the effect of BMAC–PRP on the TDSCs and to find the therapeutic effect of BMAC–PRP on the rotator cuff tendon tear. The chondrogenic and osteogenic potential of TDSCs decreased, but the adipogenic potential of TDSCs revealed no significant difference when they were cocultured with BMAC–PRP. Cell proliferation was significantly greater in TDSCs cocultured with BMAC–PRP than in TDSCs. The degree of wound closure (percentage) was different between TDSCs and TDSCs with BMAC–PRP. There was no significant difference in expression of collagen type I and type III in immunocytochemical staining in the presence of BMAC–PRP. Initial visual analog scale (VAS) score was 5.8±1.9, which changed to 5.0±2.3 at 3 weeks and 2.8±2.3 at 3 months after the BMAC–PRP injection (p<0.01). The American Shoulder Elbow Surgeon score changed from 39.4±13.0 at baseline to 52.9±22.9 at 3 weeks and 71.8±19.7 at 3 months after the injection (p<0.01). The initial torn area of the rotator cuff tendon was 30.2±24.5 mm2, and this area was reduced to 22.5±18.9 mm2 at 3 months, but the change was not significant (p > 0.05). The data indicate that BMAC–PRP enhances the proliferation and migration of TDSCs and prevents the aberrant chondrogenic and osteogenic differentiation of TDSCs, which might provide a mechanistic basis for the therapeutic benefits of BMAC–PRP for rotator cuff tendon tear. PMID:28105983

Similar Properties of Chondrocytes from Osteoarthritis Joints and Mesenchymal Stem Cells from Healthy Donors for Tissue Engineering of Articular Cartilage

PubMed Central

Fernandes, Amilton M.; Herlofsen, Sarah R.; Karlsen, Tommy A.; Küchler, Axel M.; Fløisand, Yngvar; Brinchmann, Jan E.

2013-01-01

Lesions of hyaline cartilage do not heal spontaneously, and represent a therapeutic challenge. In vitro engineering of articular cartilage using cells and biomaterials may prove to be the best solution. Patients with osteoarthritis (OA) may require tissue engineered cartilage therapy. Chondrocytes obtained from OA joints are thought to be involved in the disease process, and thus to be of insufficient quality to be used for repair strategies. Bone marrow (BM) derived mesenchymal stem cells (MSCs) from healthy donors may represent an alternative cell source. We have isolated chondrocytes from OA joints, performed cell culture expansion and tissue engineering of cartilage using a disc-shaped alginate scaffold and chondrogenic differentiation medium. We performed real-time reverse transcriptase quantitative PCR and fluorescence immunohistochemistry to evaluate mRNA and protein expression for a range of molecules involved in chondrogenesis and OA pathogenesis. Results were compared with those obtained by using BM-MSCs in an identical tissue engineering strategy. Finally the two populations were compared using genome-wide mRNA arrays. At three weeks of chondrogenic differentiation we found high and similar levels of hyaline cartilage-specific type II collagen and fibrocartilage-specific type I collagen mRNA and protein in discs containing OA and BM-MSC derived chondrocytes. Aggrecan, the dominant proteoglycan in hyaline cartilage, was more abundantly distributed in the OA chondrocyte extracellular matrix. OA chondrocytes expressed higher mRNA levels also of other hyaline extracellular matrix components. Surprisingly BM-MSC derived chondrocytes expressed higher mRNA levels of OA markers such as COL10A1, SSP1 (osteopontin), ALPL, BMP2, VEGFA, PTGES, IHH, and WNT genes, but lower levels of MMP3 and S100A4. Based on the results presented here, OA chondrocytes may be suitable for tissue engineering of articular cartilage. PMID:23671648

Biphasic Scaffolds from Marine Collagens for Regeneration of Osteochondral Defects.

PubMed

Bernhardt, Anne; Paul, Birgit; Gelinsky, Michael

2018-03-13

Collagens of marine origin are applied increasingly as alternatives to mammalian collagens in tissue engineering. The aim of the present study was to develop a biphasic scaffold from exclusively marine collagens supporting both osteogenic and chondrogenic differentiation and to find a suitable setup for in vitro chondrogenic and osteogenic differentiation of human mesenchymal stroma cells (hMSC). Biphasic scaffolds from biomimetically mineralized salmon collagen and fibrillized jellyfish collagen were fabricated by joint freeze-drying and crosslinking. Different experiments were performed to analyze the influence of cell density and TGF-β on osteogenic differentiation of the cells in the scaffolds. Gene expression analysis and analysis of cartilage extracellular matrix components were performed and activity of alkaline phosphatase was determined. Furthermore, histological sections of differentiated cells in the biphasic scaffolds were analyzed. Stable biphasic scaffolds from two different marine collagens were prepared. An in vitro setup for osteochondral differentiation was developed involving (1) different seeding densities in the phases; (2) additional application of alginate hydrogel in the chondral part; (3) pre-differentiation and sequential seeding of the scaffolds and (4) osteochondral medium. Spatially separated osteogenic and chondrogenic differentiation of hMSC was achieved in this setup, while osteochondral medium in combination with the biphasic scaffolds alone was not sufficient to reach this ambition. Biphasic, but monolithic scaffolds from exclusively marine collagens are suitable for the development of osteochondral constructs.

Spontaneous transformation of adult mesenchymal stem cells from cynomolgus macaques in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ren, Zhenhua; Key Laboratory of Neurodegeneration, Ministry of Education, Beijing; Department of Anatomy, Anhui Medical University, Hefei, 230032

2011-12-10

Mesenchymal stem cells (MSCs) have shown potential clinical utility in cell therapy and tissue engineering, due to their ability to proliferate as well as to differentiate into multiple lineages, including osteogenic, adipogenic, and chondrogenic specifications. Therefore, it is crucial to assess the safety of MSCs while extensive expansion ex vivo is a prerequisite to obtain the cell numbers for cell transplantation. Here we show that MSCs derived from adult cynomolgus monkey can undergo spontaneous transformation following in vitro culture. In comparison with MSCs, the spontaneously transformed mesenchymal cells (TMCs) display significantly different growth pattern and morphology, reminiscent of the characteristicsmore » of tumor cells. Importantly, TMCs are highly tumorigenic, causing subcutaneous tumors when injected into NOD/SCID mice. Moreover, no multiple differentiation potential of TMCs is observed in vitro or in vivo, suggesting that spontaneously transformed adult stem cells may not necessarily turn into cancer stem cells. These data indicate a direct transformation of cynomolgus monkey MSCs into tumor cells following long-term expansion in vitro. The spontaneous transformation of the cultured cynomolgus monkey MSCs may have important implications for ongoing clinical trials and for models of oncogenesis, thus warranting a more strict assessment of MSCs prior to cell therapy. -- Highlights: Black-Right-Pointing-Pointer Spontaneous transformation of cynomolgus monkey MSCs in vitro. Black-Right-Pointing-Pointer Transformed mesenchymal cells lack multipotency. Black-Right-Pointing-Pointer Transformed mesenchymal cells are highly tumorigenic. Black-Right-Pointing-Pointer Transformed mesenchymal cells do not have the characteristics of cancer stem cells.« less

Functional characteristics of mesenchymal stem cells derived from the adipose tissue of a patient with achondroplasia.

PubMed

Park, Jeong-Ran; Lee, Hanbyeol; Kim, Chung-Hyo; Hong, Seok-Ho; Ha, Kwon-Soo; Yang, Se-Ran

2016-05-01

Mesenchymal stem cells (MSCs) can be isolated from various tissues including bone marrow, adipose tissue, skin dermis, and umbilical Wharton's jelly as well as injured tissues. MSCs possess the capacity for self-renewal and the potential for differentiation into adipogenic, osteogenic, and chondrogenic lineages. However, the characteristics of MSCs in injured tissues, such as achondroplasia (ACH), are not well known. In this study, we isolated MSCs from human subcutaneous adipose (ACH-SAMSCs) tissue and circumjacent human adipose tissue of the cartilage (ACH-CAMSCs) from a patient with ACH. We then analyzed the characterization of ACH-SAMSCs and ACH-CAMSCs, compared with normal human dermis-derived MSCs (hDMSCs). In flow cytometry analysis, the isolated ACH-MSCs expressed low levels of CD73, CD90, and CD105, compared with hDMSCs. Moreover, both ACH- SAMSCs and ACH-CAMSCs had constitutionally overactive fibroblast growth factor receptor 3 (FGFR3) and exhibited significantly reduced osteogenic differentiation, compared to enhanced adipogenic differentiation. The activity of extracellular signal-regulated kinases 1/2 (ERK1/2) and p38 mitogen-activated protein kinases (p38 MAPK) was increased in ACH-MSCs. In addition, the efficacy of osteogenic differentiation was slightly restored in osteogenic differentiation medium with MAPKs inhibitors. These results suggest that they play essential roles in MSC differentiation toward adipogenesis in ACH pathology. In conclusion, the identification of the characteristics of ACH-MSCs and the favoring of adipogenic differentiation via the FGFR3/MAPK axis might help to elucidate the pathogenic mechanisms relevant to other skeletal diseases and could provide targets for therapeutic interventions.

Curcumin inhibits cellular condensation and alters microfilament organization during chondrogenic differentiation of limb bud mesenchymal cells.

PubMed

Kim, Dong Kyun; Kim, Song Ja; Kang, Shin Sung; Jin, Eun Jung

2009-09-30

Curcumin is a well known natural polyphenol product isolated from the rhizome of the plant Curcuma longa, anti-inflammatory agent for arthritis by inhibiting synthesis of inflammatory prostaglandins. However, the mechanisms by which curcumin regulates the functions of chondroprogenitor, such as proliferation, precartilage condensation, cytoskeletal organization or overall chondrogenic behavior, are largely unknown. In the present report, we investigated the effects and signaling mechanism of curcumin on the regulation of chondrogenesis. Treating chick limb bud mesenchymal cells with curcumin suppressed chondrogenesis by stimulating apoptotic cell death. It also inhibited reorganization of the actin cytoskeleton into a cortical pattern concomitant with rounding of chondrogenic competent cells and down-regulation of integrin beta1 and focal adhesion kinase (FAK) phosphorylation. Curcumin suppressed the phosphorylation of Akt leading to Akt inactivation. Activation of Akt by introducing a myristoylated, constitutively active form of Akt reversed the inhibitory actions of curcumin during chondrogenesis. In summary, for the first time, we describe biological properties of curcumin during chondrogenic differentiation of chick limb bud mesenchymal cells. Curcumin suppressed chondrogenesis by stimulating apoptotic cell death and down-regulating integrin-mediated reorganization of actin cytoskeleton via modulation of Akt signaling.

Isolation and characterization of equine endometrial mesenchymal stromal cells.

PubMed

Rink, B Elisabeth; Amilon, Karin R; Esteves, Cristina L; French, Hilari M; Watson, Elaine; Aurich, Christine; Donadeu, F Xavier

2017-07-12

Equine mesenchymal stromal/stem cells (MSCs) are most commonly harvested from bone marrow (BM) or adipose tissue, requiring the use of surgical procedures. By contrast, the uterus can be accessed nonsurgically, and may provide a more readily available cell source. While human endometrium is known to harbor mesenchymal precursor cells, MSCs have not been identified in equine endometrium. This study reports the isolation, culture, and characterization of MSCs from equine endometrium. The presence of MSC and pericyte markers in endometrial sections was determined using immunohistochemistry. Stromal cells were harvested and cultured after separation of epithelial cells from endometrial fragments using Mucin-1-bound beads. For comparison, MSCs were also harvested from BM. The expression of surface markers in endometrial and BM-derived MSCs was characterized using flow cytometry and quantitative polymerase chain reaction. MSCs were differentiated in vitro into adipogenic, chondrogenic, osteogenic, and smooth muscle lineages. Typical markers of MSCs (CD29, CD44, CD90, and CD105) and pericytes (NG2 and CD146) were localized in the equine endometrium. Both endometrial and BM MSCs grew clonally and robustly expressed MSC and pericyte markers in culture while showing greatly reduced or negligible expression of hematopoietic markers (CD45, CD34) and MHC-II. Additionally, both endometrial and BM MSCs differentiated into adipogenic, osteogenic, and chondrogenic lineages in vitro, and endometrial MSCs had a distinct ability to undergo smooth muscle differentiation. We have demonstrated for the first time the presence of cells in equine endometrium that fulfill the definition of MSCs. The equine endometrium may provide an alternative, easily accessible source of MSCs, not only for therapeutic regeneration of the uterus, but also for other tissues where MSCs from other sources are currently being used therapeutically.

Comparative studies of mesenchymal stem cells derived from different cord tissue compartments - The influence of cryopreservation and growth media.

PubMed

Dulugiac, Magda; Moldovan, Lucia; Zarnescu, Otilia

2015-10-01

We have identified some critical aspects concerning umbilical cord tissue mesenchymal stem cells: the lack of standards for cell isolation, expansion and cryopreservation, the lack of unanimous opinions upon their multilineage differentiation potential and the existence of very few results related to the functional characterization of the cells isolated from cryopreserved umbilical cord tissue. Umbilical cord tissue cryopreservation appears to be the optimal solution for umbilical cord tissue mesenchymal stem cells storage for future clinical use. Umbilical cord tissue cryopreservation allows mesenchymal stem cells isolation before expected use, according with the specific clinical applications, by different customized isolation and expansion protocols agreed by cell therapy institutions. Using an optimized protocol for umbilical cord tissue cryopreservation in autologous cord blood plasma, isolation explant method and growth media supplemented with FBS or human serum, we performed comparative studies with respect to the characteristics of mesenchymal stem cells (MSC) isolated from different compartments of the same umbilical cord tissue such as Wharton's jelly, vein, arteries, before cryopreservation (pre freeze) and after cryopreservation (post thaw). Expression of histochemical and immunohistochemical markers as well as electron microscopy observations revealed similar adipogenic, chondrogenic and osteogenic differentiation capacity for cells isolated from pre freeze and corresponding post thaw tissue fragments of Wharton's jelly, vein or arteries of the same umbilical cord tissue, regardless growth media used for cells isolation and expansion. Our efficient umbilical cord tissue cryopreservation protocol is reliable for clinical applicability of mesenchymal stem cells that could next be isolated and expanded in compliance with future accepted standards. Copyright © 2015 Elsevier Ltd. All rights reserved.

Characterization of stem and progenitor cells in the dental pulp of erupted and unerupted murine molars

PubMed Central

Balic, Anamaria; Aguila, H. Leonardo; Caimano, Melissa J.; Francone, Victor P.; Mina, Mina

2010-01-01

In the past few years there have been significant advances in the identification of putative stem cells also referred to as “mesenchymal stem cells” (MSC) in dental tissues including the dental pulp. It is thought that MSC in dental pulp share certain similarities with MSC isolated from other tissues. However, cells in dental pulp are still poorly characterized. This study focused on the characterization of progenitor and stem cells in dental pulps of erupted and unerupted mice molars. Our study showed that dental pulps from unerupted molars contain a significant number of cells expressing CD90+/CD45-, CD117+/CD45-, Sca-1+/CD45- and little if any CD45+ cells. Our in vitro functional studies showed that dental pulp cells from unerupted molars displayed extensive osteo-dentinogenic potential but were unable to differentiate into chondrocytes and adipocytes. Dental pulp from erupted molars displayed a reduced number of cells, contained higher percentage of CD45+ and lower percentage of cells expressing CD90+/CD45-, CD117+/CD45- as compared to unerupted molars. In vitro functional assays demonstrated the ability of a small fraction of cells to differentiate into odontoblasts, osteoblasts, adipocytes and chondrocytes. There was a significant reduction in the osteo-dentinogenic potential of the pulp cells derived from erupted molars compared to unerupted molars. Furthermore, the adipogenic and chondrogenic differentiation of pulp cells from erupted molars was dependent on a long induction period and infrequent. Based on these findings we propose that the dental pulp of the erupted molars contain a small population of multipotent cells, whereas the dental pulp of the unerupted molars does not contain multipotent cells but is enriched in osteo-dentinogenic progenitors engaged in the formation of coronal and radicular odontoblasts. PMID:20193787

Pax1 and Pax9 activate Bapx1 to induce chondrogenic differentiation in the sclerotome.

PubMed

Rodrigo, Isabel; Hill, Robert E; Balling, Rudi; Münsterberg, Andrea; Imai, Kenji

2003-02-01

We have previously shown that the paired-box transcription factors Pax1 and Pax9 synergistically act in the proper formation of the vertebral column. Nevertheless, downstream events of the Pax1/Pax9 action and their target genes remain to be elucidated. We show, by analyzing Pax1;Pax9 double mutant mice, that expression of Bapx1 in the sclerotome requires the presence of Pax1 and Pax9, in a gene dose-dependent manner. By using a retroviral system to overexpress Pax1 in chick presomitic mesoderm explants, we show that Pax1 can substitute for Shh in inducing Bapx1 expression and in initiating chondrogenic differentiation. Furthermore, we demonstrate that Pax1 and Pax9 can transactivate regulatory sequences in the Bapx1 promoter and that they physically interact with the Bapx1 promoter region. These results strongly suggest that Bapx1 is a direct target of Pax1 and Pax9. Together, we conclude that Pax1 and Pax9 are required and sufficient for the chondrogenic differentiation of sclerotomal cells.

Enhanced human bone marrow mesenchymal stem cell functions in novel 3D cartilage scaffolds with hydrogen treated multi-walled carbon nanotubes.

PubMed

Holmes, Benjamin; Castro, Nathan J; Li, Jian; Keidar, Michael; Zhang, Lijie Grace

2013-09-13

Cartilage tissue is a nanostructured tissue which is notoriously hard to regenerate due to its extremely poor inherent regenerative capacity and complex stratified architecture. Current treatment methods are highly invasive and may have many complications. Thus, the goal of this work is to use nanomaterials and nano/microfabrication methods to create novel biologically inspired tissue engineered cartilage scaffolds to facilitate human bone marrow mesenchymal stem cell (MSC) chondrogenesis. To this end we utilized electrospinning to design and fabricate a series of novel 3D biomimetic nanostructured scaffolds based on hydrogen (H2) treated multi-walled carbon nanotubes (MWCNTs) and biocompatible poly(L-lactic acid) (PLLA) polymers. Specifically, a series of electrospun fibrous PLLA scaffolds with controlled fiber dimension were fabricated in this study. In vitro MSC studies showed that stem cells prefer to attach in the scaffolds with smaller fiber diameter. More importantly, the MWCNT embedded scaffolds showed a drastic increase in mechanical strength and a compressive Young's modulus matching to natural cartilage. Furthermore, our MSC differentiation results demonstrated that incorporation of the H2 treated carbon nanotubes and poly-L-lysine coating can induce more chondrogenic differentiations of MSCs than controls. After two weeks of culture, PLLA scaffolds with H2 treated MWCNTs and poly-L-lysine can achieve the highest glycosaminoglycan synthesis, making them promising for further exploration for cartilage regeneration.

Enhanced human bone marrow mesenchymal stem cell functions in novel 3D cartilage scaffolds with hydrogen treated multi-walled carbon nanotubes

NASA Astrophysics Data System (ADS)

Holmes, Benjamin; Castro, Nathan J.; Li, Jian; Keidar, Michael; Zhang, Lijie Grace

2013-09-01

Cartilage tissue is a nanostructured tissue which is notoriously hard to regenerate due to its extremely poor inherent regenerative capacity and complex stratified architecture. Current treatment methods are highly invasive and may have many complications. Thus, the goal of this work is to use nanomaterials and nano/microfabrication methods to create novel biologically inspired tissue engineered cartilage scaffolds to facilitate human bone marrow mesenchymal stem cell (MSC) chondrogenesis. To this end we utilized electrospinning to design and fabricate a series of novel 3D biomimetic nanostructured scaffolds based on hydrogen (H2) treated multi-walled carbon nanotubes (MWCNTs) and biocompatible poly(L-lactic acid) (PLLA) polymers. Specifically, a series of electrospun fibrous PLLA scaffolds with controlled fiber dimension were fabricated in this study. In vitro MSC studies showed that stem cells prefer to attach in the scaffolds with smaller fiber diameter. More importantly, the MWCNT embedded scaffolds showed a drastic increase in mechanical strength and a compressive Young’s modulus matching to natural cartilage. Furthermore, our MSC differentiation results demonstrated that incorporation of the H2 treated carbon nanotubes and poly-L-lysine coating can induce more chondrogenic differentiations of MSCs than controls. After two weeks of culture, PLLA scaffolds with H2 treated MWCNTs and poly-L-lysine can achieve the highest glycosaminoglycan synthesis, making them promising for further exploration for cartilage regeneration.

Mucopolysaccharidosis enzyme production by bone marrow and dental pulp derived human mesenchymal stem cells.

PubMed

Jackson, Matilda; Derrick Roberts, Ainslie; Martin, Ellenore; Rout-Pitt, Nathan; Gronthos, Stan; Byers, Sharon

2015-04-01

Mucopolysaccharidoses (MPS) are inherited metabolic disorders that arise from a complete loss or a reduction in one of eleven specific lysosomal enzymes. MPS children display pathology in multiple cell types leading to tissue and organ failure and early death. Mesenchymal stem cells (MSCs) give rise to many of the cell types affected in MPS, including those that are refractory to current treatment protocols such as hematopoietic stem cell (HSC) based therapy. In this study we compared multiple MPS enzyme production by bone marrow derived (hBM) and dental pulp derived (hDP) MSCs to enzyme production by HSCs. hBM MSCs produce significantly higher levels of MPS I, II, IIIA, IVA, VI and VII enzyme than HSCs, while hDP MSCs produce significantly higher levels of MPS I, IIIA, IVA, VI and VII enzymes. Higher transfection efficiency was observed in MSCs (89%) compared to HSCs (23%) using a lentiviral vector. Over-expression of four different lysosomal enzymes resulted in up to 9303-fold and up to 5559-fold greater levels in MSC cell layer and media respectively. Stable, persistent transduction of MSCs and sustained over-expression of MPS VII enzyme was observed in vitro. Transduction of MSCs did not affect the ability of the cells to differentiate down osteogenic, adipogenic or chondrogenic lineages, but did partially delay differentiation down the non-mesodermal neurogenic lineage. Copyright © 2015 Elsevier Inc. All rights reserved.

Efficient transduction of equine adipose-derived mesenchymal stem cells by VSV-G pseudotyped lentiviral vectors.

PubMed

Petersen, Gayle F; Hilbert, Bryan; Trope, Gareth; Kalle, Wouter; Strappe, Padraig

2014-12-01

Equine adipose-derived mesenchymal stem cells (EADMSC) provide a unique cell-based approach for treatment of a variety of equine musculoskeletal injuries, via regeneration of diseased or damaged tissue, or the secretion of immunomodulatory molecules. These capabilities can be further enhanced by genetic modification using lentiviral vectors, which provide a safe and efficient method of gene delivery. We investigated the suitability of lentiviral vector technology for gene delivery into EADMSC, using GFP expressing lentiviral vectors pseudotyped with the G glycoprotein from the vesicular stomatitis virus (V-GFP) or, for the first time, the baculovirus gp64 envelope protein (G-GFP). In this study, we produced similarly high titre V-GFP and G-GFP lentiviral vectors. Flow cytometric analysis showed efficient transduction using V-GFP; however G-GFP exhibited a poor ability to transduce EADMSC. Transduction resulted in sustained GFP expression over four passages, with minimal effects on cell viability and doubling time, and an unaltered chondrogenic differentiation potential. Copyright © 2014 Elsevier Ltd. All rights reserved.

Behavior and biocompatibility of rabbit bone marrow mesenchymal stem cells with bacterial cellulose membrane

PubMed Central

Leite, Yulla Klinger de Carvalho; de Carvalho, Camila Ernanda Sousa; Feitosa, Matheus Levi Tajra; Alves, Michel Muálem de Moraes; Carvalho, Fernando Aécio de Amorim; Neto, Bartolomeu Cruz Viana; Miglino, Maria Angélica

2018-01-01

Background Tissue engineering has been shown to exhibit great potential for the creation of biomaterials capable of developing into functional tissues. Cellular expansion and integration depends on the quality and surface-determinant factors of the scaffold, which are required for successful biological implants. The objective of this research was to characterize and evaluate the in vitro characteristics of rabbit bone marrow mesenchymal stem cells (BM-MSCs) associated with a bacterial cellulose membrane (BCM). We assessed the adhesion, expansion, and integration of the biomaterial as well as its ability to induce macrophage activation. Finally, we evaluated the cytotoxicity and toxicity of the BCM. Methods Samples of rabbit bone marrow were collected. Mesenchymal stem cells were isolated from medullary aspirates to establish fibroblast colony-forming unit assay. Osteogenic, chondrogenic, and adipogenic differentiation was performed. Integration with the BCM was assessed by scanning electron microscopy at 1, 7, and 14 days. Cytotoxicity was assessed via the production of nitric oxide, and BCM toxicity was assessed with the MTT assay; phagocytic activity was also determined. Results The fibroblastoid colony-forming unit (CFU-F) assay showed cells with a fibroblastoid morphology organized into colonies, and distributed across the culture area surface. In the growth curve, two distinct phases, lag and log phase, were observed at 15 days. Multipotentiality of the cells was evident after induction of osteogenic, chondrogenic, and adipogenic lineages. Regarding the BM-MSCs’ bioelectrical integration with the BCM, BM-MSCs were anchored in the BCM in the first 24 h. On day 7 of culture, the cytoplasm was scattered, and on day 14, the cells were fully integrated with the biomaterial. We also observed significant macrophage activation; analysis of the MTT assay and the concentration of nitric oxide revealed no cytotoxicity of the biomaterial. Conclusion The BCM allowed the expansion and biointegration of bone marrow progenitor cells with a stable cytotoxic profile, thus presenting itself as a biomaterial with potential for tissue engineering. PMID:29736332

Direct bone morphogenetic protein 2 and Indian hedgehog gene transfer for articular cartilage repair using bone marrow coagulates.

PubMed

Sieker, J T; Kunz, M; Weißenberger, M; Gilbert, F; Frey, S; Rudert, M; Steinert, A F

2015-03-01

Bone morphogenetic protein 2 (BMP-2, encoded by BMP2) and Indian hedgehog protein (IHH, encoded by IHH) are well known regulators of chondrogenesis and chondrogenic hypertrophy. Despite being a potent chondrogenic factor BMP-2 was observed to induce chondrocyte hypertrophy in osteoarthritis (OA), growth plate cartilage and adult mesenchymal stem cells (MSCs). IHH might induce chondrogenic differentiation through different intracellular signalling pathways without inducing subsequent chondrocyte hypertrophy. The primary objective of this study is to test the efficacy of direct BMP2 and IHH gene delivery via bone marrow coagulates to influence histological repair cartilage quality in vivo. Vector-laden autologous bone marrow coagulates with 10(11) adenoviral vector particles encoding BMP2, IHH or the Green fluorescent protein (GFP) were delivered to 3.2 mm osteochondral defects in the trochlea of rabbit knees. After 13 weeks the histological repair cartilage quality was assessed using the ICRS II scoring system and the type II collagen positive area. IHH treatment resulted in superior histological repair cartilage quality than GFP controls in all of the assessed parameters (with P < 0.05 in five of 14 assessed parameters). Results of BMP2 treatment varied substantially, including severe intralesional bone formation in two of six joints after 13 weeks. IHH gene transfer is effective to improve repair cartilage quality in vivo, whereas BMP2 treatment, carried the risk intralesional bone formation. Therefore IHH protein can be considered as an attractive alternative candidate growth factor for further preclinical research and development towards improved treatments for articular cartilage defects. Copyright © 2014 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Effects of platelet rich plasma and chondrocyte co-culture on MSC chondrogenesis, hypertrophy and pathological responses

PubMed Central

Ramezanifard, Rouhallah; Kabiri, Mahboubeh; Hanaee Ahvaz, Hana

2017-01-01

Regarding the inadequate healing capability of cartilage tissue, cell-based therapy is making the future of cartilage repair and regeneration. Mesenchymal stem cells (MSC) have shown great promise in cartilage regeneration. However, a yet-unresolved issue is the emergence of hypertrophic and pathologic markers during in vitro MSC chondrogenesis. Articular chondrocytes (AC) can suppress the undesired hypertrophy when co-cultured with MSC. On the other hand, platelet rich plasma (PRP), is considered potentially effective for cartilage repair and in-vitro chondrogenesis. We thus aimed to harness chondro-promotive effects of PRP and hypertrophic-suppressive effects of AC:MSC co-culture to achieve a more functional cartilage neo-tissue. We used PRP or conventional-differentiation chondrogenic media (ConvDiff) in MSC mono-cultures and AC:MSC co-cultures. We assessed gene expression of chondrogenic and hypertrophic markers using real-time RT-PCR and immunostaining. Alkaline-phosphatase activity (ALP) and calcium content of the pellets were quantified. We also measured VEGF and TNF-α secretion via ELISA. We showed PRP had higher chondrogenic potential (in mRNA and protein level) and hypertrophic-suppressive effects than Conv-Diff (mRNA level). Co-culturing reduced ALP while PRP increased calcium deposition. In all four groups, TNF-α was down-regulated compared to MSC controls, with co-cultures receiving ConvDiff media secreting the least. Meanwhile, the only group with increased VEGF secretion was PRP-mono-cultures. We observed synergistic effects for PRP and AC:MSC co-culture in enhancing chondrogenesis. Inclusion of AC reduced hypertrophic markers and angiogenic potential in PRP groups. We thus propose that combination of PRP and co-culture would favor chondrogenesis while alleviate but not totally eradicate undesired hypertrophic and pathologic responses. PMID:28900383

Fibronectin matrix assembly is essential for cell condensation during chondrogenesis

PubMed Central

Singh, Purva; Schwarzbauer, Jean E.

2014-01-01

ABSTRACT Mesenchymal cell condensation is the initiating event in endochondral bone formation. Cell condensation is followed by differentiation into chondrocytes, which is accompanied by induction of chondrogenic gene expression. Gene mutations involved in chondrogenesis cause chondrodysplasias and other skeletal defects. Using mesenchymal stem cells (MSCs) in an in vitro chondrogenesis assay, we found that knockdown of the diastrophic dysplasia (DTD) sulfate transporter (DTDST, also known as SLC26A2), which is required for normal cartilage development, blocked cell condensation and caused a significant reduction in fibronectin matrix. Knockdown of fibronectin with small interfering RNAs (siRNAs) also blocked condensation. Fibrillar fibronectin matrix was detected prior to cell condensation, and its levels increased during and after condensation. Inhibition of fibronectin matrix assembly by use of the functional upstream domain (FUD) of adhesin F1 from Streptococcus pyogenes prevented cell condensation by MSCs and also by the chondrogenic cell line ATDC5. Our data show that cell condensation and induction of chondrogenesis depend on fibronectin matrix assembly and DTDST, and indicate that this transporter is required earlier in chondrogenesis than previously appreciated. They also raise the possibility that certain of the skeletal defects in DTD patients might derive from the link between DTDST, fibronectin matrix and condensation. PMID:25146392

Fibronectin matrix assembly is essential for cell condensation during chondrogenesis.

PubMed

Singh, Purva; Schwarzbauer, Jean E

2014-10-15

Mesenchymal cell condensation is the initiating event in endochondral bone formation. Cell condensation is followed by differentiation into chondrocytes, which is accompanied by induction of chondrogenic gene expression. Gene mutations involved in chondrogenesis cause chondrodysplasias and other skeletal defects. Using mesenchymal stem cells (MSCs) in an in vitro chondrogenesis assay, we found that knockdown of the diastrophic dysplasia (DTD) sulfate transporter (DTDST, also known as SLC26A2), which is required for normal cartilage development, blocked cell condensation and caused a significant reduction in fibronectin matrix. Knockdown of fibronectin with small interfering RNAs (siRNAs) also blocked condensation. Fibrillar fibronectin matrix was detected prior to cell condensation, and its levels increased during and after condensation. Inhibition of fibronectin matrix assembly by use of the functional upstream domain (FUD) of adhesin F1 from Streptococcus pyogenes prevented cell condensation by MSCs and also by the chondrogenic cell line ATDC5. Our data show that cell condensation and induction of chondrogenesis depend on fibronectin matrix assembly and DTDST, and indicate that this transporter is required earlier in chondrogenesis than previously appreciated. They also raise the possibility that certain of the skeletal defects in DTD patients might derive from the link between DTDST, fibronectin matrix and condensation. © 2014. Published by The Company of Biologists Ltd.

Low magnitude high frequency vibration promotes adipogenic differentiation of bone marrow stem cells via P38 MAPK signal

PubMed Central

Yu, Haiyang; Gan, Xueqi

2017-01-01

Low magnitude high frequency vibration (LMHFV) has been mainly reported for its influence on the musculoskeletal system, particularly the bone tissue. In the bone structure, osteogenic activity is the main focus of study with regards to LMHFV. However, adipogenesis, another important mode of differentiation in the bone marrow cavity that might be affected by LMHFV, is much less researched. Furthermore, the molecular mechanism of how LMHFV influences adipogenesis still needs to be understood. Here, we tested the effect of LMHFV (0.3g, 40 Hz, amplitude: 50μm), 15min/d, on multipotent stem cells (MSCs), which are the common progenitors of osteogenic, chondrogenic, adipogenic and myogenic cells. It is previously shown that LMHFV promotes osteogenesis of MSCs. In this study, we further revealed its effect on adipo-differentiation of bone marrow stem cells (BMSCs) and studied the underlying signaling pathway. We found that when treated with LMHFV, the cells showed a higher expression of PPARγ, C/EBPα, adiponectin and showed more oil droplets. After vibration, the protein expression of PPARγ increased, and the phosphorylation of p38 MAPK was enhanced. After treating cells with SB203580, a specific p38 inhibitor, both the protein level of PPARγ illustrated by immunofluorescent staining and the oil droplets number, were decreased. Altogether, this indicates that p38 MAPK is activated during adipogenesis of BMSCs, and this is promoted by LMHFV. Our results demonstrating that specific parameters of LMHFV promotes adipogenesis of MSCs and enhances osteogenesis, highlights an unbeneficial side effect of vibration therapy used for preventing obesity and osteoporosis. PMID:28253368

Extracellular Nucleotide Hydrolysis in Dermal and Limbal Mesenchymal Stem Cells: A Source of Adenosine Production.

PubMed

Naasani, Liliana I Sous; Rodrigues, Cristiano; de Campos, Rafael Paschoal; Beckenkamp, Liziane Raquel; Iser, Isabele C; Bertoni, Ana Paula Santin; Wink, Márcia R

2017-08-01

Human Limbal (L-MSCs) and Dermal Mesenchymal Stem Cell (D-MSCs) possess many properties that increase their therapeutic potential in ophthalmology and dermatology. It is known that purinergic signaling plays a role in many aspects of mesenchymal stem cells physiology. They release and respond to purinergic ligands, altering proliferation, migration, differentiation, and apoptosis. Therefore, more information on these processes would be crucial for establishing future clinical applications using their differentiation potential, but without undesirable side effects. This study evaluated and compared the expression of ecto-nucleotidases, the enzymatic activity of degradation of extracellular nucleotides and the metabolism of extracellular ATP in D-MSCs and L-MSCs, isolated from discard tissues of human skin and sclerocorneal rims. The D-MSCs and L-MSCs showed a differentiation potential into osteogenic, adipogenic, and chondrogenic lineages and the expression of markers CD105 + , CD44 + , CD14 - , CD34 - , CD45 - , as expected. Both cells hydrolyzed low levels of extracellular ATP and high levels of AMP, leading to adenosine accumulation that can regulate inflammation and tissue repair. These cells expressed mRNA for ENTPD1, 2, 3, 5 and 6, and CD73 that corresponded to the observed enzymatic activities. Thus, considering the degradation of ATP and adenosine production, limbal MSCs are very similar to dermal MSCs, indicating that from the aspect of extracellular nucleotide metabolism L-MSCs are very similar to the characterized D-MSCs. J. Cell. Biochem. 118: 2430-2442, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Cell differentiation modeled via a coupled two-switch regulatory network

NASA Astrophysics Data System (ADS)

Schittler, D.; Hasenauer, J.; Allgöwer, F.; Waldherr, S.

2010-12-01

Mesenchymal stem cells can give rise to bone and other tissue cells, but their differentiation still escapes full control. In this paper we address this issue by mathematical modeling. We present a model for a genetic switch determining the cell fate of progenitor cells which can differentiate into osteoblasts (bone cells) or chondrocytes (cartilage cells). The model consists of two switch mechanisms and reproduces the experimentally observed three stable equilibrium states: a progenitor, an osteogenic, and a chondrogenic state. Conventionally, the loss of an intermediate (progenitor) state and the entailed attraction to one of two opposite (differentiated) states is modeled as a result of changing parameters. In our model in contrast, we achieve this by distributing the differentiation process to two functional switch parts acting in concert: one triggering differentiation and the other determining cell fate. Via stability and bifurcation analysis, we investigate the effects of biochemical stimuli associated with different system inputs. We employ our model to generate differentiation scenarios on the single cell as well as on the cell population level. The single cell scenarios allow to reconstruct the switching upon extrinsic signals, whereas the cell population scenarios provide a framework to identify the impact of intrinsic properties and the limiting factors for successful differentiation.

CRISPR/Cas9-based genetic correction for recessive dystrophic epidermolysis bullosa

PubMed Central

Webber, Beau R; Osborn, Mark J; McElroy, Amber N; Twaroski, Kirk; Lonetree, Cara-lin; DeFeo, Anthony P; Xia, Lily; Eide, Cindy; Lees, Christopher J; McElmurry, Ron T; Riddle, Megan J; Kim, Chong Jai; Patel, Dharmeshkumar D; Blazar, Bruce R; Tolar, Jakub

2016-01-01

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe disorder caused by mutations to the COL7A1 gene that deactivate production of a structural protein essential for skin integrity. Haematopoietic cell transplantation can ameliorate some of the symptoms; however, significant side effects from the allogeneic transplant procedure can occur and unresponsive areas of blistering persist. Therefore, we employed genome editing in patient-derived cells to create an autologous platform for multilineage engineering of therapeutic cell types. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system facilitated correction of an RDEB-causing COL7A1 mutation in primary fibroblasts that were then used to derive induced pluripotent stem cells (iPSCs). The resulting iPSCs were subsequently re-differentiated into keratinocytes, mesenchymal stem cells (MSCs) and haematopoietic progenitor cells using defined differentiation strategies. Gene-corrected keratinocytes exhibited characteristic epithelial morphology and expressed keratinocyte-specific genes and transcription factors. iPSC-derived MSCs exhibited a spindle morphology and expression of CD73, CD90 and CD105 with the ability to undergo adipogenic, chondrogenic and osteogenic differentiation in vitro in a manner indistinguishable from bone marrow-derived MSCs. Finally, we used a vascular induction strategy to generate potent definitive haematopoietic progenitors capable of multilineage differentiation in methylcellulose-based assays. In totality, we have shown that CRISPR/Cas9 is an adaptable gene-editing strategy that can be coupled with iPSC technology to produce multiple gene-corrected autologous cell types with therapeutic potential for RDEB. PMID:28250968

Characteristics of Human Turbinate-Derived Mesenchymal Stem Cells Are Not Affected by Allergic Condition of Donor

PubMed Central

Hwang, Se Hwan; Cho, Hye Kyung; Park, Sang Hi; Lee, WeonSun; Lee, Hee Jin; Lee, Dong Chang; Park, Sun Hwa; Lim, Mi Hyun; Back, Sang A; Yun, Byeong Gon; Sun, Dong Il

2015-01-01

The characteristics of mesenchymal stem cells (MSCs) derived from human turbinates (hTMSCs) have not been investigated in allergic rhinitis. We evaluated the influence of allergic state of the donor on the characteristics, proliferation, and differentiation potential of hTMSCs, compared with hTMSCs derived from non-allergic patients. hTMSCs were isolated from five non-allergic and five allergic patients. The expression of toll-like receptors (TLRs) in hTMSCs was measured by FACS, and cell proliferation was measured using a cell counting kit. Cytokine secretion was analyzed using multiplex immunoassays. The osteogenic, chondrogenic, and adipogenic differentiation potentials of hTMSCs were evaluated by histology and gene expression analysis. In allergic patients, FACS analysis showed that TLR3 and TLR4 were more highly expressed on the surface of hTMSCs than TLR2 and TLR5. The proliferation of hTMSCs was not influenced by the presence of TLR priming. The expression of IL-6, IL-8, IL-12, IP-10, and RANTES was upregulated after the TLR4 priming. The differentiation potential of hTMSCs was not influenced by TLR priming. These characteristics of hTMSCs were similar to those of hTMSCs from non-allergic patients. We conclude that the allergic condition of the donor does not influence TLR expression, proliferation, or immunomodulatory potential of hTMSCs. PMID:26376485

Adipose tissue as a stem cell source for musculo-skeletal regeneration

PubMed Central

Gimble, Jeffrey M.; Grayson, Warren; Guilak, Farshid; Lopez, Mandi J.; Vunjak-Novakovic, Gordana

2013-01-01

Adipose tissue is an abundant, easily accessible, and reproducible cell source for musculo-skeletal regenerative medicine applications. Initial derivation steps yield a heterogeneous population of cells collectively termed the stromal vascular fraction (SVF), which consist of endothelial cells, immune cells, pericytes, and pre-adipocytes. Subsequent selection of an adherent cell subset from the SVF results in a relatively homogeneous population of adipose-derived stromal/stem cells (ASCs). Mammalian ASCs exhibit the ability to selectively differentiate into chondrogenic, myogenic, and osteogenic lineages in response to inductive stimuli in vitro (when cultured on scaffolds in bioreactors) and in vivo (when implanted in pre-clinical animal models). Unlike hematopoietic cells, ASCs do not elicit a robust lymphocyte reaction and instead generate and release immunosuppressive factors, such as prostaglandin E2. These unique immunomodulatory features suggest that both allogeneic and autologous ASCs will engraft successfully following application for tissue regeneration purposes. The differentiation and expansion potential of ASCs can be modified by growth factors like bone morphogenetic protein 6, bio-inductive scaffolds, and bioreactors providing environmental control and biophysical stimulation. Gene therapy approaches using lentiviral transduction can also be used to direct differentiation of ASCs along particular lineage pathways. We discuss here the utility of ASCs for musculo-skeletal tissue repair and some of the technologies that can be implemented to unlock the full regenerative potential of these highly valuable cells. PMID:21196358

Microengineered 3D cell-laden thermoresponsive hydrogels for mimicking cell morphology and orientation in cartilage tissue engineering.

PubMed

Mellati, Amir; Fan, Chia-Ming; Tamayol, Ali; Annabi, Nasim; Dai, Sheng; Bi, Jingxiu; Jin, Bo; Xian, Cory; Khademhosseini, Ali; Zhang, Hu

2017-01-01

Mimicking the zonal organization of native articular cartilage, which is essential for proper tissue functions, has remained a challenge. In this study, a thermoresponsive copolymer of chitosan-g-poly(N-isopropylacrylamide) (CS-g-PNIPAAm) was synthesized as a carrier of mesenchymal stem cells (MSCs) to provide a support for their proliferation and differentiation. Microengineered three-dimensional (3D) cell-laden CS-g-PNIPAAm hydrogels with different microstripe widths were fabricated to control cellular alignment and elongation in order to mimic the superficial zone of natural cartilage. Biochemical assays showed six- and sevenfold increment in secretion of glycosaminoglycans (GAGs) and total collagen from MSCs encapsulated within the synthesized hydrogel after 28 days incubation in chondrogenic medium. Chondrogenic differentiation was also verified qualitatively by histological and immunohistochemical assessments. It was found that 75 ± 6% of cells encapsulated within 50 μm wide microstripes were aligned with an aspect ratio of 2.07 ± 0.16 at day 5, which was more organized than those observed in unpatterned constructs (12 ± 7% alignment and a shape index of 1.20 ± 0.07). The microengineered constructs mimicked the cell shape and organization in the superficial zone of cartilage whiles the unpatterned one resembled the middle zone. Our results suggest that microfabrication of 3D cell-laden thermosensitive hydrogels is a promising platform for creating biomimetic structures leading to more successful multi-zonal cartilage tissue engineering. Biotechnol. Bioeng. 2017;114: 217-231. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

EFFECTS OF PLATING DENSITY AND CULTURE TIME ON BONE MARROW STROMAL CELL CHARACTERISTICS

PubMed Central

Neuhuber, Birgit; Swanger, Sharon A.; Howard, Linda; Mackay, Alastair; Fischer, Itzhak

2008-01-01

Objective Bone marrow stromal cells (MSC) are multipotent adult stem cells that have emerged as promising candidates for cell therapy in disorders including cardiac infarction, stroke and spinal cord injury. While harvesting methods used by different laboratories are relatively standard, MSC culturing protocols vary widely. This study is aimed at evaluating the effects of initial plating density and total time in culture on proliferation, cell morphology, and differentiation potential of heterogeneous MSC cultures and more homogeneous cloned subpopulations. Methods Rat MSC were plated at 20, 200 and 2000 cells/cm2 and grown to 50% confluency. The numbers of population doublings and doubling times were determined within and across multiple passages. Changes in cell morphology and differentiation potential to adipogenic, chondrogenic, and osteogenic lineages were evaluated and compared among early, intermediate and late passages, as well as between heterogeneous and cloned MSC populations. Results We found optimal cell growth at a plating density of 200 cells/cm2. Cultures derived from all plating densities developed increased proportions of flat cells over time. Assays for chondrogenesis, osteogenesis and adipogenesis showed that heterogeneous MSC plated at all densities sustained the potential for all three mesenchymal phenotypes through at least passage 5; the flat subpopulation lost adipogenic and chondrogenic potential. Conclusion Our findings suggest that the initial plating density is not critical for maintaining a well-defined, multipotent MSC population. Time in culture, however, affects cell characteristics, suggesting that cell expansion should be limited, especially until the specific characteristics of different MSC subpopulations are better understood. PMID:18495329

Enzyme-crosslinked gene-activated matrix for the induction of mesenchymal stem cells in osteochondral tissue regeneration.

PubMed

Lee, Yi-Hsuan; Wu, Hsi-Chin; Yeh, Chia-Wei; Kuan, Chen-Hsiang; Liao, Han-Tsung; Hsu, Horng-Chaung; Tsai, Jui-Che; Sun, Jui-Sheng; Wang, Tzu-Wei

2017-11-01

The development of osteochondral tissue engineering is an important issue for the treatment of traumatic injury or aging associated joint disease. However, the different compositions and mechanical properties of cartilage and subchondral bone show the complexity of this tissue interface, making it challenging for the design and fabrication of osteochondral graft substitute. In this study, a bilayer scaffold is developed to promote the regeneration of osteochondral tissue within a single integrated construct. It has the capacity to serve as a gene delivery platform to promote transfection of human mesenchymal stem cells (hMSCs) and the functional osteochondral tissues formation. For the subchondral bone layer, the bone matrix with organic (type I collagen, Col) and inorganic (hydroxyapatite, Hap) composite scaffold has been developed through mineralization of hydroxyapatite nanocrystals oriented growth on collagen fibrils. We also prepare multi-shell nanoparticles in different layers with a calcium phosphate core and DNA/calcium phosphate shells conjugated with polyethyleneimine to act as non-viral vectors for delivery of plasmid DNA encoding BMP2 and TGF-β3, respectively. Microbial transglutaminase is used as a cross-linking agent to crosslink the bilayer scaffold. The ability of this scaffold to act as a gene-activated matrix is demonstrated with successful transfection efficiency. The results show that the sustained release of plasmids from gene-activated matrix can promote prolonged transgene expression and stimulate hMSCs differentiation into osteogenic and chondrogenic lineages by spatial and temporal control within the bilayer composite scaffold. This improved delivery method may enhance the functionalized composite graft to accelerate healing process for osteochondral tissue regeneration. In this study, a gene-activated matrix (GAM) to promote the growth of both cartilage and subchondral bone within a single integrated construct is developed. It has the capacity to promote transfection of human mesenchymal stem cells (hMSCs) and the functional osteochondral tissues formation. The results show that the sustained release of plasmids including TGF-beta and BMP-2 from GAM could promote prolonged transgene expression and stimulate hMSCs differentiation into the osteogenic and chondrogenic lineages by spatial control manner. This improved delivery method should enhance the functionalized composite graft to accelerate healing process in vitro and in vivo for osteochondral tissue regeneration. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Analysis of the Material Properties of Early Chondrogenic Differentiated Adipose-Derived Stromal Cells (ASC) Using an in vitro Three-dimensional Micromass Culture System

PubMed Central

Xu, Yue; Balooch, Guive; Chiou, Michael; Bekerman, Elena; Ritchie, Robert O.; Longaker, Michael T.

2009-01-01

Cartilage is an avascular tissue with only a limited potential to heal and chondrocytes in vitro have poor proliferative capacity. Recently, adipose-derived stromal cells (ASC) have demonstrated a great potential for application to tissue engineering due to their ability to differentiate into cartilage, bone, and fat. In this study, we have utilized a high density three-dimensional (3D) micromass model system of early chondrogenesis with ASC. The material properties of these micromasses showed a significant increase in dynamic and static elastic modulus during the early chondrogenic differentiation process. These data suggest that the 3D micromass culture system represents an in vitro model of early chondrogenesis with dynamic cell signaling interactions associated with the mechanical properties of chondrocyte differentiation. PMID:17543281

Analysis of the material properties of early chondrogenic differentiated adipose-derived stromal cells (ASC) using an in vitro three-dimensional micromass culture system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Yue; Balooch, Guive; Chiou, Michael

2007-07-27

Cartilage is an avascular tissue with only a limited potential to heal and chondrocytes in vitro have poor proliferative capacity. Recently, adipose-derived stromal cells (ASC) have demonstrated a great potential for application to tissue engineering due to their ability to differentiate into cartilage, bone, and fat. In this study, we have utilized a high density three-dimensional (3D) micromass model system of early chondrogenesis with ASC. The material properties of these micromasses showed a significant increase in dynamic and static elastic modulus during the early chondrogenic differentiation process. These data suggest that the 3D micromass culture system represents an in vitromore » model of early chondrogenesis with dynamic cell signaling interactions associated with the mechanical properties of chondrocyte differentiation.« less

The Use of Blood Vessel–Derived Stem Cells for Meniscal Regeneration and Repair

PubMed Central

OSAWA, AKI; HARNER, CHRISTOPHER D.; GHARAIBEH, BURHAN; MATSUMOTO, TOMOYUKI; MIFUNE, YUTAKA; KOPF, SEBASTIAN; INGHAM, SHEILA J. M.; SCHREIBER, VERENA; USAS, ARVYDAS; HUARD, JOHNNY

2015-01-01

Purpose Surgical repairs of tears in the vascular region of the meniscus usually heal better than repairs performed in the avascular region; thus, we hypothesized that this region might possess a richer supply of vascular-derived stem cells than the avascular region. Methods In this study, we analyzed 6 menisci extracted from aborted human fetuses and 12 human lateral menisci extracted from adult human subjects undergoing total knee arthroplasty. Menisci were immunostained for CD34 (a stem cell marker) and CD146 (a pericyte marker) in situ, whereas other menisci were dissected into two regions (peripheral and inner) and used to isolate meniscus-derived cells by flow cytometry. Cell populations expressing CD34 and CD146 were tested for their multi-lineage differentiation potentials, including chondrogenic, osteogenic, and adipogenic lineages. Fetal peripheral meniscus cells were transplanted by intracapsular injection into the knee joints of an athymic rat meniscal tear model. Rat menisci were extracted and histologically evaluated after 4 wk posttransplantation. Results Immunohistochemistry and flow cytometric analyses demonstrated that a higher number of CD34- and CD146-positive cells were found in the peripheral region compared with the inner region. The CD34- and CD146-positive cells isolated from the vascular region of both fetal and adult menisci demonstrated multilineage differentiation capacities and were more potent than cells isolated from the inner (avascular) region. Fetal CD34- and CD146-positive cells transplanted into the athymic rat knee joint were recruited into the meniscal tear sites and contributed to meniscus repair. Conclusions The vascularized region of the meniscus contains more stem cells than the avascular region. These meniscal-derived stem cells were multi-potent and contributed to meniscal regeneration. PMID:23247715

miR-10a restores human mesenchymal stem cell differentiation by repressing KLF4

PubMed Central

Li, Jiao; Dong, Jun; Zhang, Zhen-hui; Zhang, Dong-Cheng; You, Xiang-Yu; Zhong, Yun; Chen, Min-Sheng; Liu, Shi-Ming

2013-01-01

miRNAs have recently been shown to play a significant role in human aging. However, data demonstrating the effects of aging-related miRNAs in human mesenchymal stem cells (hMSCs) are limited. We observed that hMSC differentiation decreased with aging. We also identified that miR-10a expression was significantly decreased with age by comparing the miRNA expression of hMSCs derived from young and aged individuals. Therefore, we hypothesized that the downregulation of miR-10a may be associated with the decreased differentiation capability of hMSCs from aged individuals. Lentiviral constructs were used to up- or downregulate miR-10a in young and old hMSCs. Upregulation of miR-10a resulted in increased differentiation to adipogenic, osteogenic, and chondrogenic lineages and in reduced cell senescence. Conversely, downregulation of miR-10a resulted in decreased cell differentiation and increased cell senescence. A chimeric luciferase reporter system was generated, tagged with the full-length 3′-UTR region of KLF4 harboring the seed-matched sequence with or without four nucleotide mutations. These constructs were cotransfected with the miR-10a mimic into cells. The luciferase activity was significantly repressed by the miR-10a mimic, proving the direct binding of miR-10a to the 3′-UTR of KLF4. Direct suppression of KLF4 in aged hMSCs increased cell differentiation and decreased cell senescence. In conclusion, miR-10a restores the differentiation capability of aged hMSCs through repression of KLF4. Aging-related miRNAs may have broad applications in the restoration of cell dysfunction caused by aging. J. Cell. Physiol. 228: 2324–2336, 2013. © The Authors. Published by Wiley Periodicals, Inc. PMID:23696417

"In vitro" and multicolor phenotypic characterization of cell subpopulations identified in fresh human adipose tissue stromal vascular fraction and in the derived mesenchymal stem cells.

PubMed

Astori, Giuseppe; Vignati, Francesca; Bardelli, Silvana; Tubio, Monica; Gola, Mauro; Albertini, Veronica; Bambi, Franco; Scali, Giancarlo; Castelli, Damiano; Rasini, Valeria; Soldati, Gianni; Moccetti, Tiziano

2007-10-31

The stromal vascular fraction (SVF) is a heterogeneous cell population derived from the adipose tissue. There is still a lack of information concerning the characterization of the cell subpopulations constituting the SVF as well as its mesenchymal and haematopoietic potential. Furthermore there are great variations in its phenotypical characterization. Composition of SVF was investigated by FACS analysis, cytological and "in vitro" assays. We studied CD34+ population by combining FACS with human CFC (colony-forming-cell haematopoietic assay). The endothelial fraction was investigated by quantifying the co-expression of specific markers (CD146, CD105, CD31 and UEA-1). Mesenchymal potential was assessed by CFU-F assay and cultured AT-MSC were characterized by a 5-color FACS analysis. The multipotent differentiation potential (osteogenic, adipogenic and chondrogenic) was investigated both at cellular and molecular level. We identified in the SVF two CD34+ populations with a marked difference in the intensity of antigen expression, the majority of the cells expressing CD34 at low intensity. Moreover, two CD146+ cell populations were clearly distinguishable in the SVF:a CD146 dim accounting for 9.9% of the total SVF cells and a CD146+ bright cell population accounting for about 39.3%. The frequency of CFC clones was comparable with the one reported for peripheral blood. Endothelial cells account for about 7.7% of the SVF cells. AT-MSC differenced in the osteogenic adipogenic and chondrogenic lineage. The SVF is not a homogeneous cell population, and its final composition could be influenced both by the flow cytometric technique analysis and the SVF extraction steps. The CFU-F frequency in the SVF was 1/4880, a value about seven times greater than the data reported for bone marrow. The antigenic profile of AT-MSC was comparable with bone-marrow derived MSC. AT-MSC were able to differentiate along the osteogenic adipogenic and chondrogenic lineages. The data here reported, further contribute to the characterization of SVF, a tissue providing an alternative as a source of MSC for clinical applications.

Cartilage Protective and Chondrogenic Capacity of WIN-34B, a New Herbal Agent, in the Collagenase-Induced Osteoarthritis Rabbit Model and in Progenitor Cells from Subchondral Bone

PubMed Central

Huh, Jeong-Eun; Park, Yeon-Cheol; Seo, Byung-Kwan; Lee, Jae-Dong; Baek, Yong-Hyeon; Choi, Do-Young; Park, Dong-Suk

2013-01-01

We sought to determine the cartilage repair capacity of WIN-34B in the collagenase-induced osteoarthritis rabbit model and in progenitor cells from subchondral bone. The cartilage protective effect of WIN-34B was measured by clinical and histological scores, cartilage area, and proteoglycan and collagen contents in the collagenase-induced osteoarthritis rabbit model. The efficacy of chondrogenic differentiation of WIN-34B was assessed by expression of CD105, CD73, type II collagen, and aggrecan in vivo and was analyzed by the surface markers of progenitor cells, the mRNA levels of chondrogenic marker genes, and the level of proteoglycan, GAG, and type II collagen in vitro. Oral administration of WIN-34B significantly increased cartilage area, and this was associated with the recovery of proteoglycan and collagen content. Moreover, WIN-34B at 200 mg/kg significantly increased the expression of CD105, CD73, type II collagen, and aggrecan compared to the vehicle group. WIN-34B markedly enhanced the chondrogenic differentiation of CD105 and type II collagen in the progenitor cells from subchondral bone. Also, we confirmed that treatment with WIN-34B strongly increased the number of SH-2(CD105) cells and expression type II collagen in subchondral progenitor cells. Moreover, WIN-34B significantly increased proteoglycan, as measured by alcian blue staining; the mRNA level of type II α1 collagen, cartilage link protein, and aggrecan; and the inhibition of cartilage matrix molecules, such as GAG and type II collagen, in IL-1β-treated progenitor cells. These findings suggest that WIN-34B could be a potential candidate for effective anti-osteoarthritic therapy with cartilage repair as well as cartilage protection via enhancement of chondrogenic differentiation in the collagenase-induced osteoarthritis rabbit model and progenitor cells from subchondral bone. PMID:23983790

Phenotypical and functional characteristics of mesenchymal stem cells from bone marrow: comparison of culture using different media supplemented with human platelet lysate or fetal bovine serum

PubMed Central

2012-01-01

Introduction Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate into several mesenchymal lineages, classically derived from bone marrow (BM) but potentially from umbilical cord blood (UCB). Although they are becoming a good tool for regenerative medicine, they usually need to be expanded in fetal bovine serum (FBS)-supplemented media. Human platelet lysate (HPL) has recently been proposed as substitute for safety reasons, but it is not yet clear how this supplement influences the properties of expanded MSCs. Methods In the present study, we compared the effect of various media combining autologous HPL with or without FBS on phenotypic, proliferative and functional (differentiation, cytokine secretion profile) characteristics of human BM-derived MSCs. Results Despite less expression of adipogenic and osteogenic markers, MSCs cultured in HPL-supplemented media fully differentiated along osteoblastic, adipogenic, chondrogenic and vascular smooth muscle lineages. The analyses of particular specific proteins expressed during osteogenic differentiation (calcium-sensing receptor (CaSR) and parathormone receptor (PTHR)) showed their decrease at D0 before any induction for MSC cultured with HPL mostly at high percentage (10%HPL). The cytokine dosage showed a clear increase of proliferation capacity and interleukin (IL)-6 and IL-8 secretion. Conclusions This study shows that MSCs can be expanded in media supplemented with HPL that can totally replace FBS. HPL-supplemented media not only preserves their phenotype as well as their differentiation capacity, but also shortens culture time by increasing their growth rate. PMID:22333342

Physiological role of urothelial cancer-associated one long noncoding RNA in human skeletogenic cell differentiation.

PubMed

Ishikawa, Takanori; Nishida, Takashi; Ono, Mitsuaki; Takarada, Takeshi; Nguyen, Ha Thi; Kurihara, Shinnosuke; Furumatsu, Takayuki; Murase, Yurika; Takigawa, Masaharu; Oohashi, Toshitaka; Kamioka, Hiroshi; Kubota, Satoshi

2018-06-01

A vast number of long-noncoding RNAs (lncRNA) are found expressed in human cells, which RNAs have been developed along with human evolution. However, the physiological functions of these lncRNAs remain mostly unknown. In the present study, we for the first time uncovered the fact that one of such lncRNAs plays a significant role in the differentiation of chondrocytes and, possibly, of osteoblasts differentiated from mesenchymal stem cells, which cells eventually construct the human skeleton. The urothelial cancer-associated 1 (UCA1) lncRNA is known to be associated with several human malignancies. Firstly, we confirmed that UCA1 was expressed in normal human chondrocytes, as well as in a human chondrocytic cell line; whereas it was not detected in human bone marrow mesenchymal stem cells (hBMSCs). Of note, although UCA1 expression was undetectable in hBMSCs, it was markedly induced along with the differentiation toward chondrocytes, suggesting its critical role in chondrogenesis. Consistent with this finding, silencing of the UCA1 gene significantly repressed the expression of chondrogenic genes in human chondrocytic cells. UCA1 gene silencing and hyper-expression also had a significant impact on the osteoblastic phenotype in a human cell line. Finally, forced expression of UCA1 in a murine chondrocyte precursor, which did not possess a UCA1 gene, overdrove its differentiation into chondrocytes. These results indicate a physiological and important role of this lncRNA in the skeletal development of humans, who require more sustained endochondral ossification and osteogenesis than do smaller vertebrates. © 2017 Wiley Periodicals, Inc.

Signaling Cascades Governing Cdc42-Mediated Chondrogenic Differentiation and Mensenchymal Condensation.

PubMed

Wang, Jirong R; Wang, Chaojun J; Xu, Chengyun Y; Wu, Xiaokai K; Hong, Dun; Shi, Wei; Gong, Ying; Chen, Haixiao X; Long, Fanxin; Wu, Ximei M

2016-03-01

Endochondral ossification consists of successive steps of chondrocyte differentiation, including mesenchymal condensation, differentiation of chondrocytes, and hypertrophy followed by mineralization and ossification. Loss-of-function studies have revealed that abnormal growth plate cartilage of the Cdc42 mutant contributes to the defects in endochondral bone formation. Here, we have investigated the roles of Cdc42 in osteogenesis and signaling cascades governing Cdc42-mediated chondrogenic differentiation. Though deletion of Cdc42 in limb mesenchymal progenitors led to severe defects in endochondral ossification, either ablation of Cdc42 in limb preosteoblasts or knockdown of Cdc42 in vitro had no obvious effects on bone formation and osteoblast differentiation. However, in Cdc42 mutant limb buds, loss of Cdc42 in mesenchymal progenitors led to marked inactivation of p38 and Smad1/5, and in micromass cultures, Cdc42 lay on the upstream of p38 to activate Smad1/5 in bone morphogenetic protein-2-induced mesenchymal condensation. Finally, Cdc42 also lay on the upstream of protein kinase B to transactivate Sox9 and subsequently induced the expression of chondrocyte differential marker in transforming growth factor-β1-induced chondrogenesis. Taken together, by using biochemical and genetic approaches, we have demonstrated that Cdc42 is involved not in osteogenesis but in chondrogenesis in which the BMP2/Cdc42/Pak/p38/Smad signaling module promotes mesenchymal condensation and the TGF-β/Cdc42/Pak/Akt/Sox9 signaling module facilitates chondrogenic differentiation. Copyright © 2016 by the Genetics Society of America.

Chondrogenically primed tonsil-derived mesenchymal stem cells encapsulated in riboflavin-induced photocrosslinking collagen-hyaluronic acid hydrogel for meniscus tissue repairs.

PubMed

Koh, Rachel H; Jin, Yinji; Kang, Byung-Jae; Hwang, Nathaniel S

2017-04-15

Current meniscus tissue repairing strategies involve partial or total meniscectomy, followed by allograft transplantation or synthetic material implantation. However, allografts and synthetic implants have major drawbacks such as the limited supply of grafts and lack of integration into host tissue, respectively. In this study, we investigated the effects of conditioned medium (CM) from meniscal fibrochondrocytes and TGF-β3 on tonsil-derived mesenchymal stem cells (T-MSCs) for meniscus tissue engineering. CM-expanded T-MSCs were encapsulated in riboflavin-induced photocrosslinked collagen-hyaluronic acid (COL-RF-HA) hydrogels and cultured in chondrogenic medium containing TGF-β3. In vitro results indicate that CM-expanded cells followed by TGF-β3 exposure stimulated the expression of fibrocartilage-related genes (COL2, SOX9, ACAN, COL1) and production of extracellular matrix components. Histological assessment of in vitro and subcutaneously implanted in vivo constructs demonstrated that CM-expanded cells followed by TGF-β3 exposure resulted in highest cell proliferation, GAG accumulation, and collagen deposition. Furthermore, when implanted into meniscus defect model, CM treatment amplified the potential of TGF-β3 and induced complete regeneration. Conditioned medium derived from chondrocytes have been reported to effectively prime mesenchymal stem cells toward chondrogenic lineage. Type I collagen is the main component of meniscus extracellular matrix and hyaluronic acid is known to promote meniscus regeneration. In this manuscript, we investigated the effects of conditioned medium (CM) and transforming growth factor-β3 (TGF-β3) on tonsil-derived mesenchymal stem cells (T-MSCs) encapsulated in riboflavin-induced photocrosslinked collagen-hyaluronic acid (COL-RF-HA) hydrogel. We employed a novel source of conditioned medium, derived from meniscal fibrochondrocytes. Our in vitro and in vivo results collectively illustrate that CM-expanded cells followed by TGF-β3 exposure have the best potential for meniscus regeneration. This manuscript highlights a novel stem cell commitment strategy combined with biomaterials designs for meniscus regeneration. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Functional characterization of cell hybrids generated by induced fusion of primary porcine mesenchymal stem cells with an immortal murine cell line.

PubMed

Islam, M Q; Ringe, J; Reichmann, E; Migotti, R; Sittinger, M; da S Meirelles, L; Nardi, N B; Magnusson, P; Islam, K

2006-10-01

Bone marrow mesenchymal stem cells (MSC) integrate into various organs and contribute to the regeneration of diverse tissues. However, the mechanistic basis of the plasticity of MSC is not fully understood. The change of cell fate has been suggested to occur through cell fusion. We have generated hybrid cell lines by polyethylene-glycol-mediated cell fusion of primary porcine MSC with the immortal murine fibroblast cell line F7, a derivative of the GM05267 cell line. The hybrid cell lines display fibroblastic morphology and proliferate like immortal cells. They contain tetraploid to hexaploid porcine chromosomes accompanied by hypo-diploid murine chromosomes. Interestingly, many hybrid cell lines also express high levels of tissue-nonspecific alkaline phosphatase, which is considered to be a marker of undifferentiated embryonic stem cells. All tested hybrid cell lines retain osteogenic differentiation, a few of them also retain adipogenic potential, but none retain chondrogenic differentiation. Conditioned media from hybrid cells enhance the proliferation of both early-passage and late-passage porcine MSC, indicating that the hybrid cells secrete diffusible growth stimulatory factors. Murine F7 cells thus have the unique property of generating immortal cell hybrids containing unusually high numbers of chromosomes derived from normal cells. These hybrid cells can be employed in various studies to improve our understanding of regenerative biology. This is the first report, to our knowledge, describing the generation of experimentally induced cell hybrids by using normal primary MSC.

Silk-ionomer and silk-tropoelastin hydrogels as charged three-dimensional culture platforms for the regulation of hMSC response.

PubMed

Calabrese, Rossella; Raia, Nicole; Huang, Wenwen; Ghezzi, Chiara E; Simon, Marc; Staii, Cristian; Weiss, Anthony S; Kaplan, David L

2017-09-01

The response of human bone marrow-derived mesenchymal stem cells (hMSCs) encapsulated in three-dimensional (3D) charged protein hydrogels was studied. Combining silk fibroin (S) with recombinant human tropoelastin (E) or silk ionomers (I) provided protein composite alloys with tunable physicochemical and biological features for regulating the bioactivity of encapsulated hMSCs. The effects of the biomaterial charges on hMSC viability, proliferation and chondrogenic or osteogenic differentiation were assessed. The silk-tropoelastin or silk-ionomers hydrogels supported hMSC viability, proliferation and differentiation. Gene expression of markers for chondrogenesis and osteogenesis, as well as biochemical and histological analysis, showed that hydrogels with different S/E and S/I ratios had different effects on cell fate. The negatively charged hydrogels upregulated hMSC chondrogenesis or osteogenesis, with or without specific differentiation media, and hydrogels with higher tropoelastin content inhibited the differentiation potential even in the presence of the differentiation media. The results provide insight on charge-tunable features of protein-based biomaterials to control hMSC differentiation in 3D hydrogels, as well as providing a new set of hydrogels for the compatible encapsulation and utility for cell functions. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Original and regenerating lizard tail cartilage contain putative resident stem/progenitor cells.

PubMed

Alibardi, Lorenzo

2015-11-01

Regeneration of cartilaginous tissues is limited in mammals but it occurs with variable extension in lizards (reptiles), including in their vertebrae. The ability of lizard vertebrae to regenerate cartilaginous tissue that is later replaced with bone has been analyzed using tritiated thymidine autoradiography and 5BrdU immunocytochemistry after single pulse or prolonged-pulse and chase experiments. The massive cartilage regeneration that can restore broad vertebral regions and gives rise to a long cartilaginous tube in the regenerating tail, depends from the permanence of some chondrogenic cells within adult vertebrae. Few cells that retain tritiated thymidine or 5-bromodeoxy-uridine for over 35 days are mainly localized in the inter-vertebral cartilage and in sparse chondrogenic regions of the neural arch of the vertebrae, suggesting that they are putative resident stem/progenitor cells. The study supports previous hypothesis indicating that the massive regeneration of the cartilaginous tissue in damaged vertebrae and in the regenerating tail of lizards derive from resident stem cells mainly present in the cartilaginous areas of the vertebrae including in the perichondrium that are retained in adult lizards as growing centers for most of their lifetime. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mesenchymal and embryonic characteristics of stem cells obtained from mouse dental pulp.

PubMed

Guimarães, Elisalva Teixeira; Cruz, Gabriela Silva; de Jesus, Alan Araújo; Lacerda de Carvalho, Acácia Fernandes; Rogatto, Silvia Regina; Pereira, Lygia da Veiga; Ribeiro-dos-Santos, Ricardo; Soares, Milena Botelho Pereira

2011-11-01

Several studies have demonstrated that human dental pulp is a source of mesenchymal stem cells. To better understand the biological properties of these cells we isolated and characterized stem cells from the dental pulp of EGFP transgenic mice. The pulp tissue was gently separated from the roots of teeth extracted from C57BL/6 mice, and cultured under appropriate conditions. Flow cytometry, RT-PCR, light microscopy (staining for alkaline phosphatase) and immunofluorescence were used to investigate the expression of stem cell markers. The presence of chromosomal abnormalities was evaluated by G banding. The mouse dental pulp stem cells (mDPSC) were highly proliferative, plastic-adherent, and exhibited a polymorphic morphology predominantly with stellate or fusiform shapes. The presence of cell clusters was observed in cultures of mDPSC. Some cells were positive for alkaline phosphatase. The karyotype was normal until the 5th passage. The Pou5f1/Oct-4 and ZFP42/Rex-1, but not Nanog transcripts were detected in mDPSC. Flow cytometry and fluorescence analyses revealed the presence of a heterogeneous population positive for embryonic and mesenchymal cell markers. Adipogenic, chondrogenic and osteogenic differentiation was achieved after two weeks of cell culture under chemically defined in vitro conditions. In addition, some elongated cells spontaneously acquired a contraction capacity. Our results reinforce that the dental pulp is an important source of adult stem cells and encourage studies on therapeutic potential of mDPSC in experimental disease models. Copyright © 2011 Elsevier Ltd. All rights reserved.

Effect of Bone Marrow Aspirate Concentrate-Platelet-Rich Plasma on Tendon-Derived Stem Cells and Rotator Cuff Tendon Tear.

PubMed

Kim, Sun Jeong; Song, Da Hyun; Park, Jong Wook; Park, Silvia; Kim, Sang Jun

2017-05-09

Bone marrow aspirate concentrates (BMACs) and platelet-rich plasma (PRP) are good sources to control the differentiation of tendon-derived stem cells (TDSCs), but there has been no study about the effect of the BMAC-PRP complex on TDSCs and tendinopathy. The aim of this study was to investigate the effect of BMAC-PRP on the TDSCs and to find the therapeutic effect of BMAC-PRP on the rotator cuff tendon tear. The chondrogenic and osteogenic potential of TDSCs decreased, but the adipogenic potential of TDSCs revealed no significant difference when they were cocultured with BMAC-PRP. Cell proliferation was significantly greater in TDSCs cocultured with BMAC-PRP than in TDSCs. The degree of wound closure (percentage) was different between TDSCs and TDSCs with BMAC-PRP. There was no significant difference in expression of collagen type I and type III in immunocytochemical staining in the presence of BMAC-PRP. Initial visual analog scale (VAS) score was 5.8 ± 1.9, which changed to 5.0 ± 2.3 at 3 weeks and 2.8 ± 2.3 at 3 months after the BMAC-PRP injection (p < 0.01). The American Shoulder Elbow Surgeon score changed from 39.4 ± 13.0 at baseline to 52.9 ± 22.9 at 3 weeks and 71.8 ± 19.7 at 3 months after the injection (p < 0.01). The initial torn area of the rotator cuff tendon was 30.2 ± 24.5 mm2, and this area was reduced to 22.5 ± 18.9 mm2 at 3 months, but the change was not significant (p > 0.05). The data indicate that BMAC-PRP enhances the proliferation and migration of TDSCs and prevents the aberrant chondrogenic and osteogenic differentiation of TDSCs, which might provide a mechanistic basis for the therapeutic benefits of BMAC-PRP for rotator cuff tendon tear.

Recloned dogs derived from adipose stem cells of a transgenic cloned beagle.

PubMed

Oh, Hyun Ju; Park, Jung Eun; Kim, Min Jung; Hong, So Gun; Ra, Jeong Chan; Jo, Jung Youn; Kang, Sung Keun; Jang, Goo; Lee, Byeong Chun

2011-04-15

A number of studies have postulated that efficiency in mammalian cloning is inversely correlated with donor cell differentiation status and may be increased by using undifferentiated cells as nuclear donors. Here, we attempted the recloning of dogs by nuclear transfer of canine adipose tissue-derived mesenchymal stem cells (cAd-MSCs) from a transgenic cloned beagle to determine if cAd-MSCs can be a suitable donor cell type. In order to isolate cAd-MSCs, adipose tissues were collected from a transgenic cloned beagle produced by somatic cell nuclear transfer (SCNT) of canine fetal fibroblasts modified genetically with a red fluorescent protein (RFP) gene. The cAd-MSCs expressed the RFP gene and cell-surface marker characteristics of MSCs including CD29, CD44 and thy1.1. Furthermore, cAd-MSCs underwent osteogenic, adipogenic, myogenic, neurogenic and chondrogenic differentiation when exposed to specific differentiation-inducing conditions. In order to investigate the developmental potential of cAd-MSCs, we carried out SCNT. Fused-couplets (82/109, 75.2%) were chemically activated and transferred into the uterine tube of five naturally estrus-synchronized surrogates. One of them (20%) maintained pregnancy and subsequently gave birth to two healthy cloned pups. The present study demonstrated for the first time the successful production of cloned beagles by nuclear transfer of cAd-MSCs. Another important outcome of the present study is the successful recloning of RFP-expressing transgenic cloned beagle pups by nuclear transfer of cells derived from a transgenic cloned beagle. In conclusion, the present study demonstrates that adipose stem cells can be a good nuclear donor source for dog cloning. Copyright © 2011 Elsevier Inc. All rights reserved.

Development of mRuby2-Transfected C3H10T1/2 Fibroblasts for Musculoskeletal Tissue Engineering

PubMed Central

Yang, Yunzhi Peter

2015-01-01

Mouse C3H10T1/2 fibroblasts are multipotent, mesenchymal stem cell (MSC)-like progenitor cells that are widely used in musculoskeletal research. In this study, we have established a clonal population of C3H10T1/2 cells stably-transfected with mRuby2, an orange-red fluorescence reporter gene. Flow cytometry analysis and fluorescence imaging confirmed successful transfection of these cells. Cell counting studies showed that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells proliferated at similar rates. Adipogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for Oil Red O and showed increased expression of adipogenic genes including adiponectin and lipoprotein lipase. Chondrogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for Alcian Blue and showed increased expression of chondrogenic genes including aggrecan. Osteogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for alkaline phosphatase (ALP) as well as Alizarin Red and showed increased expression of osteogenic genes including alp, ocn and osf-1. When seeded on calcium phosphate-based ceramic scaffolds, mRuby2-transfected C3H10T1/2 cells maintained even fluorescence labeling and osteogenic differentiation. In summary, mRuby2-transfected C3H10T1/2 cells exhibit mRuby2 fluorescence and showed little-to-no difference in terms of cell proliferation and differentiation as untransfected C3H10T1/2 cells. These cells will be available from American Type Culture Collection (ATCC; CRL-3268™) and may be a valuable tool for preclinical studies. PMID:26407291

Development of mRuby2-Transfected C3H10T1/2 Fibroblasts for Musculoskeletal Tissue Engineering.

PubMed

Ker, Dai Fei Elmer; Sharma, Rashmi; Wang, Evelyna Tsi Hsin; Yang, Yunzhi Peter

2015-01-01

Mouse C3H10T1/2 fibroblasts are multipotent, mesenchymal stem cell (MSC)-like progenitor cells that are widely used in musculoskeletal research. In this study, we have established a clonal population of C3H10T1/2 cells stably-transfected with mRuby2, an orange-red fluorescence reporter gene. Flow cytometry analysis and fluorescence imaging confirmed successful transfection of these cells. Cell counting studies showed that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells proliferated at similar rates. Adipogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for Oil Red O and showed increased expression of adipogenic genes including adiponectin and lipoprotein lipase. Chondrogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for Alcian Blue and showed increased expression of chondrogenic genes including aggrecan. Osteogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for alkaline phosphatase (ALP) as well as Alizarin Red and showed increased expression of osteogenic genes including alp, ocn and osf-1. When seeded on calcium phosphate-based ceramic scaffolds, mRuby2-transfected C3H10T1/2 cells maintained even fluorescence labeling and osteogenic differentiation. In summary, mRuby2-transfected C3H10T1/2 cells exhibit mRuby2 fluorescence and showed little-to-no difference in terms of cell proliferation and differentiation as untransfected C3H10T1/2 cells. These cells will be available from American Type Culture Collection (ATCC; CRL-3268™) and may be a valuable tool for preclinical studies.

Umbilical Cord Blood Platelet Lysate as Serum Substitute in Expansion of Human Mesenchymal Stem Cells.

PubMed

Shirzad, Negin; Bordbar, Sima; Goodarzi, Alireza; Mohammad, Monire; Khosravani, Pardis; Sayahpour, Froughazam; Baghaban Eslaminejad, Mohamadreza; Ebrahimi, Marzieh

2017-10-01

The diverse clinical applications for human mesenchymal stem cells (hMSCs) in cellular therapy and regenerative medicine warrant increased focus on developing adequate culture supplements devoid of animal-derived products. In the present study, we have investigated the feasibility of umbilical cord blood-platelet lysate (UCB-PL) as a standard substitute for fetal bovine serum (FBS) and human peripheral blood-PL (PB-PL). In this experimental study, platelet concentrates (PC) from UCB and human PB donors were frozen, melted, and sterilized to obtain PL. Quality control included platelet cell counts, sterility testing (viral and microbial), total protein concentrations, growth factor levels, and PL stability. The effects of UCB-PL and PB-PL on hMSCs proliferation and differentiation into osteocytes, chondrocytes, and adipocytes were studied and the results compared with FBS. UCB-PL contained high levels of protein content, platelet-derived growth factor- AB (PDGF-AB), and transforming growth factor (TGF) compared to PB-PL. All growth factors were stable for at least nine months post-storage at -70˚C. hMSCs proliferation enhanced following treatment with UCB-PL. With all three supplements, hMSCs could differentiate into all three lineages. PB-PL and UCB-PL both were potent in hMSCs proliferation. However, PB promoted osteoblastic differentiation and UCB-PL induced chondrogenic differentiation. Because of availability, ease of use and feasible standardization of UCB-PL, we have suggested that UCB-PL be used as an alternative to FBS and PB-PL for the cultivation and expansion of hMSCs in cellular therapy. Copyright© by Royan Institute. All rights reserved.

Isolation and characterization of the progenitor cells from the blastema tissue formed at experimentally-created rabbit ear hole.

PubMed

Baghaban Eslaminejad, Mohamadreza; Bordbar, Sima

2013-02-01

Objective(s) : Throughout evolution, mammalians have increasingly lost their ability to regenerate structures however rabbits are exceptional since they develop a blastema in their ear wound for regeneration purposes. Blastema consists of a group of undifferentiated cells capable of dividing and differentiating into the ear tissue. The objective of the present study is to isolate, culture expand, and characterize blastema progenitor cells in terms of their in vitro differentiation capacity. Five New Zealand white male rabbits were used in the present study. Using a punching apparatus, a 4-mm hole was created in the animal ears. Following 4 days, the blastema ring which was created in the periphery of primary hole in the ears was removed and cultivated. The cells migrated from the blastema were expanded through 3 successive subcultures and characterized in terms of their potential differentiation, growth characteristics, and culture requirements. The primary cultures tended to be morphologically heterogeneous having spindly-shaped fibroblast-like cells as well as flattened cells. Fibroblast-like cells survived and dominated the cultures. These cells tended to have the osteogenic, chondrogenic, and adipogenic differentiation potentials. They were highly colonogenic and maximum proliferation was achieved when the cells were plated at density of 100 cells/cm2 in a medium which contained 10% fetal bovine serum (FBS). Taken together, blastema tissue-derived stem cells from rabbit ear are of mesenchymal stem cell-like population. Studies similar to this will assist scientist better understanding the nature of blastema tissue formed at rabbit ear to regenerate the wound.

Isolation and Characterization of Human Mesenchymal Stem Cells From Facet Joints and Interspinous Ligaments.

PubMed

Kristjánsson, Baldur; Limthongkul, Worawat; Yingsakmongkol, Wicharn; Thantiworasit, Pattarawat; Jirathanathornnukul, Napaphat; Honsawek, Sittisak

2016-01-01

A descriptive in vitro study on isolation and differentiation of human mesenchymal stem cells (MSCs) derived from the facet joints and interspinous ligaments. To isolate cells from the facet joints and interspinous ligaments and investigate their surface marker profile and differentiation potentials. Lumbar spinal canal stenosis and ossification of the posterior longitudinal ligament are progressive conditions characterized by the hypertrophy and ossification of ligaments and joints within the spinal canal. MSCs are believed to play a role in the advancement of these diseases and the existence of MSCs has been demonstrated within the ligamentum flavum and posterior longitudinal ligament. The aim of this study was to investigate whether these cells could also be found within facet joints and interspinous ligaments. Samples were harvested from 10 patients undergoing spinal surgery. The MSCs from facet joints and interspinous ligaments were isolated using direct tissue explant technique. Cell surface antigen profilings were performed via flow cytometry. Their lineage differentiation potentials were analyzed. The facet joints and interspinous ligaments-derived MSCs have the tri-lineage potential to be differentiated into osteogenic, adipogenic, and chondrogenic cells under appropriate inductions. Flow cytometry analysis revealed both cell lines expressed MSCs markers. Both facet joints and interspinous ligaments-derived MSCs expressed marker genes for osteoblasts, adipocytes, and chondrocytes. The facet joints and interspinous ligaments may provide alternative sources of MSCs for tissue engineering applications. The facet joints and interspinous ligaments-derived MSCs are part of the microenvironment of the human ligaments of the spinal column and might play a crucial role in the development and progression of degenerative spine conditions.

Different culture conditions affect the growth of human tendon stem/progenitor cells (TSPCs) within a mixed tendon cells (TCs) population.

PubMed

Viganò, M; Perucca Orfei, C; Colombini, A; Stanco, D; Randelli, P; Sansone, V; de Girolamo, L

2017-12-01

Tendon resident cells (TCs) are a mixed population made of terminally differentiated tenocytes and tendon stem/progenitor cells (TSPCs). Since the enrichment of progenitors proportion could enhance the effectiveness of treatments based on these cell populations, the interest on the effect of culture conditions on the TSPCs is growing. In this study the clonal selection and the culture in presence or absence of basic fibroblast growth factor (bFGF) were used to assess their influences on the stemness properties and phenotype specific features of tendon cells. Cells cultured with the different methods were analyzed in terms of clonogenic and differentiation abilities, stem and tendon specific genes expression and immunophenotype at passage 2 and passage 4. The clonal selection allowed to isolate cells with a higher multi-differentiation potential, but at the same time a lower proliferation rate in comparison to the whole population. Moreover, the clones express a higher amounts of stemness marker OCT4 and tendon specific transcription factor Scleraxis (SCX) mRNA, but a lower level of decorin (DCN). On the other hand, the number of cells obtained by clonal selection was extremely low and most of the clones were unable to reach a high number of passages in cultures. The presence of bFGF influences TCs morphology, enhance their proliferation rate and reduce their clonogenic ability. Interestingly, the expression of CD54, a known mesenchymal stem cell marker, is reduced in presence of bFGF at early passages. Nevertheless, bFGF does not affect the chondrogenic and osteogenic potential of TCs and the expression of tendon specific markers, while it was able to downregulate the OCT4 expression. This study showed that clonal selection enhance progenitors content in TCs populations, but the extremely low number of cells produced with this method could represent an insurmountable obstacle to its application in clinical approaches. We observed that the addition of bFGF to the culture medium promotes the maintenance of a higher number of differentiated cells, reducing the proportion of progenitors within the whole population. Overall our findings demonstrated the importance of the use of specific culture protocols to obtain tendon cells for possible clinical applications.

The Effect of Desulfation of Chondroitin Sulfate on Interactions with Positively Charged Growth Factors and Upregulation of Cartilaginous Markers in Encapsulated MSCs

PubMed Central

Lim, Jeremy J.; Temenoff, Johnna S.

2013-01-01

Sulfated glycosaminoglycans (GAGs) are known to interact electrostatically with positively charged growth factors to modulate signaling. Therefore, regulating the degree of sulfation of GAGs may be a promising approach to tailor biomaterial carriers for controlled growth factor delivery and release. For this study, chondroitin sulfate (CS) was first desulfated to form chondroitin, and resulting crosslinked CS and chondroitin hydrogels were examined in vitro for release of positively charged model protein (histone) and for their effect on cartilaginous differentiation of encapsulated human mesenchymal stem cells (MSCs). Desulfation significantly increased the release of histone from chondroitin hydrogels (30.6±2.3 ìg released over 8 days, compared to natively sulfated CS with 20.2±0.8 ìg), suggesting that sulfation alone plays a significant role in modulating protein interactions with GAG hydrogels. MSCs in chondroitin hydrogels significantly upregulated gene expression of collagen II and aggrecan by day 21 in chondrogenic medium (115±100 and 23.1±7.9 fold upregulation of collagen II and aggrecan, respectively), compared to CS and PEG-based swelling controls, indicating that desulfation may actually enhance the response of MSCs to soluble chondrogenic cues, such as TGF-â1. Thus, desulfated chondroitin materials present a promising biomaterial tool to further investigate electrostatic GAG/growth factor interactions, especially for repair of cartilaginous tissues. PMID:23570717

Recapitulation of physiological spatiotemporal signals promotes in vitro formation of phenotypically stable human articular cartilage

PubMed Central

Wei, Yiyong; Zhou, Bin; Bernhard, Jonathan; Robinson, Samuel; Burapachaisri, Aonnicha; Guo, X. Edward

2017-01-01

Standard isotropic culture fails to recapitulate the spatiotemporal gradients present during native development. Cartilage grown from human mesenchymal stem cells (hMSCs) is poorly organized and unstable in vivo. We report that human cartilage with physiologic organization and in vivo stability can be grown in vitro from self-assembling hMSCs by implementing spatiotemporal regulation during induction. Self-assembling hMSCs formed cartilage discs in Transwell inserts following isotropic chondrogenic induction with transforming growth factor β to set up a dual-compartment culture. Following a switch in the basal compartment to a hypertrophic regimen with thyroxine, the cartilage discs underwent progressive deep-zone hypertrophy and mineralization. Concurrent chondrogenic induction in the apical compartment enabled the maintenance of functional and hyaline cartilage. Cartilage homeostasis, chondrocyte maturation, and terminal differentiation markers were all up-regulated versus isotropic control groups. We assessed the in vivo stability of the cartilage formed under different induction regimens. Cartilage formed under spatiotemporal regulation in vitro resisted endochondral ossification, retained the expression of cartilage markers, and remained organized following s.c. implantation in immunocompromised mice. In contrast, the isotropic control groups underwent endochondral ossification. Cartilage formed from hMSCs remained stable and organized in vivo. Spatiotemporal regulation during induction in vitro recapitulated some aspects of native cartilage development, and potentiated the maturation of self-assembling hMSCs into stable and organized cartilage resembling the native articular cartilage. PMID:28228529

Investigating the effect of peptide agonists on the chondrogenic differentiation of human mesenchymal stem cells using design of experiments.

PubMed

Renner, Julie N; Liu, Julie C

2013-01-01

Human mesenchymal stem cells (MSCs) are attractive for use in cartilage tissue engineering. Cells are often seeded in a structural scaffold containing growth factors. Peptide mimics of full-length growth factors are a promising alternative because they are less expensive and easier to manufacture. We investigated four short peptides for their effect on the chondrogenesis of human MSCs. The peptides were originally designed to mimic bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta 1 (TGF-β1), and insulin, all of which have been shown to affect MSC chondrogenesis. Previous studies demonstrated that the peptides elicited bioactivity in other cell types, but the peptides have not been investigated for their effect on chondrogenesis in human MSCs. In a preliminary investigation, peptides were added to a pellet culture of human MSCs and assayed for their effect on glycosaminoglycan (GAG) production. These experiments determined peptide concentrations used in a full-factorial experiment to investigate any interactions. The experiment revealed the BMP peptide as a robust stimulant for GAG production. . © 2013 American Institute of Chemical Engineers.

Influence of serum percentage on the behavior of Wharton's jelly mesenchymal stem cells in culture.

PubMed

Harmouch, C; El-Omar, R; Labrude, P; Decot, V; Menu, P; Kerdjoudj, H

2013-01-01

Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate into several lineages with valuable applications in regenerative medicine. MSCs differentiation is highly dependent on physicochemical properties of the culture substrate, cell density and on culture medium composition. In this study, we assessed the influence of fetal bovine serum (FBS) level on Wharton's jelly (WJ)-MSCs behavior seeded on polyelectrolyte multilayer films (PEMF) made of four bilayers of poly-allylamine hydrochloride (PAH) as polycation and poly-styrene sulfonate (PSS) as polyanion. MSCs isolated from WJ by explants method were amplified until the third passage. Their phenotypic characterization was performed by flow cytometry analyses. MSCs were seeded on PEMF, in Endothelial growth medium-2 (EGM-2) supplemented by either 5% or 2% FBS. Cell's behavior was monitored for 20 days by optical microscopy and immunofluorescence. Until 2 weeks on glass slides, no difference was observed whatever the FBS percentage. Then with 5% FBS, MSCs formed three-dimensional spheroids on PSS/PAH after 20 days of culture with a nuclear aggregate. Whereas, with 2% FBS, these spheroids did not appear and cells grown in 2D conserved the fibroblast-like morphology. The decrease of FBS percentage from 5% to 2% avoids 3D cell spheroids formation on PAH/PSS. Such results could guide bioengineering towards building 2D structures like cell layers or 3D structures by increasing the osteogenic or chondrogenic differentiation potential of MSCs.

Comparison of Simulated Microgravity and Hydrostatic Pressure for Chondrogenesis of hASC.

PubMed

Mellor, Liliana F; Steward, Andrew J; Nordberg, Rachel C; Taylor, Michael A; Loboa, Elizabeth G

2017-04-01

Cartilage tissue engineering is a growing field due to the lack of regenerative capacity of native tissue. The use of bioreactors for cartilage tissue engineering is common, but the results are controversial. Some studies suggest that microgravity bioreactors are ideal for chondrogenesis, while others show that mimicking hydrostatic pressure is crucial for cartilage formation. A parallel study comparing the effects of loading and unloading on chondrogenesis has not been performed. The goal of this study was to evaluate chondrogenesis of human adipose-derived stem cells (hASC) under two different mechanical stimuli relative to static culture: microgravity and cyclic hydrostatic pressure (CHP). Pellets of hASC were cultured for 14 d under simulated microgravity using a rotating wall vessel bioreactor or under CHP (7.5 MPa, 1 Hz, 4 h · d-1) using a hydrostatic pressure vessel. We found that CHP increased mRNA expression of Aggrecan, Sox9, and Collagen II, caused a threefold increase in sulfated glycosaminoglycan production, and resulted in stronger vimentin staining intensity and organization relative to microgravity. In addition, Wnt-signaling patterns were altered in a manner that suggests that simulated microgravity decreases chondrogenic differentiation when compared to CHP. Our goal was to compare chondrogenic differentiation of hASC using a microgravity bioreactor and a hydrostatic pressure vessel, two commonly used bioreactors in cartilage tissue engineering. Our results indicate that CHP promotes hASC chondrogenesis and that microgravity may inhibit hASC chondrogenesis. Our findings further suggest that cartilage formation and regeneration might be compromised in space due to the lack of mechanical loading.Mellor LF, Steward AJ, Nordberg RC, Taylor MA, Loboa EG. Comparison of simulated microgravity and hydrostatic pressure for chondrogenesis of hASC. Aerosp Med Hum Perform. 2017; 88(4):377-384.

Three-dimensional dynamic fabrication of engineered cartilage based on chitosan/gelatin hybrid hydrogel scaffold in a spinner flask with a special designed steel frame.

PubMed

Song, Kedong; Li, Liying; Li, Wenfang; Zhu, Yanxia; Jiao, Zeren; Lim, Mayasari; Fang, Meiyun; Shi, Fangxin; Wang, Ling; Liu, Tianqing

2015-10-01

Cartilage transplantation using in vitro tissue engineered cartilage is considered a promising treatment for articular cartilage defects. In this study, we assessed the advantages of adipose derived stem cells (ADSCs) combined with chitosan/gelatin hybrid hydrogel scaffolds, which acted as a cartilage biomimetic scaffold, to fabricate a tissue engineered cartilage dynamically in vitro and compared this with traditional static culture. Physical properties of the hydrogel scaffolds were evaluated and ADSCs were inoculated into the hydrogel at a density of 1×10(7) cells/mL and cultured in a spinner flask with a special designed steel framework and feed with chondrogenic inductive media for two weeks. The results showed that the average pore size, porosity, swelling rate and elasticity modulus of hybrid scaffolds with good biocompatibility were 118.25±19.51 μm, 82.60±2.34%, 361.28±0.47% and 61.2±0.16 kPa, respectively. ADSCs grew well in chitosan/gelatin hybrid scaffold and successfully differentiated into chondrocytes, showing that the scaffolds were suitable for tissue engineering applications in cartilage regeneration. Induced cells cultivated in a dynamic spinner flask with a special designed steel frame expressed more proteoglycans and the cell distribution was much more uniform with the scaffold being filled mostly with extracellular matrix produced by cells. A spinner flask with framework promoted proliferation and chondrogenic differentiation of ADSCs within chitosan/gelatin hybrid scaffolds and accelerated dynamic fabrication of cell-hydrogel constructs, which could be a selective and good method to construct tissue engineered cartilage in vitro. Copyright © 2015 Elsevier B.V. All rights reserved.

Hyaluronic acid enhances the effect of the PAMPS/PDMAAm double-network hydrogel on chondrogenic differentiation of ATDC5 cells

PubMed Central

2014-01-01

Background A double-network (DN) gel, which was composed of poly-(2-Acrylamido-2-methylpropanesulfonic acid) and poly-(N,N’-dimethyl acrylamide) (PAMPS/PDMAAm), has the potential to induce chondrogenesis both in vitro and in vivo. The present study investigated whether DN gel induced chondrogenic differentiation of ATDC5 cells in a maintenance medium without insulin, and whether supplementation of hyaluronic acid enhanced the chondrogenic differentiation effect of DN gel. Methods ATDC5 cells were cultured on the DN gel and the polystyrene (PS) dish in maintenance media without insulin for 21 days. Hyaluronic acid having a molecular weight of approximately 800 kDa was supplemented into the medium so that the concentration became 0.01, 0.1, or 1.0 mg/mL. The cultured cells were evaluated using immunocytochemistry for type-2 collagen and real time PCR for gene expression of type-2 collagen, aggrecan, and Sox9 at 7 and 21 days of culture. Results The cells cultured on the DN gel formed nodules and were stained with an anti-type-2 collagen antibody, and expression of type-2 collagen and aggrecan mRNA was significantly greater on the DN gel than on the PS dish surface (p < 0.05) in the hyaluronic acid-free maintenance medium. Hyaluronic acid supplementation of a high concentration (1.0 mg/mL) significantly enhanced expression of type-2 collagen and aggrecan mRNA in comparison with culture without hyaluronic acid at 21 days (p < 0.05). Conclusions The DN gel induced chondrogenic differentiation of ATDC5 cells without insulin. This effect was significantly affected by hyaluronic acid, depending on the level of concentration. There is a high possibility that hyaluronic acid plays an important role in the in vivo hyaline cartilage regeneration phenomenon induced by the DN gel. PMID:24997593

Hyaluronic acid enhances the effect of the PAMPS/PDMAAm double-network hydrogel on chondrogenic differentiation of ATDC5 cells.

PubMed

Kitamura, Nobuto; Kurokawa, Takayuki; Fukui, Takaaki; Gong, Jian P; Yasuda, Kazunori

2014-07-06

A double-network (DN) gel, which was composed of poly-(2-Acrylamido-2-methylpropanesulfonic acid) and poly-(N,N'-dimethyl acrylamide) (PAMPS/PDMAAm), has the potential to induce chondrogenesis both in vitro and in vivo. The present study investigated whether DN gel induced chondrogenic differentiation of ATDC5 cells in a maintenance medium without insulin, and whether supplementation of hyaluronic acid enhanced the chondrogenic differentiation effect of DN gel. ATDC5 cells were cultured on the DN gel and the polystyrene (PS) dish in maintenance media without insulin for 21 days. Hyaluronic acid having a molecular weight of approximately 800 kDa was supplemented into the medium so that the concentration became 0.01, 0.1, or 1.0 mg/mL. The cultured cells were evaluated using immunocytochemistry for type-2 collagen and real time PCR for gene expression of type-2 collagen, aggrecan, and Sox9 at 7 and 21 days of culture. The cells cultured on the DN gel formed nodules and were stained with an anti-type-2 collagen antibody, and expression of type-2 collagen and aggrecan mRNA was significantly greater on the DN gel than on the PS dish surface (p < 0.05) in the hyaluronic acid-free maintenance medium. Hyaluronic acid supplementation of a high concentration (1.0 mg/mL) significantly enhanced expression of type-2 collagen and aggrecan mRNA in comparison with culture without hyaluronic acid at 21 days (p < 0.05). The DN gel induced chondrogenic differentiation of ATDC5 cells without insulin. This effect was significantly affected by hyaluronic acid, depending on the level of concentration. There is a high possibility that hyaluronic acid plays an important role in the in vivo hyaline cartilage regeneration phenomenon induced by the DN gel.

Structure-function relationships in the stem cell's mechanical world A: seeding protocols as a means to control shape and fate of live stem cells.

PubMed

Zimmermann, Joshua A; Knothe Tate, Melissa L

2011-12-01

Shape and fate are intrinsic manifestations of form and function at the cell scale. Here we hypothesize that seeding density and protocol affect the form and function of live embryonic murine mesenchymal stem cells (MSCs) and their nuclei. First, the imperative for study of live cells was demonstrated in studies showing changes in cell nucleus shape that were attributable to fixation per se. Hence, we compared live cell and nuclear volume and shape between groups of a model MSC line (C3H10T1/2) seeded at, or proliferated from 5,000 cells/cm2 to one of three target densities to achieve targeted development contexts. Cell volume was shown to be dependent on initial seeding density whereas nucleus shape was shown to depend on developmental context but not seeding density. Both smaller cell volumes and flatter nuclei were found to correlate with increased expression of markers for mesenchymal condensation as well as chondrogenic and osteogenic differentiation but a decreased expression of pre-condensation and adipogenic markers. Considering the data presented here, both seeding density and protocol significantly alter the morphology of mesenchymal stem cells even at very early stages of cell culture. Thus, these design parameters may play a critical role in the success of tissue engineering strategies seeking to recreate condensation events. However, a better understanding of how these changes in cell volume and nucleus shape relate to the differentiation of MSCs is important for prescribing precise seeding conditions necessary for the development of the desired tissue type. In a companion study (Part B, following), we address the effect of concomitant volume and shape changing stresses on spatiotemporal distribution of the cytoskeletal proteins actin and tubulin. Taken together, these studies bring us one step closer to our ultimate goal of elucidating the dynamics of nucleus and cell shape change as tissue templates grow (cell proliferation) and specialize (cell differentiation).

The potential use of stem cells derived from human amniotic fluid in renal diseases.

PubMed

Noronha, Irene L; Cavaglieri, Rita C; Janz, Felipe L; Duarte, Sergio A; Lopes, Marco A B; Zugaib, Marcelo; Bydlowski, Sergio P

2011-09-01

Amniotic fluid (AF) contains a variety of cell types derived from fetal tissues that can easily grow in culture. These cells can be obtained during amniocentesis for prenatal screening of fetal genetic diseases, usually performed during the second trimester of pregnancy. Of particular interest, some expanded sub-populations derived from AF cells are capable of extensive self-renewal and maintain prolonged undifferentiated proliferation, which are defining properties of stem cells. These human AF stem cells (hAFSCs) exhibit multilineage potential and can differentiate into the three germ layers. They have high proliferation rates and express mesenchymal and embryonic markers, but do not induce tumor formation. In this study, hAFSCs derived from amniocentesis performed at 16-20 weeks of pregnancy were isolated, grown in culture, and characterized by flow cytometry and by their potential ability to differentiate into osteogenic, adipogenic, and chondrogenic lineages. After 4-7 passages, 5 × 10 5 hAFSCs were inoculated under the kidney capsule of Wistar rats that were subjected to an experimental model of chronic renal disease, the 5/6 nephrectomy model (Nx). After 30 days, Nx rats treated with hAFSCs displayed significant reductions in blood pressure, proteinuria, macrophages, and α-smooth muscle actin expression compared with Nx animals. These preliminary results suggest that hAFSCs isolated and expanded from AF obtained by routine amniocentesis can promote renoprotection in the Nx model. Considering that the AF cells not used for fetal karyotyping are usually discarded, and that their use does not raise ethical issues, they may represent an alternative source of stem cells for cell therapy and regenerative medicine.

Anisotropic Shape-Memory Alginate Scaffolds Functionalized with Either Type I or Type II Collagen for Cartilage Tissue Engineering.

PubMed

Almeida, Henrique V; Sathy, Binulal N; Dudurych, Ivan; Buckley, Conor T; O'Brien, Fergal J; Kelly, Daniel J

2017-01-01

Regenerating articular cartilage and fibrocartilaginous tissue such as the meniscus is still a challenge in orthopedic medicine. While a range of different scaffolds have been developed for joint repair, none have facilitated the development of a tissue that mimics the complexity of soft tissues such as articular cartilage. Furthermore, many of these scaffolds are not designed to function in mechanically challenging joint environments. The overall goal of this study was to develop a porous, biomimetic, shape-memory alginate scaffold for directing cartilage regeneration. To this end, a scaffold was designed with architectural cues to guide cellular and neo-tissue alignment, which was additionally functionalized with a range of extracellular matrix cues to direct stem cell differentiation toward the chondrogenic lineage. Shape-memory properties were introduced by covalent cross-linking alginate using carbodiimide chemistry, while the architecture of the scaffold was modified using a directional freezing technique. Introducing such an aligned pore structure was found to improve the mechanical properties of the scaffold, and promoted higher levels of sulfated glycosaminoglycans (sGAG) and collagen deposition compared to an isotropic (nonaligned) pore geometry when seeded with adult human stem cells. Functionalization with collagen improved stem cell recruitment into the scaffold and facilitated more homogenous cartilage tissue deposition throughout the construct. Incorporating type II collagen into the scaffolds led to greater cell proliferation, higher sGAG and collagen accumulation, and the development of a stiffer tissue compared to scaffolds functionalized with type I collagen. The results of this study demonstrate how both scaffold architecture and composition can be tailored in a shape-memory alginate scaffold to direct stem cell differentiation and support the development of complex cartilaginous tissues.

Cell source-dependent in vivo immunosuppressive properties of mesenchymal stem cells derived from the bone marrow and synovial fluid of minipigs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Won-Jae; Hah, Young-Sool; Ock, Sun-A.

The in vitro differentiation and immunosuppressive capacity of mesenchymal stem cells (MSCs) derived from synovial fluid (SF-MSCs) and bone marrow extract (BM-MSCs) in an isogenic background of minipigs were comparatively analyzed in a collagen-induced arthritis (CIA) mouse model of rheumatoid arthritis (RA). The proliferation capacity and expression of pluripotent transcription factors (Oct3/4 and Sox2) were significantly (P<0.05) higher in SF-MSCs than in BM-MSCs. The differentiation capacity of SF-MSCs into adipocytes, osteocytes and neurocytes was significantly (P<0.05) lower than that of BM-MSCs, and the differentiation capacity of SF-MSCs into chondrocytes was significantly (P<0.05) higher than that of BM-MSCs. Systemic injection ofmore » BM- and SF-MSCs significantly (P<0.05) ameliorated the clinical symptoms of CIA mice, with SF-MSCs having significantly (P<0.05) higher clinical and histopathological recovery scores than BM-MSCs. Furthermore, the immunosuppressive properties of SF-MSCs in CIA mice were associated with increased levels of the anti-inflammatory cytokine interleukin (IL)-10, and decreased levels of the pro-inflammatory cytokine IL-1β and osteoclast-related sRANKL. In conclusion, SF-MSCs exhibited eminent pluripotency and differentiation capacity into chondrocytes, addition to substantial in vivo immunosuppressive capacity by elevating IL-10 and reducing IL-1β levels in CIA mice. - Highlights: • Immunosuppressive capacity of BM-, SM-, and SF-MSCs was evaluated in an RA model. • Proliferation, pluripotency and chondrogenic differentiation capacity were higher in SF-MSCs. • SF-MSCs exhibited improved therapeutic effects than BM-MSCs. • SF-MSCs may have applications as immunosuppressive therapy in autoimmune diseases.« less

Cryopreservation Method for the Effective Collection of Dental Pulp Stem Cells.

PubMed

Takebe, Yusuke; Tatehara, Seiko; Fukushima, Tatsuhiro; Tokuyama-Toda, Reiko; Yasuhara, Rika; Mishima, Kenji; Satomura, Kazuhito

2017-05-01

Dental pulp stem cells (DPSCs) are an attractive cell source for use in cell-based therapy, regenerative medicine, and tissue engineering because DPSCs have a high cell proliferation ability and multidifferentiation capacity. However, several problems are associated with the collection and preservation of DPSCs for use in future cell-based therapy. In particular, the isolation of DPSCs for cryopreservation is time consuming and expensive. In this study, we developed a novel cryopreservation method (NCM) for dental pulp tissues to isolate suitable DPSCs after thawing cryopreserved tissue. Using the NCM, dental pulp tissues were cultured on adhesion culture dishes for 5 days and then cryopreserved. After thawing, the cryopreserved dental pulp tissue fragments exhibited cell migration. We evaluated each property of DPSCs isolated using the NCM (DPSCs-NCM) and the explant method alone without cryopreservation (DPSCs-C). DPSCs-NCM had the same proliferation capacity as DPSCs-C. Flow cytometry (FACS) analysis indicated that both DPSCs-NCM and DPSCs-C were positive for mesenchymal stem cell markers at the same level but negative for hematopoietic cell markers. Moreover, both DPSCs-NCM and DPSCs-C could differentiate into osteogenic, chondrogenic, and adipogenic cells during culture in each induction medium. These results suggest that DPSCs-NCM may be mesenchymal stem cells. Therefore, our novel method might facilitate the less expensive cryopreservation of DPSCs, thereby providing suitable DPSCs for use in patients in future cell-based therapies.

Repair of Osteochondral Defects Using Human Umbilical Cord Wharton's Jelly-Derived Mesenchymal Stem Cells in a Rabbit Model

PubMed Central

Jia, Yanhui; Yuan, Mei; Guo, Weimin; Huang, Jingxiang; Zhao, Bin; Xu, Wenjing; Lu, Shibi

2017-01-01

Umbilical cord Wharton's jelly-derived mesenchymal stem cell (WJMSC) is a new-found mesenchymal stem cell in recent years with multiple lineage potential. Due to its abundant resources, no damage procurement, and lower immunogenicity than other adult MSCs, WJMSC promises to be a good xenogenous cell candidate for tissue engineering. This in vivo pilot study explored the use of human umbilical cord Wharton's jelly mesenchymal stem cells (hWJMSCs) containing a tissue engineering construct xenotransplant in rabbits to repair full-thickness cartilage defects in the femoral patellar groove. We observed orderly spatial-temporal remodeling of hWJMSCs into cartilage tissues during repair over 16 months, with characteristic architectural features, including a hyaline-like neocartilage layer with good surface regularity, complete integration with adjacent host cartilage, and regenerated subchondral bone. No immune rejection was detected when xenograft hWJMSCs were implanted into rabbit cartilage defects. The repair results using hWJMSCs were superior to those of chondrogenically induced hWJMSCs after assessing gross appearance and histological grading scores. These preliminary results suggest that using novel undifferentiated hWJMSCs as seed cells might be a better approach than using transforming growth factor-β-induced differentiated hWJMSCs for in vivo tissue engineering treatment of cartilage defects. hWJMSC allografts may be promising for clinical applications. PMID:28261617

Chondrogenic potential of physically treated bovine cartilage matrix derived porous scaffolds on human dermal fibroblast cells.

PubMed

Moradi, Ali; Ataollahi, Forough; Sayar, Katayoun; Pramanik, Sumit; Chong, Pan-Pan; Khalil, Alizan Abdul; Kamarul, Tunku; Pingguan-Murphy, Belinda

2016-01-01

Extracellular matrices have drawn attention in tissue engineering as potential biomaterials for scaffold fabrication because of their bioactive components. Noninvasive techniques of scaffold fabrication and cross-linking treatments are believed to maintain the integrity of bioactive molecules while providing proper architectural and mechanical properties. Cartilage matrix derived scaffolds are designed to support the maintenance of chondrocytes and provide proper signals for differentiation of chondroinducible cells. Chondroinductive potential of bovine articular cartilage matrix derived porous scaffolds on human dermal fibroblasts and the effect of scaffold shrinkage on chondrogenesis were investigated. An increase in sulfated glycosaminoglycans production along with upregulation of chondrogenic genes confirmed that physically treated cartilage matrix derived scaffolds have chondrogenic potential on human dermal fibroblasts. © 2015 Wiley Periodicals, Inc.

FGFR3 induces degradation of BMP type I receptor to regulate skeletal development.

PubMed

Qi, Huabing; Jin, Min; Duan, Yaqi; Du, Xiaolan; Zhang, Yuanquan; Ren, Fangli; Wang, Yinyin; Tian, Qingyun; Wang, Xiaofeng; Wang, Quan; Zhu, Ying; Xie, Yangli; Liu, Chuanju; Cao, Xu; Mishina, Yuji; Chen, Di; Deng, Chu-xia; Chang, Zhijie; Chen, Lin

2014-07-01

Fibroblast growth factors (FGFs) and their receptors (FGFRs) play significant roles in vertebrate organogenesis and morphogenesis. FGFR3 is a negative regulator of chondrogenesis and multiple mutations with constitutive activity of FGFR3 result in achondroplasia, one of the most common dwarfisms in humans, but the molecular mechanism remains elusive. In this study, we found that chondrocyte-specific deletion of BMP type I receptor a (Bmpr1a) rescued the bone overgrowth phenotype observed in Fgfr3 deficient mice by reducing chondrocyte differentiation. Consistently, using in vitro chondrogenic differentiation assay system, we demonstrated that FGFR3 inhibited BMPR1a-mediated chondrogenic differentiation. Furthermore, we showed that FGFR3 hyper-activation resulted in impaired BMP signaling in chondrocytes of mouse growth plates. We also found that FGFR3 inhibited BMP-2- or constitutively activated BMPR1-induced phosphorylation of Smads through a mechanism independent of its tyrosine kinase activity. We found that FGFR3 facilitates BMPR1a to degradation through Smurf1-mediated ubiquitination pathway. We demonstrated that down-regulation of BMP signaling by BMPR1 inhibitor dorsomorphin led to the retardation of chondrogenic differentiation, which mimics the effect of FGF-2 on chondrocytes and BMP-2 treatment partially rescued the retarded growth of cultured bone rudiments from thanatophoric dysplasia type II mice. Our findings reveal that FGFR3 promotes the degradation of BMPR1a, which plays an important role in the pathogenesis of FGFR3-related skeletal dysplasia. Copyright © 2014 Elsevier B.V. All rights reserved.

Perivascular Mesenchymal Stem Cells in Sheep: Characterization and Autologous Transplantation in a Model of Articular Cartilage Repair.

PubMed

Hindle, Paul; Baily, James; Khan, Nusrat; Biant, Leela C; Simpson, A Hamish R; Péault, Bruno

2016-11-01

Previous research has indicated that purified perivascular stem cells (PSCs) have increased chondrogenic potential compared to conventional mesenchymal stem cells (MSCs) derived in culture. This study aimed to develop an autologous large animal model for PSC transplantation and to specifically determine if implanted cells are retained in articular cartilage defects. Immunohistochemistry and fluorescence-activated cell sorting were used to ascertain the reactivity of anti-human and anti-ovine antibodies, which were combined and used to identify and isolate pericytes (CD34 - CD45 - CD146 + ) and adventitial cells (CD34 + CD45 - CD146 - ). The purified cells demonstrated osteogenic, adipogenic, and chondrogenic potential in culture. Autologous ovine PSCs (oPSCs) were isolated, cultured, and efficiently transfected using a green fluorescence protein (GFP) encoding lentivirus. The cells were implanted into articular cartilage defects on the medial femoral condyle using hydrogel and collagen membranes. Four weeks following implantation, the condyle was explanted and confocal laser scanning microscopy demonstrated the presence of oPSCs in the defect repaired with the hydrogel. These data suggest the testability in a large animal of native MSC autologous grafting, thus avoiding possible biases associated with xenotransplantation. Such a setting will be used in priority for indications in orthopedics, at first to model articular cartilage repair.

Influence of three-dimensional hyaluronic acid microenvironments on mesenchymal stem cell chondrogenesis.

PubMed

Chung, Cindy; Burdick, Jason A

2009-02-01

Mesenchymal stem cells (MSCs) are multipotent progenitor cells whose plasticity and self-renewal capacity have generated significant interest for applications in tissue engineering. The objective of this study was to investigate MSC chondrogenesis in photo-cross-linked hyaluronic acid (HA) hydrogels. Because HA is a native component of cartilage, and MSCs may interact with HA via cell surface receptors, these hydrogels could influence stem cell differentiation. In vitro and in vivo cultures of MSC-laden HA hydrogels permitted chondrogenesis, measured by the early gene expression and production of cartilage-specific matrix proteins. For in vivo culture, MSCs were encapsulated with and without transforming growth factor beta-3 (TGF-beta3) or pre-cultured for 2 weeks in chondrogenic medium before implantation. Up-regulation of type II collagen, aggrecan, and sox 9 was observed for all groups over MSCs at the time of encapsulation, and the addition of TGF-beta3 further enhanced the expression of these genes. To assess the influence of scaffold chemistry on chondrogenesis, HA hydrogels were compared with relatively inert poly(ethylene glycol) (PEG) hydrogels and showed enhanced expression of cartilage-specific markers. Differences between HA and PEG hydrogels in vivo were most noticeable for MSCs and polymer alone, indicating that hydrogel chemistry influences the commitment of MSCs to undergo chondrogenesis (e.g., approximately 43-fold up-regulation of type II collagen of MSCs in HA over PEG hydrogels). Although this study investigated only early markers of tissue regeneration, these results emphasize the importance of material cues in MSC differentiation microenvironments, potentially through interactions between scaffold materials and cell surface receptors.

Hypoxia-cultured human adipose-derived mesenchymal stem cells are non-oncogenic and have enhanced viability, motility, and tropism to brain cancer

PubMed Central

Feng, Y; Zhu, M; Dangelmajer, S; Lee, Y M; Wijesekera, O; Castellanos, C X; Denduluri, A; Chaichana, K L; Li, Q; Zhang, H; Levchenko, A; Guerrero-Cazares, H; Quiñones-Hinojosa, A

2014-01-01

Adult human adipose-derived mesenchymal stem cells (hAMSCs) are multipotent cells, which are abundant, easily collected, and bypass the ethical concerns that plague embryonic stem cells. Their utility and accessibility have led to the rapid development of clinical investigations to explore their autologous and allogeneic cellular-based regenerative potential, tissue preservation capabilities, anti-inflammatory properties, and anticancer properties, among others. hAMSCs are typically cultured under ambient conditions with 21% oxygen. However, physiologically, hAMSCs exist in an environment of much lower oxygen tension. Furthermore, hAMSCs cultured in standard conditions have shown limited proliferative and migratory capabilities, as well as limited viability. This study investigated the effects hypoxic culture conditions have on primary intraoperatively derived hAMSCs. hAMSCs cultured under hypoxia (hAMSCs-H) remained multipotent, capable of differentiation into osteogenic, chondrogenic, and adipogenic lineages. In addition, hAMSCs-H grew faster and exhibited less cell death. Furthermore, hAMSCs-H had greater motility than normoxia-cultured hAMSCs and exhibited greater homing ability to glioblastoma (GBM) derived from brain tumor-initiating cells from our patients in vitro and in vivo. Importantly, hAMSCs-H did not transform into tumor-associated fibroblasts in vitro and were not tumorigenic in vivo. Rather, hAMSCs-H promoted the differentiation of brain cancer cells in vitro and in vivo. These findings suggest an alternative culturing technique that can enhance the function of hAMSCs, which may be necessary for their use in the treatment of various pathologies including stroke, myocardial infarction, amyotrophic lateral sclerosis, and GBM. PMID:25501828

Highly Efficient In Vitro Reparative Behaviour of Dental Pulp Stem Cells Cultured with Standardised Platelet Lysate Supplementation.

PubMed

Marrazzo, Pasquale; Paduano, Francesco; Palmieri, Francesca; Marrelli, Massimo; Tatullo, Marco

2016-01-01

Dental pulp is an accessible source of multipotent mesenchymal stromal cells (MSCs). The perspective role of dental pulp stem cells (DPSCs) in regenerative medicine demands an in vitro expansion and in vivo delivery which must deal with the safety issues about animal serum, usually required in cell culture practice. Human platelet lysate (PL) contains autologous growth factors and has been considered as valuable alternative to fetal bovine serum (FBS) in cell cultures. The optimum concentration to be added of such supplement is highly dependent on its preparation whose variability limits comparability of results. By in vitro experiments, we aimed to evaluate a standardised formulation of pooled PL. A low selected concentration of PL (1%) was able to support the growth and maintain the viability of the DPSCs. The use of PL in cell cultures did not impair cell surface signature typically expressed by MSCs and even upregulated the transcription of Sox2. Interestingly, DPSCs cultured in presence of PL exhibited a higher healing rate after injury and are less susceptible to toxicity mediated by exogenous H 2 O 2 than those cultured with FBS. Moreover, PL addition was shown as a suitable option for protocols promoting osteogenic and chondrogenic differentiation of DPSCs. Taken together, our results indicated that PL is a valid substitute of FBS to culture and differentiate DPSCs for clinical-grade use.

Highly Efficient In Vitro Reparative Behaviour of Dental Pulp Stem Cells Cultured with Standardised Platelet Lysate Supplementation

PubMed Central

Palmieri, Francesca; Marrelli, Massimo

2016-01-01

Dental pulp is an accessible source of multipotent mesenchymal stromal cells (MSCs). The perspective role of dental pulp stem cells (DPSCs) in regenerative medicine demands an in vitro expansion and in vivo delivery which must deal with the safety issues about animal serum, usually required in cell culture practice. Human platelet lysate (PL) contains autologous growth factors and has been considered as valuable alternative to fetal bovine serum (FBS) in cell cultures. The optimum concentration to be added of such supplement is highly dependent on its preparation whose variability limits comparability of results. By in vitro experiments, we aimed to evaluate a standardised formulation of pooled PL. A low selected concentration of PL (1%) was able to support the growth and maintain the viability of the DPSCs. The use of PL in cell cultures did not impair cell surface signature typically expressed by MSCs and even upregulated the transcription of Sox2. Interestingly, DPSCs cultured in presence of PL exhibited a higher healing rate after injury and are less susceptible to toxicity mediated by exogenous H2O2 than those cultured with FBS. Moreover, PL addition was shown as a suitable option for protocols promoting osteogenic and chondrogenic differentiation of DPSCs. Taken together, our results indicated that PL is a valid substitute of FBS to culture and differentiate DPSCs for clinical-grade use. PMID:27774106

Combinatory approach for developing silk fibroin scaffolds for cartilage regeneration.

PubMed

Ribeiro, Viviana P; da Silva Morais, Alain; Maia, F Raquel; Canadas, Raphael F; Costa, João B; Oliveira, Ana L; Oliveira, Joaquim M; Reis, Rui L

2018-05-01

Several processing technologies and engineering strategies have been combined to create scaffolds with superior performance for efficient tissue regeneration. Cartilage tissue is a good example of that, presenting limited self-healing capacity together with a high elasticity and load-bearing properties. In this work, novel porous silk fibroin (SF) scaffolds derived from horseradish peroxidase (HRP)-mediated crosslinking of highly concentrated aqueous SF solution (16 wt%) in combination with salt-leaching and freeze-drying methodologies were developed for articular cartilage tissue engineering (TE) applications. The HRP-crosslinked SF scaffolds presented high porosity (89.3 ± 0.6%), wide pore distribution and high interconnectivity (95.9 ± 0.8%). Moreover, a large swelling capacity and favorable degradation rate were observed up to 30 days, maintaining the porous-like structure and β-sheet conformational integrity obtained with salt-leaching and freeze-drying processing. The in vitro studies supported human adipose-derived stem cells (hASCs) adhesion, proliferation, and high glycosaminoglycans (GAGs) synthesis under chondrogenic culture conditions. Furthermore, the chondrogenic differentiation of hASCs was assessed by the expression of chondrogenic-related markers (collagen type II, Sox-9 and Aggrecan) and deposition of cartilage-specific extracellular matrix for up to 28 days. The cartilage engineered constructs also presented structural integrity as their mechanical properties were improved after chondrogenic culturing. Subcutaneous implantation of the scaffolds in CD-1 mice demonstrated no necrosis or calcification, and deeply tissue ingrowth. Collectively, the structural properties and biological performance of these porous HRP-crosslinked SF scaffolds make them promising candidates for cartilage regeneration. In cartilage tissue engineering (TE), several processing technologies have been combined to create scaffolds for efficient tissue repair. In our study, we propose novel silk fibroin (SF) scaffolds derived from enzymatically crosslinked SF hydrogels processed by salt-leaching and freeze-drying technologies, for articular cartilage applications. Though these scaffolds, we were able to combine the elastic properties of hydrogel-based systems, with the stability, resilience and controlled porosity of scaffolds processed via salt-leaching and freeze-drying technologies. SF protein has been extensively explored for TE applications, as a result of its mechanical strength, elasticity, biocompatibility, and biodegradability. Thus, the structural, mechanical and biological performance of the proposed scaffolds potentiates their use as three-dimensional matrices for cartilage regeneration. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Nanostructured 3D constructs based on chitosan and chondroitin sulphate multilayers for cartilage tissue engineering.

PubMed

Silva, Joana M; Georgi, Nicole; Costa, Rui; Sher, Praveen; Reis, Rui L; Van Blitterswijk, Clemens A; Karperien, Marcel; Mano, João F

2013-01-01

Nanostructured three-dimensional constructs combining layer-by-layer technology (LbL) and template leaching were processed and evaluated as possible support structures for cartilage tissue engineering. Multilayered constructs were formed by depositing the polyelectrolytes chitosan (CHT) and chondroitin sulphate (CS) on either bidimensional glass surfaces or 3D packet of paraffin spheres. 2D CHT/CS multi-layered constructs proved to support the attachment and proliferation of bovine chondrocytes (BCH). The technology was transposed to 3D level and CHT/CS multi-layered hierarchical scaffolds were retrieved after paraffin leaching. The obtained nanostructured 3D constructs had a high porosity and water uptake capacity of about 300%. Dynamical mechanical analysis (DMA) showed the viscoelastic nature of the scaffolds. Cellular tests were performed with the culture of BCH and multipotent bone marrow derived stromal cells (hMSCs) up to 21 days in chondrogenic differentiation media. Together with scanning electronic microscopy analysis, viability tests and DNA quantification, our results clearly showed that cells attached, proliferated and were metabolically active over the entire scaffold. Cartilaginous extracellular matrix (ECM) formation was further assessed and results showed that GAG secretion occurred indicating the maintenance of the chondrogenic phenotype and the chondrogenic differentiation of hMSCs.

Hypothermia reduces VEGF-165 expression, but not osteogenic differentiation of human adipose stem cells under hypoxia

PubMed Central

Bakker, Astrid D.; Hogervorst, Jolanda M. A.; Nolte, Peter A.; Klein-Nulend, Jenneke

2017-01-01

Cryotherapy is successfully used in the clinic to reduce pain and inflammation after musculoskeletal damage, and might prevent secondary tissue damage under the prevalent hypoxic conditions. Whether cryotherapy reduces mesenchymal stem cell (MSC) number and differentiation under hypoxic conditions, causing impaired callus formation is unknown. We aimed to determine whether hypothermia modulates proliferation, apoptosis, nitric oxide production, VEGF gene and protein expression, and osteogenic/chondrogenic differentiation of human MSCs under hypoxia. Human adipose MSCs were cultured under hypoxia (37°C, 1% O2), hypothermia and hypoxia (30°C, 1% O2), or control conditions (37°C, 20% O2). Total DNA, protein, nitric oxide production, alkaline phosphatase activity, gene expression, and VEGF protein concentration were measured up to day 8. Hypoxia enhanced KI67 expression at day 4. The combination of hypothermia and hypoxia further enhanced KI67 gene expression compared to hypoxia alone, but was unable to prevent the 1.2-fold reduction in DNA amount caused by hypoxia at day 4. Addition of hypothermia to hypoxic cells did not alter the effect of hypoxia alone on BAX-to-BCL-2 ratio, alkaline phosphatase activity, gene expression of SOX9, COL1, or osteocalcin, or nitric oxide production. Hypothermia decreased the stimulating effect of hypoxia on VEGF-165 gene expression by 6-fold at day 4 and by 2-fold at day 8. Hypothermia also decreased VEGF protein expression under hypoxia by 2.9-fold at day 8. In conclusion, hypothermia decreased VEGF-165 gene and protein expression, but did not affect differentiation, or apoptosis of MSCs cultured under hypoxia. These in vitro results implicate that hypothermia treatment in vivo, applied to alleviate pain and inflammation, is not likely to harm early stages of callus formation. PMID:28166273

Effect of different freezing rates during cryopreservation of rat mesenchymal stem cells using combinations of hydroxyethyl starch and dimethylsulfoxide

PubMed Central

2012-01-01

Background Mesenchymal stem cells (MSCs) are increasingly used as therapeutic agents as well as research tools in regenerative medicine. Development of technologies which allow storing and banking of MSC with minimal loss of cell viability, differentiation capacity, and function is required for clinical and research applications. Cryopreservation is the most effective way to preserve cells long term, but it involves potentially cytotoxic compounds and processing steps. Here, we investigate the effect of decreasing dimethyl sulfoxide (DMSO) concentrations in cryosolution by substituting with hydroxyethyl starch (HES) of different molecular weights using different freezing rates. Post-thaw viability, phenotype and osteogenic differentiation capacity of MSCs were analysed. Results The study confirms that, for rat MSC, cryopreservation effects need to be assessed some time after, rather than immediately after thawing. MSCs cryopreserved with HES maintain their characteristic cell surface marker expression as well as the osteogenic, adipogenic and chondrogenic differentiation potential. HES alone does not provide sufficient cryoprotection for rat MSCs, but provides good cryoprotection in combination with DMSO, permitting the DMSO content to be reduced to 5%. There are indications that such a combination would seem useful not just for the clinical disadvantages of DMSO but also based on a tendency for reduced osteogenic differentiation capacity of rat MSC cryopreserved with high DMSO concentration. HES molecular weight appears to play only a minor role in its capacity to act as a cryopreservation solution for MSC. The use of a ‘straight freeze’ protocol is no less effective in maintaining post-thaw viability of MSC compared to controlled rate freezing methods. Conclusion A 5% DMSO / 5% HES solution cryopreservation solution using a ‘straight freeze’ approach can be recommended for rat MSC. PMID:22889198

Use of Rat Mature Adipocyte-Derived Dedifferentiated Fat Cells as a Cell Source for Periodontal Tissue Regeneration

PubMed Central

Akita, Daisuke; Kano, Koichiro; Saito-Tamura, Yoko; Mashimo, Takayuki; Sato-Shionome, Momoko; Tsurumachi, Niina; Yamanaka, Katsuyuki; Kaneko, Tadashi; Toriumi, Taku; Arai, Yoshinori; Tsukimura, Naoki; Matsumoto, Taro; Ishigami, Tomohiko; Isokawa, Keitaro; Honda, Masaki

2016-01-01

Lipid-free fibroblast-like cells, known as dedifferentiated fat (DFAT) cells, can be generated from mature adipocytes with a large single lipid droplet. DFAT cells can re-establish their active proliferation ability and can transdifferentiate into various cell types under appropriate culture conditions. The first objective of this study was to compare the multilineage differentiation potential of DFAT cells with that of adipose-derived stem cells (ASCs) on mesenchymal stem cells. We obtained DFAT cells and ASCs from inbred rats and found that rat DFAT cells possess higher osteogenic differentiation potential than rat ASCs. On the other hand, DFAT cells show similar adipogenic differentiation, and chondrogenic differentiation potential in comparison with ASCs. The second objective of this study was to assess the regenerative potential of DFAT cells combined with novel solid scaffolds composed of PLGA (Poly d, l-lactic-co-glycolic acid) on periodontal tissue, and to compare this with the regenerative potential of ASCs combined with PLGA scaffolds. Cultured DFAT cells and ASCs were seeded onto PLGA scaffolds (DFAT/PLGA and ASCs/PLGA) and transplanted into periodontal fenestration defects in rat mandible. Micro computed tomography analysis revealed a significantly higher amount of bone regeneration in the DFAT/PLGA group compared with that of ASCs/PLGA and PLGA-alone groups at 2, 3, and 5 weeks after transplantation. Similarly, histomorphometric analysis showed that DFAT/PLGA groups had significantly greater width of cementum, periodontal ligament and alveolar bone than ASCs/PLGA and PLGA-alone groups. In addition, transplanted fluorescent-labeled DFAT cells were observed in the periodontal ligament beside the newly formed bone and cementum. These findings suggest that DFAT cells have a greater potential for enhancing periodontal tissue regeneration than ASCs. Therefore, DFAT cells are a promising cell source for periodontium regeneration. PMID:26941649

Wharton's Jelly Derived Mesenchymal Stem Cells: Comparing Human and Horse.

PubMed

Merlo, Barbara; Teti, Gabriella; Mazzotti, Eleonora; Ingrà, Laura; Salvatore, Viviana; Buzzi, Marina; Cerqueni, Giorgia; Dicarlo, Manuela; Lanci, Aliai; Castagnetti, Carolina; Iacono, Eleonora

2018-08-01

Wharton's jelly (WJ) is an important source of mesenchymal stem cells (MSCs) both in human and other animals. The aim of this study was to compare human and equine WJMSCs. Human and equine WJMSCs were isolated and cultured using the same protocols and culture media. Cells were characterized by analysing morphology, growth rate, migration and adhesion capability, immunophenotype, differentiation potential and ultrastructure. Results showed that human and equine WJMSCs have similar ultrastructural details connected with intense synthetic and metabolic activity, but differ in growth, migration, adhesion capability and differentiation potential. In fact, at the scratch assay and transwell migration assay, the migration ability of human WJMSCs was higher (P < 0.05) than that of equine cells, while the volume of spheroids obtained after 48 h of culture in hanging drop was larger than the volume of equine ones (P < 0.05), demonstrating a lower cell adhesion ability. This can also revealed in the lower doubling time of equine cells (3.5 ± 2.4 days) as compared to human (6.5 ± 4.3 days) (P < 0.05), and subsequently in the higher number of cell doubling after 44 days of culture observed for the equine (20.3 ± 1.7) as compared to human cells (8.7 ± 2.4) (P < 0.05), and to the higher (P < 0.05) ability to form fibroblast colonies at P3. Even if in both species tri-lineage differentiation was achieved, equine cells showed an higher chondrogenic and osteogenic differentiation ability (P < 0.05). Our findings indicate that, although the ultrastructure demonstrated a staminal phenotype in human and equine WJMSCs, they showed different properties reflecting the different sources of MSCs.

3D Printing of Cytocompatible Water-Based Light-Cured Polyurethane with Hyaluronic Acid for Cartilage Tissue Engineering Applications

PubMed Central

Shie, Ming-You; Chang, Wen-Ching; Wei, Li-Ju; Huang, Yu-Hsin; Chen, Chien-Han; Shih, Cheng-Ting; Chen, Yi-Wen; Shen, Yu-Fang

2017-01-01

Diseases in articular cartilages have affected millions of people globally. Although the biochemical and cellular composition of articular cartilages is relatively simple, there is a limitation in the self-repair ability of the cartilage. Therefore, developing strategies for cartilage repair is very important. Here, we report on a new liquid resin preparation process of water-based polyurethane based photosensitive materials with hyaluronic acid with application of the materials for 3D printed customized cartilage scaffolds. The scaffold has high cytocompatibility and is one that closely mimics the mechanical properties of articular cartilages. It is suitable for culturing human Wharton’s jelly mesenchymal stem cells (hWJMSCs) and the cells in this case showed an excellent chondrogenic differentiation capacity. We consider that the 3D printing hybrid scaffolds may have potential in customized tissue engineering and also facilitate the development of cartilage tissue engineering. PMID:28772498

The role of laminins in cartilaginous tissues: from development to regeneration.

PubMed

Sun, Y; Wang, T L; Toh, W S; Pei, M

2017-07-21

As a key molecule of the extracellular matrix, laminin provides a delicate microenvironment for cell functions. Recent findings suggest that laminins expressed by cartilage-forming cells (chondrocytes, progenitor cells and stem cells) could promote chondrogenesis. However, few papers outline the effect of laminins on providing a favorable matrix microenvironment for cartilage regeneration. In this review, we delineated the expression of laminins in hyaline cartilage, fibrocartilage and cartilage-like tissue (nucleus pulposus) throughout several developmental stages. We also examined the effect of laminins on the biological activities of chondrocytes, including adhesion, migration and survival. Furthermore, we scrutinized the potential influence of various laminin isoforms on cartilage-forming cells' proliferation and chondrogenic differentiation. With this information, we hope to facilitate the understanding of the spatial and temporal interactions between cartilage-forming cells and laminin microenvironment to eventually advance cell-based cartilage engineering and regeneration.

Lineage mapping and characterization of the native progenitor population in cellular allograft.

PubMed

Neman, Josh; Duenas, Vincent; Kowolik, Claudia; Hambrecht, Amanda; Chen, Mike; Jandial, Rahul

2013-02-01

The gold standard for bone grafting remains the autograft. However, the attractiveness of autograft is counterbalanced by donor site morbidity. To mimic autograft-and its fundamental properties of osteoconductivity, osteoinductivity, and osteogenicity-novel bone grafting materials such as cellular allograft (Osteocel Plus) are composed of allograft in which the progenitor cells are preserved. However, the true identity of these cells remains obscure largely due to the lack of specific bona fide antigenic markers for stem versus progenitor cells. To characterize the stem and progenitor population in cellular allograft, Osteocel Plus. To determine whether cells endogenous to a cellular allograft undergo extensive self-renewal (a functional hallmark of stem cells), we employed a novel use of lineage mapping using a modern and refined replication incompetent lentiviral library with high complexity to uniquely label single cells with indelible genetic tags faithfully passed on to all progeny, allowing identification of highly proliferative clones. We used genetic and proteomic profiling as well as functional assays to show that these cells are capable of multipotential differentiation (the second functional hallmark of stem cells). Use of these two functional hallmarks enabled us to establish the existence of a stem and progenitor cell population in cellular allografts. Specifically, we employed (1) cellular dissociation and (2) in vitro expansion and differentiation capacity of cells released from cellular allograft. We determined differential gene expression profiling of a bona fide human mesenchymal stem cell line and cells from cellular allograft using focused PCR arrays mesenchymal stem cell (MSC) and osteogenesis associated. Proteomic profiling of cells from cellular allograft was performed using (1) immunofluorescence for BMP-2, Runx2 SMADs, CD44, Stro-1, Collagen, RANKL, Osterix Osteocalcin, and Ki67; (2) flow cytometry for Ki67, CD44, Stro-1, Thy1, CD146, and Osteocalcin; and (3) enzyme-linked immunosorbent assays (ELISA) for BMP-2, Osteocalcin, RANKL, Osteoprotegrin, and Osteocalcin. Clonal analysis of cells from cellular allograft was performed utilizing advance lentivirus lineage mapping techniques and massive parallel sequencing. Alizarin Red, Alcian Blue, and Oil red O staining assessed tripotential differentiation capacity. Serial trypsinization of allograft cellular bone matrix yielded approximately 1×105 cells per mL with viability greater than 90%. Cells expressed a panel of 84 MSC-associated genes in a pattern similar to but not identical to pure MSCs; specifically, 59 of 84 genes showed less than a 2.5-fold change in both cell types. Protein analysis showed that cellular allograft -derived cells maintained in nondifferentiation media expressed the early osteo-progenitor markers BMP-2, SMADs, and Runx2. Corresponding flow cytometry data for MSC markers revealed the presence of Stro-1 (49%), CD44 (99%), CD90 (42%), and CD146 (97%). Lineage mapping indicated that 62% of clones persisted and generated progeny through 10 passages, strongly suggesting the presence of bona fide stem cells. Passage 10 clones also exhibited tri-lineage differentiation capacity into osteogenic (Alizarin Red with H&E counterstain), chondrogenic (Alcian Blue), and adipogenic (Oil red O). Cells that did not proliferate through 10 passages presumably differentiated along an osteo-progenitor lineage. These data indicate that cellular allograft (Osteocel Plus) contains a heterogeneous population of cells with most cells demonstrating the capacity for extensive self-renewal and multipotential differentiation, which are hallmarks of stem cells. Whether stem cell-enriched allografts function comparably to autograft will require further studies, and their efficacy in facilitating arthrodesis will depend on randomized clinical studies. Copyright © 2013 Elsevier Inc. All rights reserved.

Development of collagen peptide-based biomaterials for tissue engineering applications

NASA Astrophysics Data System (ADS)

Hernandez Gordillo, Victor

The transition from in vitro to in vivo use of stem cells in regenerative medicine requires biomaterial scaffolds that can maintain stem cell viability and at the same time allow cell differentiation. We have previously reported the design of a collagen mimetic peptide (CMP) that assembles into a mesh-like three-dimensional (3D) structure upon the addition of metal ions and its potential for the culture of human cells. The addition of a chelating solution, such as EDTA, results in disassembly of the 3D structure, demonstrating the flexibility in the assembly/disassembly process. In the second chapter of this dissertation, we report the design of CMPs that can be functionalized with His-tagged cargoes within the 3D scaffold, via metal coordination. We show that the addition of GFP-His8 and human epidermal growth factor (hEGF-His6) has minimal effect in the assembly process. Additionally, we show that the bound hEGF-His6 can be released gradually in vitro for 5 days and induces cell proliferation in an EGF-dependent cell line. Furthermore, we functionalized the CMPs with the cell adhesion sequence (RGDS) to promote cell differentiation of two human non-tumorigenic cells lines, MCF10A and 3522-S1. In the third chapter, we evaluated the possibility of using the collagen mimetic-peptide-based (CMP) scaffolds for cell encapsulation and differentiation of human mesenchymal stem cells (hMSC). We show that hMSC encapsulated within the CMP scaffold are viable for up to 24 days post encapsulation. Moreover, hMSC at days 1, 4 and 8 days after encapsulation can be recovered from the scaffold and retain their stemness properties when analyzed for in vitro differentiation. We also demonstrate by real time PCR (RT-PCR) that genes important for osteogenic and chondrogenic differentiation are over-expressed in the absence of stimulating factors when the cells are encapsulated in the 3D scaffold at 8 and 24 days post encapsulation. Lastly, the incorporation of the cell adhesion sequence (RGDS) was shown to influence the scaffold-cell interaction. hMSCs within these RGDS-modified scaffold adopted spindle shape morphology and a complex cell organization at the outermost layer of the scaffold. In contrast, in the scaffold lacking the RGDS sequence hSMCs formed cell aggregates.

The effect of desulfation of chondroitin sulfate on interactions with positively charged growth factors and upregulation of cartilaginous markers in encapsulated MSCs.

PubMed

Lim, Jeremy J; Temenoff, Johnna S

2013-07-01

Sulfated glycosaminoglycans (GAGs) are known to interact electrostatically with positively charged growth factors to modulate signaling. Therefore, regulating the degree of sulfation of GAGs may be a promising approach to tailor biomaterial carriers for controlled growth factor delivery and release. For this study, chondroitin sulfate (CS) was first desulfated to form chondroitin, and resulting crosslinked CS and chondroitin hydrogels were examined in vitro for release of positively charged model protein (histone) and for their effect on cartilaginous differentiation of encapsulated human mesenchymal stem cells (MSCs). Desulfation significantly increased the release of histone from chondroitin hydrogels (30.6 ± 2.3 μg released over 8 days, compared to natively sulfated CS with 20.2 ± 0.8 μg), suggesting that sulfation alone plays a significant role in modulating protein interactions with GAG hydrogels. MSCs in chondroitin hydrogels significantly upregulated gene expression of collagen II and aggrecan by day 21 in chondrogenic medium (115 ± 100 and 23.1 ± 7.9 fold upregulation of collagen II and aggrecan, respectively), compared to CS hydrogels and PEG-based swelling controls, indicating that desulfation may actually enhance the response of MSCs to soluble chondrogenic cues, such as TGF-β1. Thus, desulfated chondroitin materials present a promising biomaterial tool to further investigate electrostatic GAG/growth factor interactions, especially for repair of cartilaginous tissues. Copyright © 2013 Elsevier Ltd. All rights reserved.

Biomimetic microbeads containing a chondroitin sulfate/chitosan polyelectrolyte complex for cell-based cartilage therapy.

PubMed

Daley, Ethan Lh; Coleman, Rhima M; Stegemann, Jan P

2015-10-28

Articular cartilage has a limited healing capacity that complicates the treatment of joint injuries and osteoarthritis. Newer repair strategies have focused on the use of cells and biomaterials to promote cartilage regeneration. In the present study, we developed and characterized bioinspired materials designed to mimic the composition of the cartilage extracellular matrix. Chondroitin sulfate (CS) and chitosan (CH) were used to form physically cross-linked macromolecular polyelectrolyte complexes (PEC) without the use of additional crosslinkers. A single-step water-in-oil emulsification process was used to either directly embed mesenchymal stem cells (MSC) in PEC particles created with a various concentrations of CS and CH, or to co-embed MSC with PEC in agarose-based microbeads. Direct embedding of MSC in PEC resulted in high cell viability but irregular and large particles. Co-embedding of PEC particles with MSC in agarose (Ag) resulted in uniform microbeads 80-90 μm in diameter that maintained high cell viability over three weeks in culture. Increased serum content resulted in more uniform PEC distribution within the microbead matrix, and both high and low CS:CH ratios resulted in more homogeneous microbeads than 1:1 formulations. Under chondrogenic conditions, expression of sulfated GAG and collagen type II was increased in 10:1 CS:CH PEC-Ag microbeads compared to pure Ag beads, indicating a chondrogenic influence of the PEC component. Such PEC-Ag microbeads may have utility in the directed differentiation and delivery of progenitor cell populations for cartilage repair.

Utilizing two-photon fluorescence and second harmonic generation microscopy to study human bone marrow mesenchymal stem cell morphogenesis in chitosan scaffold

NASA Astrophysics Data System (ADS)

Su, Ping-Jung; Huang, Chi-Hsiu; Huang, Yi-You; Lee, Hsuan-Sue; Dong, Chen-Yuan

2008-02-01

A major goal of tissue engineering is to cultivate the cartilage in vitro. One approach is to implant the human bone marrow mesenchymal stem cells into the three dimensional biocompatible and biodegradable material. Through the action of the chondrogenic factor TGF-β3, the stem cells can be induced to secrete collagen. In this study, mesenchymal stem cells are implanted on the chitosan scaffold and TGF-β3 was added to produce the cartilage tissue and TP autofluorescence and SHG microscopy was used to image the process of chondrogenesis. With additional development, multiphoton microscopy can be developed into an effective tool for evaluating the quality of tissue engineering products.

R-spondin 2 facilitates differentiation of proliferating chondrocytes into hypertrophic chondrocytes by enhancing Wnt/β-catenin signaling in endochondral ossification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takegami, Yasuhiko; Department of Orthopaedic Surgery, Nagoya University School of Medicine, Nagoya; Ohkawara, Bisei

Endochondral ossification is a crucial process for longitudinal growth of bones. Differentiating chondrocytes in growth cartilage form four sequential zones of proliferation, alignment into column, hypertrophy, and substitution of chondrocytes with osteoblasts. Wnt/β-catenin signaling is essential for differentiation of proliferating chondrocytes into hypertrophic chondrocytes in growth cartilage. R-spondin 2 (Rspo2), a member of R-spondin family, is an agonist for Wnt signaling, but its role in chondrocyte differentiation remains unknown. Here we report that growth cartilage of Rspo2-knockout mice shows a decreased amount of β-catenin and increased amounts collagen type II (CII) and Sox9 in the abnormally extended proliferating zone. Inmore » contrast, expression of collagen type X (CX) in the hypertrophic zone remains unchanged. Differentiating chondrogenic ATDC5 cells, mimicking proliferating chondrocytes, upregulate Rspo2 and its putative receptor, Lgr5, in parallel. Addition of recombinant human Rspo2 to differentiating ATDC5 cells decreases expressions of Col2a1, Sox9, and Acan, as well as production of proteoglycans. In contrast, lentivirus-mediated knockdown of Rspo2 has the opposite effect. The effect of Rspo2 on chondrogenic differentiation is mediated by Wnt/β-catenin signaling, and not by Wnt/PCP or Wnt/Ca{sup 2+} signaling. We propose that Rspo2 activates Wnt/β-catenin signaling to reduce Col2a1 and Sox9 and to facilitate differentiation of proliferating chondrocytes into hypertrophic chondrocytes in growth cartilage. - Highlights: • Rspo2 is a secreted activator of Wnt, and its knockout shows extended proliferating chondrocytes in endochondral ossification. • In proliferating chondrocytes of Rspo2-knockout mice, Sox9 and collagen type 2 are increased and β-catenin is decreased. • Rspo2 and its receptor Lgr5, as well as Sox9 and collagen type 2, are expressed in differentiating ATDC5 chondrogenic cells. • In ATDC5 cells, Rspo2 decreases expressions of Sox9, collagen type 2, and aggrecan through Wnt/β-catenin signaling. • We propose that Rspo2 activates Wnt/β-catenin to facilitate chondrocyte differentiation in endochondral ossification.« less

Biologic resurfacing of the patella: current status.

PubMed

Scapinelli, Raphaele; Aglietti, Paolo; Baldovin, Marino; Giron, Francesco; Teitge, Robert

2002-07-01

The techniques of biologic resurfacing of the patella, like other joint surfaces, are still evolving. Currently none of them is free from criticism. In this regard it is our hope that progress in the basic science will offer in the near future new and more optimistic therapeutic possibilities (i.e., the restoration of a reparative cartilage that is structurally and functionally comparable to the native one). The greater expectancies come perhaps from the present experimental investigations about the combined use of tissue-engineered implants embedded with staminal cells and growth factors. Many problems remain to be solved, however, before reliable applicability in humans. From a general point of view, stem cells obtained from various sources (e.g., adult bone marrow, umbilical cord) offer the same finalities as the embryonic stem cells, without the ethical obstacles related to the latter. Therefore, it may be that restoration of part or all of the articular surface of a joint will be possible by way of these mesenchymal progenitors that have the ability to differentiate into the chondrogenic and osteogenic lines, which is required for the restoration of the various layers of a normal articular cartilage and subchondral bone.

Preclinical and clinical data for the use of mesenchymal stem cells in articular cartilage tissue engineering.

PubMed

Tang, Quen Oak; Carasco, Clare Francesca; Gamie, Zakareya; Korres, Nectarios; Mantalaris, Athanasios; Tsiridis, Eleftherios

2012-10-01

With an ageing population, the prevalence of osteoarthritis (OA) has increased. Mesenchymal Stem Cells (MSCs) have been proposed to be an attractive alternative candidate in the tissue engineering of articular cartilage primarily due to its abundant source, reduced cartilage donor site morbidity, and strong capacity for proliferation and potential to differentiate toward a chondrogenic phenotype. A current overview of human, in vivo, and in vitro evidence on the use of MSCs in cartilage tissue engineering. We demonstrate robust evidence that MSCs have the potential to regenerate articular cartilage. We also identify the complexity of designing a suitable preclinical model and the challenges in considering its clinical application such as type of MSC, scaffold, culture construct and the method by which growth factors are delivered. Of great interest is further characterization of the factors that may prevent MSC-derived chondrocytes to undergo premature hypertrophy and to understand what enables the terminal developmental pathway for permanent hyaline cartilage regeneration. Despite this, there is an abundance of evidence suggesting that MSCs are a desirable cell source and will have significant impact in tissue engineering of cartilage in the future.

Temporomandibular Joint Regenerative Medicine

PubMed Central

Van Bellinghen, Xavier; Idoux-Gillet, Ysia; Pugliano, Marion; Strub, Marion; Bornert, Fabien; Clauss, Francois; Schwinté, Pascale; Keller, Laetitia; Benkirane-Jessel, Nadia; Lutz, Jean Christophe; Fioretti, Florence

2018-01-01

The temporomandibular joint (TMJ) is an articulation formed between the temporal bone and the mandibular condyle which is commonly affected. These affections are often so painful during fundamental oral activities that patients have lower quality of life. Limitations of therapeutics for severe TMJ diseases have led to increased interest in regenerative strategies combining stem cells, implantable scaffolds and well-targeting bioactive molecules. To succeed in functional and structural regeneration of TMJ is very challenging. Innovative strategies and biomaterials are absolutely crucial because TMJ can be considered as one of the most difficult tissues to regenerate due to its limited healing capacity, its unique histological and structural properties and the necessity for long-term prevention of its ossified or fibrous adhesions. The ideal approach for TMJ regeneration is a unique scaffold functionalized with an osteochondral molecular gradient containing a single stem cell population able to undergo osteogenic and chondrogenic differentiation such as BMSCs, ADSCs or DPSCs. The key for this complex regeneration is the functionalization with active molecules such as IGF-1, TGF-β1 or bFGF. This regeneration can be optimized by nano/micro-assisted functionalization and by spatiotemporal drug delivery systems orchestrating the 3D formation of TMJ tissues. PMID:29393880

The role of environmental factors in regulating the development of cartilaginous grafts engineered using osteoarthritic human infrapatellar fat pad-derived stem cells.

PubMed

Liu, Yurong; Buckley, Conor T; Downey, Richard; Mulhall, Kevin J; Kelly, Daniel J

2012-08-01

Engineering functional cartilaginous grafts using stem cells isolated from osteoarthritic human tissue is of fundamental importance if autologous tissue engineering strategies are to be used in the treatment of diseased articular cartilage. It has previously been demonstrated that human infrapatellar fat pad (IFP)-derived stem cells undergo chondrogenesis in pellet culture; however, the ability of such cells to generate functional cartilaginous grafts has not been adequately addressed. The objective of this study was to explore how environmental conditions regulate the functional development of cartilaginous constructs engineered using diseased human IFP-derived stem cells (FPSCs). FPSCs were observed to display a diminished chondrogenic potential upon encapsulation in a three-dimensional hydrogel compared with pellet culture, synthesizing significantly lower levels of glycosaminoglycan and collagen on a per cell basis. To engineer more functional cartilaginous grafts, we next explored whether additional biochemical and biophysical stimulations would enhance chondrogenesis within the hydrogels. Serum stimulation was observed to partially recover the diminished chondrogenic potential within hydrogel culture. Over 42 days, stem cells that had first been expanded in a low-oxygen environment proliferated extensively on the outer surface of the hydrogel in response to serum stimulation, assembling a dense type II collagen-positive cartilaginous tissue resembling that formed in pellet culture. The application of hydrostatic pressure did not further enhance extracellular matrix synthesis within the hydrogels, but did appear to alter the spatial accumulation of extracellular matrix leading to the formation of a more compact tissue with superior mechanically functionality. Further work is required in order to recapitulate the environmental conditions present during pellet culture within scaffolds or hydrogels in order to engineer more functional cartilaginous grafts using human osteoarthritic FPSCs.

Engineering endostatin-producing cartilaginous constructs for cartilage repair using nonviral transfection of chondrocyte-seeded and mesenchymal-stem-cell-seeded collagen scaffolds.

PubMed

Jeng, Lily; Olsen, Bjorn R; Spector, Myron

2010-10-01

Although there is widespread recognition of the importance of angiogenesis in tissue repair, there is little work on the inhibition of angiogenesis in the context of tissue engineering of naturally avascular tissues, like articular cartilage. The objective was to engineer a collagen-scaffold-based cartilaginous construct overexpressing a potent antiangiogenic factor, endostatin, using nonviral transfection. Endostatin-plasmid-supplemented collagen scaffolds were seeded with mesenchymal stem cells and chondrocytes and cultured for 20–22 days. The effects of the following variables on endostatin expression and chondrogenesis were examined: collagen scaffold material, method of nonviral vector incorporation, plasmid load, culture medium, and oxygen tension. An increase and peak of endostatin protein was observed during the first week of culture, followed by a decrease to low levels, suggesting that overexpression of endostatin could be sustained for several days using the nonviral vector. The amount of endostatin produced was tunable with the external factors. Chondrogenesis was observed in the engineered constructs cultured in chondrogenic medium at the 3-week time point, demonstrating that endostatin did not inhibit the chondrogenic potential of mesenchymal stem cells or the general viability of the cells. The ability to engineer endostatin-expressing cartilaginous constructs will be of value for future work exercising regulatory control of angiogenesis in cartilage repair.

Heterogeneous Human Periodontal Ligament-Committed Progenitor and Stem Cell Populations Exhibit a Unique Cementogenic Property Under In Vitro and In Vivo Conditions.

PubMed

Shinagawa-Ohama, Rei; Mochizuki, Mai; Tamaki, Yuichi; Suda, Naoto; Nakahara, Taka

2017-05-01

An undesirable complication that arises during dental treatments is external apical-root resorption, which causes root-cementum and root-dentin loss. To induce de novo cementogenesis, stem cell therapy is required. Cementum-forming cells (cementoblasts) are known to be differentiated from periodontal-lineage mesenchymal stem cells (MSCs), which are derived from the dental follicle (DF) in developing tissues and the periodontal ligament (PDL) in adult tissues, but the periodontal-lineage MSC type that is optimal for inducing de novo cementogenesis remains unidentified, as does the method to isolate these cells from harvested tissues. Thus, we investigated the cementogenic potential of DF- and PDL-derived MSCs that were isolated by using two widely used cell-isolation methods: enzymatic digestion and outgrowth (OG) methods. DF- and PDL-derived cells isolated by using both methods proliferated actively, and all four isolated cell types showed MSC gene/protein expression phenotype and ability to differentiate into adipogenic and chondrogenic lineages. Furthermore, cementogenic-potential analysis revealed that all cell types produced alizarin red S-positive mineralized materials in in vitro cultures. However, PDL-OG cells presented unique cementogenic features, such as nodular formation of mineralized deposits displaying a cellular intrinsic fiber cementum-like structure, as well as a higher expression of cementoblast-specific genes than in the other cell types. Moreover, in in vivo transplantation experiments, PDL-OG cells formed cellular cementum-like hard tissue containing embedded osteocalcin-positive cells, whereas the other cells formed acellular cementum-like materials. Given that the root-cementum defect is likely regenerated through cellular cementum deposition, PDL-OG cell-based therapies might potentially facilitate the de novo cellular cementogenesis required for regenerating the root defect.

A Magnetically Actuated Microscaffold Containing Mesenchymal Stem Cells for Articular Cartilage Repair.

PubMed

Go, Gwangjun; Han, Jiwon; Zhen, Jin; Zheng, Shaohui; Yoo, Ami; Jeon, Mi-Jeong; Park, Jong-Oh; Park, Sukho

2017-07-01

This study proposes a magnetically actuated microscaffold with the capability of targeted mesenchymal stem cell (MSC) delivery for articular cartilage regeneration. The microscaffold, as a 3D porous microbead, is divided into body and surface portions according to its materials and fabrication methods. The microscaffold body, which consists of poly(lactic-co-glycolic acid) (PLGA), is formed through water-in-oil-in-water emulsion templating, and its surface is coated with amine functionalized magnetic nanoparticles (MNPs) via amino bond formation. The porous PLGA structure of the microscaffold can assist in cell adhesion and migration, and the MNPs on the microscaffold can make it possible to steer using an electromagnetic actuation system that provides external magnetic fields for the 3D locomotion of the microscaffold. As a fundamental test of the magnetic response of the microscaffold, it is characterized in terms of the magnetization curve, velocity, and 3D locomotion of a single microscaffold. In addition, its function with a cargo of MSCs for cartilage regeneration is demonstrated from the proliferation, viability, and chondrogenic differentiation of D1 mouse MSCs that are cultured on the microscaffold. For the feasibility tests for cartilage repair, 2D/3D targeting of multiple microscaffolds with the MSCs is performed to demonstrate targeted stem cell delivery using the microscaffolds and their swarm motion. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genome‐Wide MicroRNA and Gene Analysis of Mesenchymal Stem Cell Chondrogenesis Identifies an Essential Role and Multiple Targets for miR‐140‐5p

PubMed Central

Tselepi, Maria; Gómez, Rodolfo; Woods, Steven; Hui, Wang; Smith, Graham R.; Shanley, Daryl P.; Clark, Ian M.; Young, David A.

2015-01-01

Abstract microRNAs (miRNAs) are abundantly expressed in development where they are critical determinants of cell differentiation and phenotype. Accordingly miRNAs are essential for normal skeletal development and chondrogenesis in particular. However, the question of which miRNAs are specific to the chondrocyte phenotype has not been fully addressed. Using microarray analysis of miRNA expression during mesenchymal stem cell chondrogenic differentiation and detailed examination of the role of essential differentiation factors, such as SOX9, TGF‐β, and the cell condensation phase, we characterize the repertoire of specific miRNAs involved in chondrocyte development, highlighting in particular miR‐140 and miR‐455. Further with the use of mRNA microarray data we integrate miRNA expression and mRNA expression during chondrogenesis to underline the particular importance of miR‐140, especially the ‐5p strand. We provide a detailed identification and validation of direct targets of miR‐140‐5p in both chondrogenesis and adult chondrocytes with the use of microarray and 3′UTR analysis. This emphasizes the diverse array of targets and pathways regulated by miR‐140‐5p. We are also able to confirm previous experimentally identified targets but, additionally, identify a novel positive regulation of the Wnt signaling pathway by miR‐140‐5p. Wnt signaling has a complex role in chondrogenesis and skeletal development and these findings illustrate a previously unidentified role for miR‐140‐5p in regulation of Wnt signaling in these processes. Together these developments further highlight the role of miRNAs during chondrogenesis to improve our understanding of chondrocyte development and guide cartilage tissue engineering. Stem Cells 2015;33:3266–3280 PMID:26175215

Properties and usefulness of aggregates of synovial mesenchymal stem cells as a source for cartilage regeneration

PubMed Central

2012-01-01

Introduction Transplantation of mesenchymal stem cells (MSCs) derived from synovium is a promising therapy for cartilage regeneration. For clinical application, improvement of handling operation, enhancement of chondrogenic potential, and increase of MSCs adhesion efficiency are needed to achieve a more successful cartilage regeneration with a limited number of MSCs without scaffold. The use of aggregated MSCs may be one of the solutions. Here, we investigated the handling, properties and effectiveness of aggregated MSCs for cartilage regeneration. Methods Human and rabbit synovial MSCs were aggregated using the hanging drop technique. The gene expression changes after aggregation of synovial MSCs were analyzed by microarray and real time RT-PCR analyses. In vitro and in vivo chondrogenic potential of aggregates of synovial MSCs was examined. Results Aggregates of MSCs cultured for three days became visible, approximately 1 mm in diameter and solid and durable by manipulation; most of the cells were viable. Microarray analysis revealed up-regulation of chondrogenesis-related, anti-inflammatory and anti-apoptotic genes in aggregates of MSCs. In vitro studies showed higher amounts of cartilage matrix synthesis in pellets derived from aggregates of MSCs compared to pellets derived from MSCs cultured in a monolayer. In in vivo studies in rabbits, aggregates of MSCs could adhere promptly on the osteochondral defects by surface tension, and stay without any loss. Transplantation of aggregates of MSCs at relatively low density achieved successful cartilage regeneration. Contrary to our expectation, transplantation of aggregates of MSCs at high density failed to regenerate cartilage due to cell death and nutrient deprivation of aggregates of MSCs. Conclusions Aggregated synovial MSCs were a useful source for cartilage regeneration considering such factors as easy preparation, higher chondrogenic potential and efficient attachment. PMID:22676383

Transplantation of Scaffold-Free Cartilage-Like Cell-Sheets Made from Human Bone Marrow Mesenchymal Stem Cells for Cartilage Repair: A Preclinical Study.

PubMed

Itokazu, Maki; Wakitani, Shigeyuki; Mera, Hisashi; Tamamura, Yoshihiro; Sato, Yasushi; Takagi, Mutsumi; Nakamura, Hiroaki

2016-10-01

The object of this study was to determine culture conditions that create stable scaffold-free cartilage-like cell-sheets from human bone marrow-derived mesenchymal stem cells (hBMSCs) and to assess their effects after transplantation into osteochondral defects in nude rats. (Experiment 1) The hBMSCs were harvested from 3 males, the proliferative and chondrogenic capacities were assessed at passage 1, and the cells were expanded in 3 different culture conditions: (1) 5% fetal bovine serum (FBS), (2) 10% FBS, and (3) 5% FBS with fibroblast growth factor 2 (FGF-2). The cells were harvested and made chondrogenic pellet culture. The cell proliferation rate, glycosaminoglycan/DNA ratio, and safranin-O staining intensity of pellets cultured condition 3 were higher than those of conditions 1 and 2. (Experiment 2) The hBMSCs were expanded and passaged 3 times under culture condition 3, and fabricate the cell-sheets in chondrogenic medium either with or without FBS. The cell-sheets fabricated with FBS maintained their size with flat edges. (Experiment 3) The cell-sheets were transplanted into osteochondral defects in nude rats. Histological analysis was performed at 2, 4, and 12 weeks after surgery. The osteochondral repair was better after sheet transplantation than in the control group and significantly improved Wakitani score. Immunostaining with human-specific vimentin antibody showed that the transplanted cells became fewer and disappeared at 12 weeks. These results indicate that culture with FGF-2 may help to quickly generate sufficient numbers of cells to create stable and reliable scaffold-free cartilage-like cell-sheets, which contribute to the regeneration of osteochondral defects.

Large, stratified, and mechanically functional human cartilage grown in vitro by mesenchymal condensation

PubMed Central

Bhumiratana, Sarindr; Eton, Ryan E.; Oungoulian, Sevan R.; Wan, Leo Q.; Ateshian, Gerard A.; Vunjak-Novakovic, Gordana

2014-01-01

The efforts to grow mechanically functional cartilage from human mesenchymal stem cells have not been successful. We report that clinically sized pieces of human cartilage with physiologic stratification and biomechanics can be grown in vitro by recapitulating some aspects of the developmental process of mesenchymal condensation. By exposure to transforming growth factor-β, mesenchymal stem cells were induced to condense into cellular bodies, undergo chondrogenic differentiation, and form cartilagenous tissue, in a process designed to mimic mesenchymal condensation leading into chondrogenesis. We discovered that the condensed mesenchymal cell bodies (CMBs) formed in vitro set an outer boundary after 5 d of culture, as indicated by the expression of mesenchymal condensation genes and deposition of tenascin. Before setting of boundaries, the CMBs could be fused into homogenous cellular aggregates giving rise to well-differentiated and mechanically functional cartilage. We used the mesenchymal condensation and fusion of CMBs to grow centimeter-sized, anatomically shaped pieces of human articular cartilage over 5 wk of culture. For the first time to our knowledge biomechanical properties of cartilage derived from human mesenchymal cells were comparable to native cartilage, with the Young’s modulus of >800 kPa and equilibrium friction coeffcient of <0.3. We also demonstrate that CMBs have capability to form mechanically strong cartilage–cartilage interface in an in vitro cartilage defect model. The CMBs, which acted as “lego-like” blocks of neocartilage, were capable of assembling into human cartilage with physiologic-like structure and mechanical properties. PMID:24778247

Mechanical fibrinogen-depletion supports heparin-free mesenchymal stem cell propagation in human platelet lysate.

PubMed

Laner-Plamberger, Sandra; Lener, Thomas; Schmid, Doris; Streif, Doris A; Salzer, Tina; Öller, Michaela; Hauser-Kronberger, Cornelia; Fischer, Thorsten; Jacobs, Volker R; Schallmoser, Katharina; Gimona, Mario; Rohde, Eva

2015-11-10

Pooled human platelet lysate (pHPL) is an efficient alternative to xenogenic supplements for ex vivo expansion of mesenchymal stem cells (MSCs) in clinical studies. Currently, porcine heparin is used in pHPL-supplemented medium to prevent clotting due to plasmatic coagulation factors. We therefore searched for an efficient and reproducible medium preparation method that avoids clot formation while omitting animal-derived heparin. We established a protocol to deplete fibrinogen by clotting of pHPL in medium, subsequent mechanical hydrogel disruption and removal of the fibrin pellet. After primary culture, bone-marrow and umbilical cord derived MSCs were tested for surface markers by flow cytometry and for trilineage differentiation capacity. Proliferation and clonogenicity were analyzed for three passages. The proposed clotting procedure reduced fibrinogen more than 1000-fold, while a volume recovery of 99.5 % was obtained. All MSC types were propagated in standard and fibrinogen-depleted medium. Flow cytometric phenotype profiles and adipogenic, osteogenic and chondrogenic differentiation potential in vitro were independent of MSC-source or medium type. Enhanced proliferation of MSCs was observed in the absence of fibrinogen but presence of heparin compared to standard medium. Interestingly, this proliferative response to heparin was not detected after an initial contact with fibrinogen during the isolation procedure. Here, we present an efficient, reproducible and economical method in compliance to good manufacturing practice for the preparation of MSC media avoiding xenogenic components and suitable for clinical studies.

Platelet lysate enhances synovial fluid multipotential stromal cells functions: Implications for therapeutic use.

PubMed

Altaie, Ala; Baboolal, Thomas G; Wall, Owen; Jones, Elena; McGonagle, Dennis

2018-03-01

Although intra-articular injection of platelet products is increasingly used for joint regenerative approaches, there are few data on their biological effects on joint-resident multipotential stromal cells (MSCs), which are directly exposed to the effects of these therapeutic strategies. Therefore, this study investigated the effect of platelet lysate (PL) on synovial fluid-derived MSCs (SF-MSCs), which in vivo have direct access to sites of cartilage injury. SF-MSCs were obtained during knee arthroscopic procedures (N = 7). Colony forming unit-fibroblast (CFU-F), flow-cytometric phenotyping, carboxyfluorescein succinimidyl ester-based immunomodulation for T-cell and trilineage differentiation assays were performed using PL and compared with standard conditions. PL-enhanced SF-MSC (PL-MSC) proliferation as CFU-F colonies was 1.4-fold larger, and growing cultures had shorter population-doubling times. PL-MSCs and fetal calf serum (FCS)-MSCs had the same immunophenotype and similar immunomodulation activities. In chondrogenic and osteogenic differentiation assays, PL-MSCs produced 10% more sulfated-glycosaminoglycan (sGAG) and 45% less Ca ++ compared with FCS-MSCs, respectively. Replacing chondrogenic medium transforming growth factor-β3 with 20% or 50% PL further increased sGAG production of PL-MSCs by 69% and 95%, respectively, compared with complete chondrogenic medium. Also, Dulbecco's Modified Eagle's Medium high glucose (HG-DMEM) plus 50% PL induced more chondrogenesis compared with HG-DMEM plus 10% FCS and was comparable to complete chondrogenic medium. This is the first study to assess SF-MSC responses to PL and provides biological support to the hypothesis that PL may be capable of modulating multiple functional aspects of joint resident MSCs with direct access to injured cartilage. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Human Cartilage-Derived Progenitor Cells From Committed Chondrocytes for Efficient Cartilage Repair and Regeneration

PubMed Central

Jiang, Yangzi; Cai, Youzhi; Zhang, Wei; Yin, Zi; Hu, Changchang; Tong, Tong; Lu, Ping; Zhang, Shufang; Neculai, Dante

2016-01-01

Articular cartilage is not a physiologically self-renewing tissue. Injury of cartilage often progresses from the articular surface to the subchondral bone, leading to pathogenesis of tissue degenerative diseases, such as osteoarthritis. Therapies to treat cartilage defects using autologous chondrocyte-based tissue engineering have been developed and used for more than 20 years; however, the challenge of chondrocyte expansion in vitro remains. A promising cell source, cartilage stem/progenitor cells (CSPCs), has attracted recent attention. Because their origin and identity are still unclear, the application potential of CSPCs is under active investigation. Here we have captured the emergence of a group of stem/progenitor cells derived from adult human chondrocytes, highlighted by dynamic changes in expression of the mature chondrocyte marker, COL2, and mesenchymal stromal/stem cell (MSC) marker, CD146. These cells are termed chondrocyte-derived progenitor cells (CDPCs). The stem cell-like potency and differentiation status of CDPCs were determined by physical and biochemical cues during culture. A low-density, low-glucose 2-dimensional culture condition (2DLL) was critical for the emergence and proliferation enhancement of CDPCs. CDPCs showed similar phenotype as bone marrow mesenchymal stromal/stem cells but exhibited greater chondrogenic potential. Moreover, the 2DLL-cultured CDPCs proved efficient in cartilage formation both in vitro and in vivo and in repairing large knee cartilage defects (6–13 cm2) in 15 patients. These findings suggest a phenotype conversion between chondrocytes and CDPCs and provide conditions that promote the conversion. These insights expand our understanding of cartilage biology and may enhance the success of chondrocyte-based therapies. Significance Injury of cartilage, a non-self-repairing tissue, often progresses to pathogenesis of degenerative joint diseases, such as osteoarthritis. Although tissue-derived stem cells have been shown to contribute to tissue renewal and homeostasis, the derivation, biological function, and application potential of stem/progenitor cells found in adult human articular cartilage are incompletely understood. This study reports the derivation of a population of cartilage stem/progenitor cells from fully differentiated chondrocytes under specific culture conditions, which have the potential to reassume their chondrocytic phenotype for efficient cartilage regeneration. These findings support the possibility of using in vitro amplified chondrocyte-derived progenitor cells for joint cartilage repair. PMID:27130221

Human Cartilage-Derived Progenitor Cells From Committed Chondrocytes for Efficient Cartilage Repair and Regeneration.

PubMed

Jiang, Yangzi; Cai, Youzhi; Zhang, Wei; Yin, Zi; Hu, Changchang; Tong, Tong; Lu, Ping; Zhang, Shufang; Neculai, Dante; Tuan, Rocky S; Ouyang, Hong Wei

2016-06-01

Articular cartilage is not a physiologically self-renewing tissue. Injury of cartilage often progresses from the articular surface to the subchondral bone, leading to pathogenesis of tissue degenerative diseases, such as osteoarthritis. Therapies to treat cartilage defects using autologous chondrocyte-based tissue engineering have been developed and used for more than 20 years; however, the challenge of chondrocyte expansion in vitro remains. A promising cell source, cartilage stem/progenitor cells (CSPCs), has attracted recent attention. Because their origin and identity are still unclear, the application potential of CSPCs is under active investigation. Here we have captured the emergence of a group of stem/progenitor cells derived from adult human chondrocytes, highlighted by dynamic changes in expression of the mature chondrocyte marker, COL2, and mesenchymal stromal/stem cell (MSC) marker, CD146. These cells are termed chondrocyte-derived progenitor cells (CDPCs). The stem cell-like potency and differentiation status of CDPCs were determined by physical and biochemical cues during culture. A low-density, low-glucose 2-dimensional culture condition (2DLL) was critical for the emergence and proliferation enhancement of CDPCs. CDPCs showed similar phenotype as bone marrow mesenchymal stromal/stem cells but exhibited greater chondrogenic potential. Moreover, the 2DLL-cultured CDPCs proved efficient in cartilage formation both in vitro and in vivo and in repairing large knee cartilage defects (6-13 cm(2)) in 15 patients. These findings suggest a phenotype conversion between chondrocytes and CDPCs and provide conditions that promote the conversion. These insights expand our understanding of cartilage biology and may enhance the success of chondrocyte-based therapies. Injury of cartilage, a non-self-repairing tissue, often progresses to pathogenesis of degenerative joint diseases, such as osteoarthritis. Although tissue-derived stem cells have been shown to contribute to tissue renewal and homeostasis, the derivation, biological function, and application potential of stem/progenitor cells found in adult human articular cartilage are incompletely understood. This study reports the derivation of a population of cartilage stem/progenitor cells from fully differentiated chondrocytes under specific culture conditions, which have the potential to reassume their chondrocytic phenotype for efficient cartilage regeneration. These findings support the possibility of using in vitro amplified chondrocyte-derived progenitor cells for joint cartilage repair. ©AlphaMed Press.

CRISPR/Cas9-mediated reversibly immortalized mouse bone marrow stromal stem cells (BMSCs) retain multipotent features of mesenchymal stem cells (MSCs).

PubMed

Hu, Xue; Li, Li; Yu, Xinyi; Zhang, Ruyi; Yan, Shujuan; Zeng, Zongyue; Shu, Yi; Zhao, Chen; Wu, Xingye; Lei, Jiayan; Li, Yasha; Zhang, Wenwen; Yang, Chao; Wu, Ke; Wu, Ying; An, Liping; Huang, Shifeng; Ji, Xiaojuan; Gong, Cheng; Yuan, Chengfu; Zhang, Linghuan; Liu, Wei; Huang, Bo; Feng, Yixiao; Zhang, Bo; Haydon, Rex C; Luu, Hue H; Reid, Russell R; Lee, Michael J; Wolf, Jennifer Moriatis; Yu, Zebo; He, Tong-Chuan

2017-12-19

Mesenchymal stem cells (MSCs) are multipotent non-hematopoietic progenitor cells that can undergo self-renewal and differentiate into multi-lineages. Bone marrow stromal stem cells (BMSCs) represent one of the most commonly-used MSCs. In order to overcome the technical challenge of maintaining primary BMSCs in long-term culture, here we seek to establish reversibly immortalized mouse BMSCs (imBMSCs). By exploiting CRISPR/Cas9-based homology-directed-repair (HDR) mechanism, we target SV40T to mouse Rosa26 locus and efficiently immortalize mouse BMSCs (i.e., imBMSCs). We also immortalize BMSCs with retroviral vector SSR #41 and establish imBMSC41 as a control line. Both imBMSCs and imBMSC41 exhibit long-term proliferative capability although imBMSC41 cells have a higher proliferation rate. SV40T mRNA expression is 130% higher in imBMSC41 than that in imBMSCs. However, FLP expression leads to 86% reduction of SV40T expression in imBMSCs, compared with 63% in imBMSC41 cells. Quantitative genomic PCR analysis indicates that the average copy number of SV40T and hygromycin is 1.05 for imBMSCs and 2.07 for imBMSC41, respectively. Moreover, FLP expression removes 92% of SV40T in imBMSCs at the genome DNA level, compared with 58% of that in imBMSC41 cells, indicating CRISPR/Cas9 HDR-mediated immortalization of BMSCs can be more effectively reversed than that of retrovirus-mediated random integrations. Nonetheless, both imBMSCs and imBMSC41 lines express MSC markers and are highly responsive to BMP9-induced osteogenic, chondrogenic and adipogenic differentiation in vitro and in vivo . Thus, the engineered imBMSCs can be used as a promising alternative source of primary MSCs for basic and translational research in the fields of MSC biology and regenerative medicine.

Behaviour of adipose-derived canine mesenchymal stem cells after superparamagnetic iron oxide nanoparticles labelling for magnetic resonance imaging.

PubMed

Kolecka, Malgorzata Anna; Arnhold, Stefan; Schmidt, Martin; Reich, Christine; Kramer, Martin; Failing, Klaus; von Pückler, Kerstin

2017-02-24

Therapy with mesenchymal stem cells (MSCs) has been reported to provide beneficial effects in the treatment of neurological and orthopaedic disorders in dogs. The exact mechanism of action is poorly understood. Magnetic resonance imaging (MRI) gives the opportunity to observe MSCs after clinical administration. To visualise MSCs with the help of MRI, labelling with an MRI contrast agent is necessary. However, it must be clarified whether there is any negative influence on cell function and viability after labelling prior to clinical administration. For the purpose of the study, seven samples with canine adipose-derived stem cells were incubated with superparamagnetic iron oxide nanoparticles (SPIO: 319.2 μg/mL Fe) for 24 h. The internalisation of the iron particles occurred via endocytosis. SPIO particles were localized as free clusters in the cytoplasm or within lysosomes depending on the time of investigation. The efficiency of the labelling was investigated using Prussian blue staining and MACS assay. After 3 weeks the percentage of SPIO labelled canine stem cells decreased. Phalloidin staining showed no negative effect on the cytoskeleton. Labelled cells underwent osteogenic and adipogenic differentiation. Chondrogenic differentiation occurred to a lesser extent compared with a control sample. MTT-Test and wound healing assay showed no influence of labelling on the proliferation. The duration of SPIO labelling was assessed using a 1 Tesla clinical MRI scanner and T2 weighted turbo spin echo and T2 weighted gradient echo MRI sequences 1, 2 and 3 weeks after labelling. The hypointensity caused by SPIO lasted for 3 weeks in both sequences. An Endorem labelling concentration of 319.2 μg/mL Fe (448 μg/mL SPIO) had no adverse effects on the viability of canine ASCs. Therefore, this contrast agent could be used as a model for iron oxide labelling agents. However, the tracking ability in vivo has to be evaluated in further studies.

CRISPR/Cas9-mediated reversibly immortalized mouse bone marrow stromal stem cells (BMSCs) retain multipotent features of mesenchymal stem cells (MSCs)

PubMed Central

Hu, Xue; Li, Li; Yu, Xinyi; Zhang, Ruyi; Yan, Shujuan; Zeng, Zongyue; Shu, Yi; Zhao, Chen; Wu, Xingye; Lei, Jiayan; Li, Yasha; Zhang, Wenwen; Yang, Chao; Wu, Ke; Wu, Ying; An, Liping; Huang, Shifeng; Ji, Xiaojuan; Gong, Cheng; Yuan, Chengfu; Zhang, Linghuan; Liu, Wei; Huang, Bo; Feng, Yixiao; Zhang, Bo; Haydon, Rex C.; Luu, Hue H.; Reid, Russell R.; Lee, Michael J.; Wolf, Jennifer Moriatis; Yu, Zebo; He, Tong-Chuan

2017-01-01

Mesenchymal stem cells (MSCs) are multipotent non-hematopoietic progenitor cells that can undergo self-renewal and differentiate into multi-lineages. Bone marrow stromal stem cells (BMSCs) represent one of the most commonly-used MSCs. In order to overcome the technical challenge of maintaining primary BMSCs in long-term culture, here we seek to establish reversibly immortalized mouse BMSCs (imBMSCs). By exploiting CRISPR/Cas9-based homology-directed-repair (HDR) mechanism, we target SV40T to mouse Rosa26 locus and efficiently immortalize mouse BMSCs (i.e., imBMSCs). We also immortalize BMSCs with retroviral vector SSR #41 and establish imBMSC41 as a control line. Both imBMSCs and imBMSC41 exhibit long-term proliferative capability although imBMSC41 cells have a higher proliferation rate. SV40T mRNA expression is 130% higher in imBMSC41 than that in imBMSCs. However, FLP expression leads to 86% reduction of SV40T expression in imBMSCs, compared with 63% in imBMSC41 cells. Quantitative genomic PCR analysis indicates that the average copy number of SV40T and hygromycin is 1.05 for imBMSCs and 2.07 for imBMSC41, respectively. Moreover, FLP expression removes 92% of SV40T in imBMSCs at the genome DNA level, compared with 58% of that in imBMSC41 cells, indicating CRISPR/Cas9 HDR-mediated immortalization of BMSCs can be more effectively reversed than that of retrovirus-mediated random integrations. Nonetheless, both imBMSCs and imBMSC41 lines express MSC markers and are highly responsive to BMP9-induced osteogenic, chondrogenic and adipogenic differentiation in vitro and in vivo. Thus, the engineered imBMSCs can be used as a promising alternative source of primary MSCs for basic and translational research in the fields of MSC biology and regenerative medicine. PMID:29340096

Ultrasound-Assisted Liposuction Does Not Compromise the Regenerative Potential of Adipose-Derived Stem Cells

PubMed Central

Duscher, Dominik; Atashroo, David; Maan, Zeshaan N.; Luan, Anna; Brett, Elizabeth A.; Barrera, Janos; Khong, Sacha M.; Zielins, Elizabeth R.; Whittam, Alexander J.; Hu, Michael S.; Walmsley, Graham G.; Pollhammer, Michael S.; Schmidt, Manfred; Schilling, Arndt F.; Machens, Hans-Günther; Huemer, Georg M.; Wan, Derrick C.; Longaker, Michael T.

2016-01-01

Human mesenchymal stem cells (MSCs) have recently become a focus of regenerative medicine, both for their multilineage differentiation capacity and their excretion of proregenerative cytokines. Adipose-derived mesenchymal stem cells (ASCs) are of particular interest because of their abundance in fat tissue and the ease of harvest via liposuction. However, little is known about the impact of different liposuction methods on the functionality of ASCs. Here we evaluate the regenerative abilities of ASCs harvested via a third-generation ultrasound-assisted liposuction (UAL) device versus ASCs obtained via standard suction-assisted lipoaspiration (SAL). Lipoaspirates were sorted using fluorescent assisted cell sorting based on an established surface-marker profile (CD34+/CD31−/CD45−), to obtain viable ASCs. Yield and viability were compared and the differentiation capacities of the ASCs were assessed. Finally, the regenerative potential of ASCs was examined using an in vivo model of tissue regeneration. UAL- and SAL-derived samples demonstrated equivalent ASC yield and viability, and UAL ASCs were not impaired in their osteogenic, adipogenic, or chondrogenic differentiation capacity. Equally, quantitative real-time polymerase chain reaction showed comparable expression of most osteogenic, adipogenic, and key regenerative genes between both ASC groups. Cutaneous regeneration and neovascularization were significantly enhanced in mice treated with ASCs obtained by either UAL or SAL compared with controls, but there were no significant differences in healing between cell-therapy groups. We conclude that UAL is a successful method of obtaining fully functional ASCs for regenerative medicine purposes. Cells harvested with this alternative approach to liposuction are suitable for cell therapy and tissue engineering applications. Significance Adipose-derived mesenchymal stem cells (ASCs) are an appealing source of therapeutic progenitor cells because of their multipotency, diverse cytokine profile, and ease of harvest via liposuction. Alternative approaches to classical suction-assisted liposuction are gaining popularity; however, little evidence exists regarding the impact of different liposuction methods on the regenerative functionality of ASCs. Human ASC characteristics and regenerative capacity were assessed when harvested via ultrasound-assisted (UAL) versus standard suction-assisted liposuction. ASCs obtained via UAL were of equal quality when directly compared with the current gold standard harvest method. UAL is an adjunctive source of fully functional mesenchymal stem cells for applications in basic research and clinical therapy. PMID:26702129

Ultrasound-Assisted Liposuction Does Not Compromise the Regenerative Potential of Adipose-Derived Stem Cells.

PubMed

Duscher, Dominik; Atashroo, David; Maan, Zeshaan N; Luan, Anna; Brett, Elizabeth A; Barrera, Janos; Khong, Sacha M; Zielins, Elizabeth R; Whittam, Alexander J; Hu, Michael S; Walmsley, Graham G; Pollhammer, Michael S; Schmidt, Manfred; Schilling, Arndt F; Machens, Hans-Günther; Huemer, Georg M; Wan, Derrick C; Longaker, Michael T; Gurtner, Geoffrey C

2016-02-01

Human mesenchymal stem cells (MSCs) have recently become a focus of regenerative medicine, both for their multilineage differentiation capacity and their excretion of proregenerative cytokines. Adipose-derived mesenchymal stem cells (ASCs) are of particular interest because of their abundance in fat tissue and the ease of harvest via liposuction. However, little is known about the impact of different liposuction methods on the functionality of ASCs. Here we evaluate the regenerative abilities of ASCs harvested via a third-generation ultrasound-assisted liposuction (UAL) device versus ASCs obtained via standard suction-assisted lipoaspiration (SAL). Lipoaspirates were sorted using fluorescent assisted cell sorting based on an established surface-marker profile (CD34+/CD31-/CD45-), to obtain viable ASCs. Yield and viability were compared and the differentiation capacities of the ASCs were assessed. Finally, the regenerative potential of ASCs was examined using an in vivo model of tissue regeneration. UAL- and SAL-derived samples demonstrated equivalent ASC yield and viability, and UAL ASCs were not impaired in their osteogenic, adipogenic, or chondrogenic differentiation capacity. Equally, quantitative real-time polymerase chain reaction showed comparable expression of most osteogenic, adipogenic, and key regenerative genes between both ASC groups. Cutaneous regeneration and neovascularization were significantly enhanced in mice treated with ASCs obtained by either UAL or SAL compared with controls, but there were no significant differences in healing between cell-therapy groups. We conclude that UAL is a successful method of obtaining fully functional ASCs for regenerative medicine purposes. Cells harvested with this alternative approach to liposuction are suitable for cell therapy and tissue engineering applications. Significance: Adipose-derived mesenchymal stem cells (ASCs) are an appealing source of therapeutic progenitor cells because of their multipotency, diverse cytokine profile, and ease of harvest via liposuction. Alternative approaches to classical suction-assisted liposuction are gaining popularity; however, little evidence exists regarding the impact of different liposuction methods on the regenerative functionality of ASCs. Human ASC characteristics and regenerative capacity were assessed when harvested via ultrasound-assisted (UAL) versus standard suction-assisted liposuction. ASCs obtained via UAL were of equal quality when directly compared with the current gold standard harvest method. UAL is an adjunctive source of fully functional mesenchymal stem cells for applications in basic research and clinical therapy. ©AlphaMed Press.

Tissues from equine cadaver ligaments up to 72 hours of post-mortem: a promising reservoir of stem cells.

PubMed

Shikh Alsook, Mohamad Khir; Gabriel, Annick; Piret, Joëlle; Waroux, Olivier; Tonus, Céline; Connan, Delphine; Baise, Etienne; Antoine, Nadine

2015-12-18

Mesenchymal stem cells (MSCs) harvested from cadaveric tissues represent a promising approach for regenerative medicine. To date, no study has investigated whether viable MSCs could survive in cadaveric tissues from tendon or ligament up to 72 hours of post-mortem. The purpose of the present work was to find out if viable MSCs could survive in cadaveric tissues from adult equine ligaments up to 72 hours of post-mortem, and to assess their ability (i) to remain in an undifferentiated state and (ii) to divide and proliferate in the absence of any specific stimulus. MSCs were isolated from equine cadaver (EC) suspensory ligaments within 48-72 hours of post-mortem. They were evaluated for viability, proliferation, capacity for tri-lineage differentiation, expression of cell surface markers (CD90, CD105, CD73, CD45), pluripotent transcription factor (OCT-4), stage-specific embryonic antigen-1 (SSEA-1), neuron-specific class III beta-tubulin (TUJ-1), and glial fibrillary acidic protein (GFAP). As well, they were characterized by transmission electron microscope (TEM). EC-MSCs were successfully isolated and maintained for 20 passages with high cell viability and proliferation. Phase contrast microscopy revealed that cells with fibroblast-like appearance were predominant in the culture. Differentiation assays proved that EC-MSCs are able to differentiate towards mesodermal lineages (osteogenic, adipogenic, chondrogenic). Flow cytometry analysis demonstrated that EC-MSCs expressed CD90, CD105, and CD73, while being negative for the leukocyte common antigen CD45. Immunofluorescence analysis showed a high percentage of positive cells for OCT-4 and SSEA-1. Surprisingly, in absence of any stimuli, some adherent cells closely resembling neuronal and glial morphology were also observed. Interestingly, our results revealed that approximately 15 % of the cell populations were TUJ-1 positive, whereas GFAP expression was detected in only a few cells. Furthermore, TEM analysis confirmed the stemness of EC-MSCs and identified some cells with a typical neuronal morphology. Our findings raise the prospect that the tissues harvested from equine ligaments up to 72 hours of post-mortem represent an available reservoir of specific stem cells. EC-MSCs could be a promising alternative source for tissue engineering and stem cell therapy in equine medicine.

Hydrostatic pressure-driven three-dimensional cartilage induction using human adipose-derived stem cells and collagen gels.

PubMed

Ogawa, Rei; Orgill, Dennis P; Murphy, George F; Mizuno, Shuichi

2015-01-01

The chondrogenic potential of adipose-derived stem cells (ASCs) has been previously demonstrated, although several reports have indicated that ASCs produce less cartilage-specific matrix than bone marrow-derived mesenchymal stem cells. In this study, we intended to improve chondrogenic phenotypes of ASCs using hydrostatic pressure (HP), without utilizing any growth factors other than the transforming growth factor-β1. Human ASCs (CD13(+), 44(+), 90(+), 14(-), 31(-), 34(-)) were harvested and cultured. After three passages, the cells were suspended in 0.3% neutralized collagen type I solution and injected into semipermeable membrane tubes, from which 66 pouches were constructed. After a day of incubation, the 66 pouches were divided into three groups. Group HP1: Pouches were incubated for 1 week with treatment of cyclic HP at 0-0.5 MPa (4.93 atm), 0.5 Hz, with a medium replenishment rate of 0.1 mL/min at 37°C, 3% O2, and 5% CO2 in air using a bioprocessor. This was followed by 3 weeks with no HP and without pouches. Group HP2: Pouches were incubated for the first and third week (2 total weeks) with the same condition of Group HP1. No HP was applied in the second and fourth week. Group AP: Pouches with one end opened were incubated without HP. We evaluated the cell constructs histologically and immunohistochemically, as well as for specific gene expression. Accumulation of the matrix in the HP1 and HP2 groups was much denser than AP groups, particularly after 2 weeks. Cell numbers in the HP groups increased gradually in the middle zone and peaked at 1 week after incubation, maintaining their numbers for the entire course on the surface layer of the construct. In the genomic study results, COL 2A1, COL 10A1, ACAN, SOX9, MMP3, and MMP13 were upregulated and COL 1A1, ITGB1, and PCNA were downregulated by HP. There were no significant differences between HP1 and HP2 gene expression. It was suggested that HP is especially beneficial in the early stage of chondrogenesis of ASCs. Moreover, the expression profile of genes related to chondrocyte differentiation/proliferation was significantly enhanced by HP loading compared with the AP control.

Decellularized adipose tissue microcarriers as a dynamic culture platform for human adipose-derived stem/stromal cell expansion.

PubMed

Yu, Claire; Kornmuller, Anna; Brown, Cody; Hoare, Todd; Flynn, Lauren E

2017-03-01

With the goal of designing a clinically-relevant expansion strategy for human adipose-derived stem/stromal cells (ASCs), methods were developed to synthesize porous microcarriers derived purely from human decellularized adipose tissue (DAT). An electrospraying approach was applied to generate spherical DAT microcarriers with an average diameter of 428 ± 41 μm, which were soft, compliant, and stable in long-term culture without chemical crosslinking. Human ASCs demonstrated enhanced proliferation on the DAT microcarriers relative to commercially-sourced Cultispher-S microcarriers within a spinner culture system over 1 month. ASC immunophenotype was maintained post expansion, with a trend for reduced expression of the cell adhesion receptors CD73, CD105, and CD29 under dynamic conditions. Upregulation of the early lineage-specific genes PPARγ, LPL, and COMP was observed in the ASCs expanded on the DAT microcarriers, but the cells retained their multilineage differentiation capacity. Comparison of adipogenic and osteogenic differentiation in 2-D cultures prepared with ASCs pre-expanded on the DAT microcarriers or Cultispher-S microcarriers revealed similar adipogenic and enhanced osteogenic marker expression in the DAT microcarrier group, which had undergone a higher population fold change. Further, histological staining results suggested a more homogeneous differentiation response in the ASCs expanded on the DAT microcarriers as compared to either Cultispher-S microcarriers or tissue culture polystyrene. A pilot chondrogenesis study revealed higher levels of chondrogenic gene and protein expression in the ASCs expanded on the DAT microcarriers relative to all other groups, including the baseline controls. Overall, this study demonstrates the promise of applying dynamic culture with tissue-specific DAT microcarriers as a means of deriving regenerative cell populations. Copyright © 2016 Elsevier Ltd. All rights reserved.

Profiling of human epigenetic regulators using a semi-automated real-time qPCR platform validated by next generation sequencing

PubMed Central

Dudakovic, Amel; Gluscevic, Martina; Paradise, Christopher R.; Dudakovic, Halil; Khani, Farzaneh; Thaler, Roman; Ahmed, Farah S.; Li, Xiaodong; Dietz, Allan B.; Stein, Gary S.; Montecino, Martin A.; Deyle, David R.; Westendorf, Jennifer J.; van Wijnen, Andre J.

2017-01-01

Epigenetic mechanisms control phenotypic commitment of mesenchymal stromal/stem cells (MSCs) into osteogenic, chondrogenic or adipogenic lineages. To investigate enzymes and chromatin binding proteins controlling the epigenome, we developed a hybrid expression screening strategy that combines semi-automatic real-time qPCR (RT-qPCR), next generation RNA sequencing (RNA-seq), and a novel data management application (FileMerge). This strategy was used to interrogate expression of a large cohort (n>300) of human epigenetic regulators (EpiRegs) that generate, interpret and/or edit the histone code. We find that EpiRegs with similar enzymatic functions are variably expressed and specific isoforms dominate over others in human MSCs. This principle is exemplified by analysis of key histone acetyl transferases (HATs) and deacetylases (HDACs), H3 lysine methyl transferases (e.g., EHMTs) and demethylases (KDMs), as well as bromodomain (BRDs) and chromobox (CBX) proteins. Our results show gender-specific expression of H3 lysine 9 [H3K9] demethylases (e.g., KDM5D and UTY) as expected and upregulation of distinct EpiRegs (n>30) during osteogenic differentiation of MSCs (e.g., HDAC5 and HDAC7). The functional significance of HDACs in osteogenic lineage commitment of MSCs was functionally validated using panobinostat (LBH-589). This pan-deacetylase inhibitor suppresses osteoblastic differentiation as evidenced by reductions in bone-specific mRNA markers (e.g., ALPL), alkaline phosphatase activity and calcium deposition (i.e., Alizarin Red staining). Thus, our RT-qPCR platform identifies candidate EpiRegs by expression screening, predicts biological outcomes of their corresponding inhibitors, and enables manipulation of the human epigenome using molecular or pharmacological approaches to control stem cell differentiation. PMID:28132772

Layering PLGA-based electrospun membranes and cell sheets for engineering cartilage-bone transition.

PubMed

Mouthuy, P-A; El-Sherbini, Y; Cui, Z; Ye, H

2016-04-01

It is now widely acknowledged that implants that have been designed with an effort towards reconstructing the transition between tissues might improve their functionality and integration in vivo. This paper contributes to the development of improved treatment for articular cartilage repair by exploring the potential of the combination of electrospinning technology and cell sheet engineering to create cartilage tissue. Poly(lactic-co-glycolic acid) (PLGA) was used to create the electrospun membranes. The focus being on the cartilage-bone transition, collagen type I and hydroxyapatite (HA) were also added to the scaffolds to increase the histological biocompatibility. Human mesenchymal stem cells (hMSCs) were cultured in thermoresponsive dishes to allow non-enzymatic removal of an intact cell layer after reaching confluence. The tissue constructs were created by layering electrospun membranes with sheets of hMSCs and were cultured under chondrogenic conditions for up to 21 days. High viability was found to be maintained in the multilayered construct. Under chondrogenic conditions, reverse-transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry have shown high expression levels of collagen type X, a form of collagen typically found in the calcified zone of articular cartilage, suggesting an induction of chondrocyte hypertrophy in the PLGA-based scaffolds. To conclude, this paper suggests that layering electrospun scaffolds and cell sheets is an efficient approach for the engineering of tissue transitions, and in particular the cartilage-bone transition. The use of PLGA-based scaffold might be particularly useful for the bone-cartilage reconstruction, since the differentiated tissue constructs seem to show characteristics of calcified cartilage. Copyright © 2013 John Wiley & Sons, Ltd.

Live imaging flow bioreactor for the simulation of articular cartilage regeneration after treatment with bioactive hydrogel.

PubMed

Bar, Assaf; Ruvinov, Emil; Cohen, Smadar

2018-06-05

Osteochondral defects (OCDs) are conditions affecting both cartilage and the underlying bone. Since cartilage is not spontaneously regenerated, our group has recently developed a strategy of injecting bioactive alginate hydrogel into the defect for promoting endogenous regeneration of cartilage via presentation of affinity-bound transforming growth factor β1 (TGF-β1). As in vivo model systems often provide only limited insights as for the mechanism behind regeneration processes, here we describe a novel flow bioreactor for the in vitro modeling of the OCD microenvironment, designed to promote cell recruitment from the simulated bone marrow compartment into the hydrogel, under physiological flow conditions. Computational fluid dynamics modeling confirmed that the bioreactor operates in a relevant slow-flowing regime. Using a chemotaxis assay, it was shown that TGF-β1 does not affect human mesenchymal stem cell (hMSC) chemotaxis in 2D culture. Accessible through live imaging, the bioreactor enabled monitoring and discrimination between erosion rates and profiles of different alginate hydrogel compositions, using green fluorescent protein-expressing cells. Mathematical modeling of the erosion front progress kinetics predicted the erosion rate in the bioreactor up to 7 days postoperation. Using quantitative real-time polymerase chain reaction of early chondrogenic markers, the onset of chondrogenic differentiation in hMSCs was detected after 7 days in the bioreactor. In conclusion, the designed bioreactor presents multiple attributes, making it an optimal device for mechanistical studies, serving as an investigational tool for the screening of other biomaterial-based, tissue engineering strategies. © 2018 Wiley Periodicals, Inc.

Discovery of novel differentiation markers in the early stage of chondrogenesis by glycoform-focused reverse proteomics and genomics.

PubMed

Ishihara, Takeshi; Kakiya, Kiyoshi; Takahashi, Koji; Miwa, Hiroto; Rokushima, Masatomo; Yoshinaga, Tomoyo; Tanaka, Yoshikazu; Ito, Takaomi; Togame, Hiroko; Takemoto, Hiroshi; Amano, Maho; Iwasaki, Norimasa; Minami, Akio; Nishimura, Shin-Ichiro

2014-01-01

Osteoarthritis (OA) is one of the most common chronic diseases among adults, especially the elderly, which is characterized by destruction of the articular cartilage. Despite affecting more than 100 million individuals all over the world, therapy is currently limited to treating pain, which is a principal symptom of OA. New approaches to the treatment of OA that induce regeneration and repair of cartilage are strongly needed. To discover potent markers for chondrogenic differentiation, glycoform-focused reverse proteomics and genomics were performed on the basis of glycoblotting-based comprehensive approach. Expression levels of high-mannose type N-glycans were up-regulated significantly at the late stage of differentiation of the mouse chondroprogenitor cells. Among 246 glycoproteins carrying this glycotype identified by ConA affinity chromatography and LC/MS, it was demonstrated that 52% are classified as cell surface glycoproteins. Gene expression levels indicated that mRNAs for 15 glycoproteins increased distinctly in the earlier stages during differentiation compared with Type II collagen. The feasibility of mouse chondrocyte markers in human chondrogenesis model was demonstrated by testing gene expression levels of these 15 glycoproteins during differentiation in human mesenchymal stem cells. The results showed clearly an evidence of up-regulation of 5 genes, ectonucleotide pyrophosphatase/phosphodiesterase family member 1, collagen alpha-1(III) chain, collagen alpha-1(XI) chain, aquaporin-1, and netrin receptor UNC5B, in the early stages of differentiation. These cell surface 5 glycoproteins become highly sensitive differentiation markers of human chondrocytes that contribute to regenerative therapies, and development of novel therapeutic reagents. © 2013.

Chondrosarcoma: A Rare Misfortune in Aging Human Cartilage? The Role of Stem and Progenitor Cells in Proliferation, Malignant Degeneration and Therapeutic Resistance

PubMed Central

Boehme, Karen A.; Schleicher, Sabine B.; Rolauffs, Bernd

2018-01-01

Unlike other malignant bone tumors including osteosarcomas and Ewing sarcomas with a peak incidence in adolescents and young adults, conventional and dedifferentiated chondrosarcomas mainly affect people in the 4th to 7th decade of life. To date, the cell type of chondrosarcoma origin is not clearly defined. However, it seems that mesenchymal stem and progenitor cells (MSPC) in the bone marrow facing a pro-proliferative as well as predominantly chondrogenic differentiation milieu, as is implicated in early stage osteoarthritis (OA) at that age, are the source of chondrosarcoma genesis. But how can MSPC become malignant? Indeed, only one person in 1,000,000 will develop a chondrosarcoma, whereas the incidence of OA is a thousandfold higher. This means a rare coincidence of factors allowing escape from senescence and apoptosis together with induction of angiogenesis and migration is needed to generate a chondrosarcoma. At early stages, chondrosarcomas are still assumed to be an intermediate type of tumor which rarely metastasizes. Unfortunately, advanced stages show a pronounced resistance both against chemo- and radiation-therapy and frequently metastasize. In this review, we elucidate signaling pathways involved in the genesis and therapeutic resistance of chondrosarcomas with a focus on MSPC compared to signaling in articular cartilage (AC). PMID:29361725

Bioreactor mechanically guided 3D mesenchymal stem cell chondrogenesis using a biocompatible novel thermo-reversible methylcellulose-based hydrogel.

PubMed

Cochis, A; Grad, S; Stoddart, M J; Farè, S; Altomare, L; Azzimonti, B; Alini, M; Rimondini, L

2017-03-23

Autologous chondrocyte implantation for cartilage repair represents a challenge because strongly limited by chondrocytes' poor expansion capacity in vitro. Mesenchymal stem cells (MSCs) can differentiate into chondrocytes, while mechanical loading has been proposed as alternative strategy to induce chondrogenesis excluding the use of exogenous factors. Moreover, MSC supporting material selection is fundamental to allow for an active interaction with cells. Here, we tested a novel thermo-reversible hydrogel composed of 8% w/v methylcellulose (MC) in a 0.05 M Na 2 SO 4 solution. MC hydrogel was obtained by dispersion technique and its thermo-reversibility, mechanical properties, degradation and swelling were investigated, demonstrating a solution-gelation transition between 34 and 37 °C and a low bulk degradation (<20%) after 1 month. The lack of any hydrogel-derived immunoreaction was demonstrated in vivo by mice subcutaneous implantation. To induce in vitro chondrogenesis, MSCs were seeded into MC solution retained within a porous polyurethane (PU) matrix. PU-MC composites were subjected to a combination of compression and shear forces for 21 days in a custom made bioreactor. Mechanical stimulation led to a significant increase in chondrogenic gene expression, while histological analysis detected sulphated glycosaminoglycans and collagen II only in loaded specimens, confirming MC hydrogel suitability to support load induced MSCs chondrogenesis.

Variations in chondrogenesis of human bone marrow-derived mesenchymal stem cells in fibrin/alginate blended hydrogels

PubMed Central

Ma, Kun; Titan, Ashley L.; Stafford, Melissa; Zheng, Chun hua; Levenston, Marc E.

2012-01-01

Fibrin and alginate hydrogels have been widely used to support chondrogenesis of bone marrow-derived mesenchymal stem cells (BM-MSCs) for articular cartilage and fibrocartilage tissue engineering, with distinct advantages and disadvantages to each material. Attempting to produce a gel scaffold exhibiting beneficial characteristics of both materials, we fabricated fibrin/alginate blended hydrogels at various blend ratios and evaluated the gel morphology, mechanical properties and their support for BM-MSC chondrogenesis. Results show that when the fibrin/alginate ratio decreased, the fibrin architecture transitioned from uniform to interconnected fibrous and finally to disconnected islands against an alginate background, with opposing trends in the alginate architecture. Fibrin maintained gel extensibility and promoted cell proliferation, while alginate improved the gel biostability and better supported glycosaminoglycan and collagen II production and chondrogenic gene expression. Blended gels had physical and biological characteristics intermediate between fibrin and alginate. Of the blends examined, FA 40:8 (40 mg/mL fibrinogen blended with 8 mg/mL alginate) was found to be the most appropriate group for future studies on tension-driven BM-MSC fibrochondrogenesis. As BM-MSC differentiation appeared to vary between fibrin and alginate regions of blended scaffolds, this study also highlighted the potential to develop spatially heterogeneous tissues through manipulating the heterogeneity of scaffold composition. PMID:22750738

Extracellular Matrix (ECM) Multilayer Membrane as a Sustained Releasing Growth Factor Delivery System for rhTGF-β3 in Articular Cartilage Repair

PubMed Central

Park, Sang-Hyug; Kim, Moon Suk; Kim, Young Jick; Choi, Byung Hyune; Lee, Chun Tek; Park, So Ra; Min, Byoung-Hyun

2016-01-01

Recombinant human transforming growth factor beta-3 (rhTGF-β3) is a key regulator of chondrogenesis in stem cells and cartilage formation. We have developed a novel drug delivery system that continuously releases rhTGF-β3 using a multilayered extracellular matrix (ECM) membrane. We hypothesize that the sustained release of rhTGF-β3 could activate stem cells and result in enhanced repair of cartilage defects. The properties and efficacy of the ECM multilayer-based delivery system (EMLDS) are investigated using rhTGF-β3 as a candidate drug. The bioactivity of the released rhTGF-ß3 was evaluated through chondrogenic differentiation of mesenchymal stem cells (MSCs) using western blot and circular dichroism (CD) analyses in vitro. The cartilage reparability was evaluated through implanting EMLDS with endogenous and exogenous MSC in both in vivo and ex vivo models, respectively. In the results, the sustained release of rhTGF-ß3 was clearly observed over a prolonged period of time in vitro and the released rhTGF-β3 maintained its structural stability and biological activity. Successful cartilage repair was also demonstrated when rabbit MSCs were treated with rhTGF-β3-loaded EMLDS ((+) rhTGF-β3 EMLDS) in an in vivo model and when rabbit chondrocytes and MSCs were treated in ex vivo models. Therefore, the multilayer ECM membrane could be a useful drug delivery system for cartilage repair. PMID:27258120

Synergistic promoting effects of bone morphogenetic protein 12/connective tissue growth factor on functional differentiation of tendon derived stem cells and patellar tendon window defect regeneration.

PubMed

Xu, Kang; Sun, Yanjun; Kh Al-Ani, Mohanad; Wang, Chunli; Sha, Yongqiang; Sung, Kl Paul; Dong, Nianguo; Qiu, Xuefeng; Yang, Li

2018-01-03

Current study investigated bone morphogenetic protein 12 (BMP12) and connective tissue growth factor (CTGF) activate tendon derived stem cells (TDSCs) tenogenic differentiation, and promotion of injured tendon regeneration. TDSCs were transfected with BMP12 and CTGF via recombinant adenovirus (Ad) infection. Gene transfection efficiency, cell viability and cytotoxicity, tenogenic gene expression, collagen I/III synthesis were evaluated in vitro. For the in vivo study, the transfected cells were transplanted into the rat patellar tendon window defect. At weeks 2 and 8 of post-surgery, the repaired tendon tissues were harvested for histological and biomechanical examinations. The transfected TDSCs revealed relatively stable transfection efficiency (80-90%) with active cell viability means while rare cytotoxicity in each group. During days 1 and 5, BMP12 and CTGF transfection caused tenogenic differentiation genes activation in TDSCs: type I/III collagen, tenascin-C, and scleraxis were all up-regulated, whereas osteogenic, adipogenic, and chondrogenic markers were all down-regulated respectively. In addition, BMP12 and CTGF overexpression significantly promote type I/III collagen synthesis. After in vivo transplantation, at 2 and 8 weeks post-surgery, BMP12, CTGF and co-transfection groups showed more integrated tendon tissue structure versus control, meanwhile, the ultimate failure loads and Young's were all higher than control. Remarkably, at 8 weeks post-surgery, the biomechanical properties of co-transfection group was approaching to normal rat patellar tendon, moreover, the ratio of type III/I collagen maintained about 20% in each transfection group, meanwhile, the type I collagen were significantly increased with co-transfection treatment. In conclusion, BMP12 and CTGF transfection stimulate tenogenic differentiation of TDSCs. The synergistic effects of simultaneous transfection of both may significantly promoted rat patellar tendon window defect regeneration. Copyright © 2017 Elsevier Ltd. All rights reserved.

Integrating biologically inspired nanomaterials and table-top stereolithography for 3D printed biomimetic osteochondral scaffolds

NASA Astrophysics Data System (ADS)

Castro, Nathan J.; O'Brien, Joseph; Zhang, Lijie Grace

2015-08-01

The osteochondral interface of an arthritic joint is notoriously difficult to regenerate due to its extremely poor regenerative capacity and complex stratified architecture. Native osteochondral tissue extracellular matrix is composed of numerous nanoscale organic and inorganic constituents. Although various tissue engineering strategies exist in addressing osteochondral defects, limitations persist with regards to tissue scaffolding which exhibit biomimetic cues at the nano to micro scale. In an effort to address this, the current work focused on 3D printing biomimetic nanocomposite scaffolds for improved osteochondral tissue regeneration. For this purpose, two biologically-inspired nanomaterials have been synthesized consisting of (1) osteoconductive nanocrystalline hydroxyapatite (nHA) (primary inorganic component of bone) and (2) core-shell poly(lactic-co-glycolic) acid (PLGA) nanospheres encapsulated with chondrogenic transforming growth-factor β1 (TGF-β1) for sustained delivery. Then, a novel table-top stereolithography 3D printer and the nano-ink (i.e., nHA + nanosphere + hydrogel) were employed to fabricate a porous and highly interconnected osteochondral scaffold with hierarchical nano-to-micro structure and spatiotemporal bioactive factor gradients. Our results showed that human bone marrow-derived mesenchymal stem cell adhesion, proliferation, and osteochondral differentiation were greatly improved in the biomimetic graded 3D printed osteochondral construct in vitro. The current work served to illustrate the efficacy of the nano-ink and current 3D printing technology for efficient fabrication of a novel nanocomposite hydrogel scaffold. In addition, tissue-specific growth factors illustrated a synergistic effect leading to increased cell adhesion and directed stem cell differentiation.

Chondrogenic differentiation of ATDC5-cells under the influence of Mg and Mg alloy degradation.

PubMed

Martinez Sanchez, Adela H; Feyerabend, Frank; Laipple, Daniel; Willumeit-Römer, Regine; Weinberg, Annelie; Luthringer, Bérengère J C

2017-03-01

Biodegradable magnesium (Mg)-based materials are a potential alternative to permanent implants for application in children. Nevertheless effects of those materials on growth plate cartilage and chondrogenesis have not been previously evaluated. In vitro differentiation of ATDC5 cells was evaluated under the influence of pure Mg (PMg), Mg with 10wt% of gadolinium (Mg-10Gd) and Mg with 2wt% of silver (Mg-2Ag) degradation products (extracts) and direct cell culture on the materials. Gene expression showed an inhibitory effect on ATDC5 mineralization with the three extracts and a chondrogenic potential of Mg-10Gd. Cells cultured in Mg-10Gd and Mg-2Ag extracts showed the same proliferation and morphology than cells cultured in growth conditions. Mg-10Gd induced an increase in production of ECM and a bigger cell size, similar to the effects found with differentiation conditions. An increased metabolic activity was observed in cells cultured under the influence of Mg-10Gd extracts, indicated by an acidic pH during most of the culture period. After 7days of culture on the materials, ATDC5 growth, distribution and ECM synthesis were higher on Mg-10Gd samples, followed by Mg-2Ag and PMg, which was influenced by the homogeneity and composition of the degradation layer. This study confirmed the tolerance of ATDC5 cells to Mg-based materials and a chondrogenic effect of Mg-10Gd. Further studies in vitro and in vivo are necessary to evaluate cell reactions to those materials, as well as the effects on bone growth and the biocompatibility of the alloying system in the body. Copyright © 2016 Elsevier B.V. All rights reserved.

A small population of resident limb bud mesenchymal cells express few MSC-associated markers, but the expression of these markers is increased immediately after cell culture.

PubMed

Marín-Llera, Jessica Cristina; Chimal-Monroy, Jesús

2018-05-01

Skeletal progenitors are derived from resident limb bud mesenchymal cells of the vertebrate embryos. However, it remains poorly understood if they represent stem cells, progenitors, or multipotent mesenchymal stromal cells (MSC). Derived-MSC of different adult tissues under in vitro experimental conditions can differentiate into the same cellular lineages that are present in the limb. Here, comparing non-cultured versus cultured mesenchymal limb bud cells, we determined the expression of MSC-associated markers, the in vitro differentiation capacity and their gene expression profile. Results showed that in freshly isolated limb bud mesenchymal cells, the proportion of cells expressing Sca1, CD44, CD105, CD90, and CD73 is very low and a low expression of lineage-specific genes was observed. However, recently seeded limb bud mesenchymal cells acquired Sca1 and CD44 markers and the expression of the key differentiation genes Runx2 and Sox9, while Scx and Pparg genes decreased. Also, their chondrogenic differentiation capacity decreased through cellular passages while the osteogenic increased. Our findings suggest that the modification of the cell adhesion process through the in vitro method changed the limb mesenchymal cell immunophenotype leading to the expression and maintenance of common MSC-associated markers. These findings could have a significant impact on MSC study and isolation strategy because they could explain common variations observed in the MSC immunophenotype in different tissues. © 2018 International Federation for Cell Biology.

PPARD is an Inhibitor of Cartilage Growth in External Ears.

PubMed

Zhang, Zhen; Duan, Yanyu; Wu, Zhongping; Zhang, Hui; Ren, Jun; Huang, Lusheng

2017-01-01

Peroxisome proliferator-activated receptor beta/delta (PPARD) is an important determinant of multiple biological processes. Our previous studies identified a missense mutation in the PPARD gene that significantly reduces its transcription activity, and consequently causes enlarged external ears in pigs. However, the mechanisms underlying the causality has remained largely unknown. Here, we show that PPARD retards the development of auricular cartilage by accelerating the apoptosis of cartilage stem/progenitor cells (CSPCs), the terminal differentiation of cartilage cells and the degradation of cartilage extracellular matrix in the auricle. At the transcription level, PPARD upregulates a set of genes that are associated with CSPCs apoptosis and chondrogenic differentiation, chondroblast differentiation and extracellular matrix degradation. ChIP-seq identified direct target genes of PPARD, including a well-documented gene for cartilage development: PPARG . We further show that compared to wild-type PPARD, the G32E mutant up-regulates the expression of PPARG and subsequently leads to the downregulation of critical genes that inhibit cartilage growth. These findings allow us to conclude that PPARD is an inhibitor of auricular cartilage growth in pigs. The causative mutation (G32E) in the PPARD gene attenuates the PPARD-mediated retardation of cartilage growth in the auricle, contributing to enlarged ears in pigs. The findings advance our understanding of the mechanisms underlying auricular development in mammals, and shed insight into the studies of innate pinna disorders and cartilage regeneration medicine in humans.

Leptin differentially regulates chondrogenesis in mouse vertebral and tibial growth plates.

PubMed

Yu, Bo; Jiang, Kaibiao; Chen, Bin; Wang, Hantao; Li, Xinfeng; Liu, Zude

2017-05-31

Leptin plays an important role in mediating chondrogenesis of limb growth plate. Previous studies suggest that bone structures and development of spine and limb are different. The expression of Ob-Rb, the gene that encodes leptin receptors, is vertebral and appendicular region-specific, suggesting the regulation of leptin on VGP and TGP chondrogenesis may be very different. The aim of the present study was to investigate the differential regulation of leptin on the chondrogenesis of vertebral growth plate (VGP) and tibial growth plate (TGP). We compared the VGP and TGP from wild type (C57BL/6) and leptin-deficient (ob/ob) mice. We then generated primary cultures of TGP and VGP chondrocytes. By treating the primary cells with different concentrations of leptin in vitro, we analyzed proliferation and apoptosis of the primary chondrocytes from TGP and VGP. We further measured expression of chondrogenic-related genes in these cells that had been incubated with different doses of leptin. Leptin-deficient mice of 8-week-old had shorter tibial and longer vertebral lengths than the wide type mice. Disturbed columnar structure was observed for TGP but not for VGP. In primary chondrocyte cultures, leptin inhibited VGP chondrocyte proliferation but promoted their apoptosis. Collagen IIA and aggrecan mRNA, and the protein levels of proliferation- and chondrogenesis-related markers, including PCNA, Sox9, and Smad4, were downregulated by leptin in a dose-dependent manner. In contrast, leptin stimulated the proliferation and chondrogenic differentiation of TGP chondrocytes at physiological levels (i.e., 10 and 50 ng/mL) but not at high levels (i.e., 100 and 1000 ng/mL). Leptin exerts a stimulatory effect on the proliferation and chondrogenic differentiation of the long bone growth plate but an inhibitory effect on the spine growth plate. The ongoing study will shed light on the regulatory mechanisms of leptin in bone development and metabolism.

Sol-gel derived lithium-releasing glass for cartilage regeneration.

PubMed

Li, Siwei; Maçon, Anthony L B; Jacquemin, Manon; Stevens, Molly M; Jones, Julian R

2017-07-01

Wnt-signalling cascade is one of the crucial pathways involved in the development and homeostasis of cartilage. Influencing this pathway can potentially contribute to improved cartilage repair or regeneration. One key molecular regulator of the Wnt pathway is the glycogen synthase kinase-3 enzyme, the inhibition of which allows initiation of the signalling pathway. This study aims to utilise a binary SiO 2 -Li 2 O sol-gel derived glass for controlled delivery of lithium, a known glycogen synthase kinase-3 antagonist. The effect of the dissolution products of the glass on chondrogenic differentiation in an in vitro 3D pellet culture model is reported. Dissolution products that contained 5 mM lithium and 3.5 mM silicon were capable of inducing chondrogenic differentiation and hyaline cartilaginous matrix formation without the presence of growth factors such as TGF-β3. The results suggest that sol-gel derived glass has the potential to be used as a delivery vehicle for therapeutic lithium ions in cartilage regeneration applications.

Infrapatellar fat pad-derived stem cells maintain their chondrogenic capacity in disease and can be used to engineer cartilaginous grafts of clinically relevant dimensions.

PubMed

Liu, Yurong; Buckley, Conor Timothy; Almeida, Henrique V; Mulhall, Kevin J; Kelly, Daniel John

2014-11-01

A therapy for regenerating large cartilaginous lesions within the articular surface of osteoarthritic joints remains elusive. While tissue engineering strategies such as matrix-assisted autologous chondrocyte implantation can be used in the repair of focal cartilage defects, extending such approaches to the treatment of osteoarthritis will require a number of scientific and technical challenges to be overcome. These include the identification of an abundant source of chondroprogenitor cells that maintain their chondrogenic capacity in disease, as well as the development of novel approaches to engineer scalable cartilaginous grafts that could be used to resurface large areas of damaged joints. In this study, it is first demonstrated that infrapatellar fat pad-derived stem cells (FPSCs) isolated from osteoarthritic (OA) donors possess a comparable chondrogenic capacity to FPSCs isolated from patients undergoing ligament reconstruction. In a further validation of their functionality, we also demonstrate that FPSCs from OA donors respond to the application of physiological levels of cyclic hydrostatic pressure by increasing aggrecan gene expression and the production of sulfated glycosaminoglycans. We next explored whether cartilaginous grafts could be engineered with diseased human FPSCs using a self-assembly or scaffold-free approach. After examining a range of culture conditions, it was found that continuous supplementation with both transforming growth factor-β3 (TGF-β3) and bone morphogenic protein-6 (BMP-6) promoted the development of tissues rich in proteoglycans and type II collagen. The final phase of the study sought to scale-up this approach to engineer cartilaginous grafts of clinically relevant dimensions (≥2 cm in diameter) by assembling FPSCs onto electrospun PLLA fiber membranes. Over 6 weeks in culture, it was possible to generate robust, flexible cartilage-like grafts of scale, opening up the possibility that tissues engineered using FPSCs derived from OA patients could potentially be used to resurface large areas of joint surfaces damaged by trauma or disease.

Infrapatellar Fat Pad-Derived Stem Cells Maintain Their Chondrogenic Capacity in Disease and Can be Used to Engineer Cartilaginous Grafts of Clinically Relevant Dimensions

PubMed Central

Liu, Yurong; Buckley, Conor Timothy; Almeida, Henrique V.; Mulhall, Kevin J.

2014-01-01

A therapy for regenerating large cartilaginous lesions within the articular surface of osteoarthritic joints remains elusive. While tissue engineering strategies such as matrix-assisted autologous chondrocyte implantation can be used in the repair of focal cartilage defects, extending such approaches to the treatment of osteoarthritis will require a number of scientific and technical challenges to be overcome. These include the identification of an abundant source of chondroprogenitor cells that maintain their chondrogenic capacity in disease, as well as the development of novel approaches to engineer scalable cartilaginous grafts that could be used to resurface large areas of damaged joints. In this study, it is first demonstrated that infrapatellar fat pad-derived stem cells (FPSCs) isolated from osteoarthritic (OA) donors possess a comparable chondrogenic capacity to FPSCs isolated from patients undergoing ligament reconstruction. In a further validation of their functionality, we also demonstrate that FPSCs from OA donors respond to the application of physiological levels of cyclic hydrostatic pressure by increasing aggrecan gene expression and the production of sulfated glycosaminoglycans. We next explored whether cartilaginous grafts could be engineered with diseased human FPSCs using a self-assembly or scaffold-free approach. After examining a range of culture conditions, it was found that continuous supplementation with both transforming growth factor-β3 (TGF-β3) and bone morphogenic protein-6 (BMP-6) promoted the development of tissues rich in proteoglycans and type II collagen. The final phase of the study sought to scale-up this approach to engineer cartilaginous grafts of clinically relevant dimensions (≥2 cm in diameter) by assembling FPSCs onto electrospun PLLA fiber membranes. Over 6 weeks in culture, it was possible to generate robust, flexible cartilage-like grafts of scale, opening up the possibility that tissues engineered using FPSCs derived from OA patients could potentially be used to resurface large areas of joint surfaces damaged by trauma or disease. PMID:24785365

Subchondral mesenchymal stem cells from osteoarthritic knees display high osteogenic differentiation capacity through microRNA-29a regulation of HDAC4.

PubMed

Lian, Wei-Shiung; Wu, Ren-Wen; Lee, Mel S; Chen, Yu-Shan; Sun, Yi-Chih; Wu, Shing-Long; Ke, Huei-Jing; Ko, Jih-Yang; Wang, Feng-Sheng

2017-12-01

Subchondral bone deterioration and osteophyte formation attributable to excessive mineralization are prominent features of end-stage knee osteoarthritis (OA). The cellular events underlying subchondral integrity diminishment remained elusive. This study was undertaken to characterize subchondral mesenchymal stem cells (SMSCs) isolated from patients with end-stage knee OA who required total knee arthroplasty. The SMSCs expressed surface antigens CD29, CD44, CD73, CD90, CD105, and CD166 and lacked CD31, CD45, and MHCII expression. The cell cultures exhibited higher proliferation and greater osteogenesis and chondrogenesis potencies, whereas their population-doubling time and adipogenic lineage commitment were lower than those of bone marrow MSCs (BMMSCs). They also displayed higher expressions of embryonic stem cell marker OCT3/4 and osteogenic factors Wnt3a, β-catenin, and microRNA-29a (miR-29a), concomitant with lower expressions of joint-deleterious factors HDAC4, TGF-β1, IL-1β, TNF-α, and MMP3, in comparison with those of BMMSCs. Knockdown of miR-29a lowered Wnt3a expression and osteogenic differentiation of the SMSCs through elevating HDAC4 translation, which directly regulated the 3'-untranslated region of HDAC4. Likewise, transgenic mice that overexpressed miR-29a in osteoblasts exhibited a high bone mass in the subchondral region. SMSCs in the transgenic mice showed a higher osteogenic differentiation and lower HDAC4 signaling than those in wild-type mice. Taken together, high osteogenesis potency existed in the SMSCs in the osteoarthritic knee. The miR-29a modulation of HDAC4 and Wnt3a signaling was attributable to the increase in osteogenesis. This study shed an emerging light on the characteristics of SMSCs and highlighted the contribution of SMSCs in the exacerbation of subchondral integrity in end-stage knee OA. Subchondral MSCs (SMSCs) from OA knee expressed embryonic stem cell marker Oct3/4. The SMSCs showed high proliferation and osteogenic and chondrogenic potencies. miR-29a regulated osteogenesis of the SMSCs through modulation of HDAC4 and Wnt3a. A high osteogenic potency of the SMSCs existed in mice overexpressing miR-29a in bone. Aberrant osteogenesis in SMSCs provides a new insight to subchondral damage in OA.

LncRNA-HIT Functions as an Epigenetic Regulator of Chondrogenesis through Its Recruitment of p100/CBP Complexes.

PubMed

Carlson, Hanqian L; Quinn, Jeffrey J; Yang, Yul W; Thornburg, Chelsea K; Chang, Howard Y; Stadler, H Scott

2015-12-01

Gene expression profiling in E 11 mouse embryos identified high expression of the long noncoding RNA (lncRNA), LNCRNA-HIT in the undifferentiated limb mesenchyme, gut, and developing genital tubercle. In the limb mesenchyme, LncRNA-HIT was found to be retained in the nucleus, forming a complex with p100 and CBP. Analysis of the genome-wide distribution of LncRNA-HIT-p100/CBP complexes by ChIRP-seq revealed LncRNA-HIT associated peaks at multiple loci in the murine genome. Ontological analysis of the genes contacted by LncRNA-HIT-p100/CBP complexes indicate a primary role for these loci in chondrogenic differentiation. Functional analysis using siRNA-mediated reductions in LncRNA-HIT or p100 transcripts revealed a significant decrease in expression of many of the LncRNA-HIT-associated loci. LncRNA-HIT siRNA treatments also impacted the ability of the limb mesenchyme to form cartilage, reducing mesenchymal cell condensation and the formation of cartilage nodules. Mechanistically the LncRNA-HIT siRNA treatments impacted pro-chondrogenic gene expression by reducing H3K27ac or p100 activity, confirming that LncRNA-HIT is essential for chondrogenic differentiation in the limb mesenchyme. Taken together, these findings reveal a fundamental epigenetic mechanism functioning during early limb development, using LncRNA-HIT and its associated proteins to promote the expression of multiple genes whose products are necessary for the formation of cartilage.

LncRNA-HIT Functions as an Epigenetic Regulator of Chondrogenesis through Its Recruitment of p100/CBP Complexes

PubMed Central

Carlson, Hanqian L.; Quinn, Jeffrey J.; Yang, Yul W.; Thornburg, Chelsea K.; Chang, Howard Y.; Stadler, H. Scott

2015-01-01

Gene expression profiling in E 11 mouse embryos identified high expression of the long noncoding RNA (lncRNA), LNCRNA-HIT in the undifferentiated limb mesenchyme, gut, and developing genital tubercle. In the limb mesenchyme, LncRNA-HIT was found to be retained in the nucleus, forming a complex with p100 and CBP. Analysis of the genome-wide distribution of LncRNA-HIT-p100/CBP complexes by ChIRP-seq revealed LncRNA-HIT associated peaks at multiple loci in the murine genome. Ontological analysis of the genes contacted by LncRNA-HIT-p100/CBP complexes indicate a primary role for these loci in chondrogenic differentiation. Functional analysis using siRNA-mediated reductions in LncRNA-HIT or p100 transcripts revealed a significant decrease in expression of many of the LncRNA-HIT-associated loci. LncRNA-HIT siRNA treatments also impacted the ability of the limb mesenchyme to form cartilage, reducing mesenchymal cell condensation and the formation of cartilage nodules. Mechanistically the LncRNA-HIT siRNA treatments impacted pro-chondrogenic gene expression by reducing H3K27ac or p100 activity, confirming that LncRNA-HIT is essential for chondrogenic differentiation in the limb mesenchyme. Taken together, these findings reveal a fundamental epigenetic mechanism functioning during early limb development, using LncRNA-HIT and its associated proteins to promote the expression of multiple genes whose products are necessary for the formation of cartilage. PMID:26633036

Human umbilical cord mesenchymal stromal cells in a sandwich approach for osteochondral tissue engineering

PubMed Central

Wang, Limin; Zhao, Liang; Detamore, Michael S.

2013-01-01

Cell sources and tissue integration between cartilage and bone regions are critical to successful osteochondral regeneration. In this study, human umbilical cord mesenchymal stromal cells (hUCMSCs), derived from Wharton’s jelly, were introduced to the field of osteochondral tissue engineering and a new strategy for osteochondral integration was developed by sandwiching a layer of cells between chondrogenic and osteogenic constructs before suturing them together. Specifically, hUCMSCs were cultured in biodegradable poly-l-lactic acid scaffolds for 3 weeks in either chondrogenic or osteogenic medium to differentiate cells toward cartilage or bone lineages, respectively. A highly concentrated cell solution containing undifferentiated hUCMSCs was pasted onto the surface of the bone layer at week 3 and the two layers were then sutured together to form an osteochondral composite for another 3 week culture period. Chondrogenic and osteogenic differentiation was initiated during the first 3 weeks, as evidenced by the expression of type II collagen and runt-related transcription factor 2 genes, respectively, and continued with the increase of extracellular matrix during the last 3 weeks. Histological and immunohistochemical staining, such as for glycosaminoglycans, type I collagen and calcium, revealed better integration and transition of these matrices between two layers in the composite group containing sandwiched cells compared to other control composites. These results suggest that hUCMSCs may be a suitable cell source for osteochondral regeneration, and the strategy of sandwiching cells between two layers may facilitate scaffold and tissue integration. PMID:21953869

Intra-articular delivery of purified mesenchymal stem cells from C57BL/6 or MRL/MpJ superhealer mice prevents posttraumatic arthritis.

PubMed

Diekman, Brian O; Wu, Chia-Lung; Louer, Craig R; Furman, Bridgette D; Huebner, Janet L; Kraus, Virginia B; Olson, Steven A; Guilak, Farshid

2013-01-01

Joint injury dramatically enhances the onset of osteoarthritis (OA) and is responsible for an estimated 12% of OA. Posttraumatic arthritis (PTA) is especially common after intra-articular fracture, and no disease-modifying therapies are currently available. We hypothesized that the delivery of mesenchymal stem cells (MSCs) would prevent PTA by altering the balance of inflammation and regeneration after fracture of the mouse knee. Additionally, we examined the hypothesis that MSCs from the MRL/MpJ (MRL) "superhealer" mouse strain would show increased multilineage and therapeutic potentials as compared to those from C57BL/6 (B6) mice, as MRL mice have shown exceptional in vivo regenerative abilities. A highly purified population of MSCs was prospectively isolated from bone marrow using cell surface markers (CD45-/TER119-/PDGFRα+/Sca-1+). B6 MSCs expanded greater than 100,000-fold in 3 weeks when cultured at 2% oxygen and displayed greater adipogenic, osteogenic, and chondrogenic differentiation as compared to MRL MSCs. Mice receiving only a control saline injection after fracture demonstrated PTA after 8 weeks, but the delivery of 10,000 B6 or MRL MSCs to the joint prevented the development of PTA. Cytokine levels in serum and synovial fluid were affected by treatment with stem cells, including elevated systemic interleukin-10 at several time points. The delivery of MSCs did not reduce the degree of synovial inflammation but did show increased bone volume during repair. This study provides evidence that intra-articular stem cell therapy can prevent the development of PTA after fracture and has implications for possible clinical interventions after joint injury before evidence of significant OA.

Amniotic Mesenchymal Stromal Cells Exhibit Preferential Osteogenic and Chondrogenic Differentiation and Enhanced Matrix Production Compared With Adipose Mesenchymal Stromal Cells.

PubMed

Topoluk, Natasha; Hawkins, Richard; Tokish, John; Mercuri, Jeremy

2017-09-01

Therapeutic efficacy of various mesenchymal stromal cell (MSC) types for orthopaedic applications is currently being investigated. While the concept of MSC therapy is well grounded in the basic science of healing and regeneration, little is known about individual MSC populations in terms of their propensity to promote the repair and/or regeneration of specific musculoskeletal tissues. Two promising MSC sources, adipose and amnion, have each demonstrated differentiation and extracellular matrix (ECM) production in the setting of musculoskeletal tissue regeneration. However, no study to date has directly compared the differentiation potential of these 2 MSC populations. To compare the ability of human adipose- and amnion-derived MSCs to undergo osteogenic and chondrogenic differentiation. Controlled laboratory study. MSC populations from the human term amnion were quantified and characterized via cell counting, histologic assessment, and flow cytometry. Differentiation of these cells in comparison to commercially purchased human adipose-derived mesenchymal stromal cells (hADSCs) in the presence and absence of differentiation media was evaluated via reverse transcription polymerase chain reaction (PCR) for bone and cartilage gene transcript markers and histology/immunohistochemistry to examine ECM production. Analysis of variance and paired t tests were performed to compare results across all cell groups investigated. The authors confirmed that the human term amnion contains 2 primary cell types demonstrating MSC characteristics-(1) human amniotic epithelial cells (hAECs) and (2) human amniotic mesenchymal stromal cells (hAMSCs)-and each exhibited more than 90% staining for MSC surface markers (CD90, CD105, CD73). Average viable hAEC and hAMSC yields at harvest were 2.3 × 10 6 ± 3.7 × 10 5 and 1.6 × 10 6 ± 4.7 × 10 5 per milliliter of amnion, respectively. As well, hAECs and hAMSCs demonstrated significantly greater osteocalcin ( P = .025), aggrecan ( P < .0001), and collagen type 2 ( P = .044) gene expression compared with hADSCs, respectively, after culture in differentiation medium. Moreover, both hAECs and hAMSCs produced significantly greater quantities of mineralized ( P < .0001) and cartilaginous ( P = .0004) matrix at earlier time points compared with hADSCs when cultured under identical osteogenic and chondrogenic differentiation conditions, respectively. Amnion-derived MSCs demonstrate a greater differentiation potential toward bone and cartilage compared with hADSCs. Amniotic MSCs may be the source of choice in the regenerative treatment of bone or osteochondral musculoskeletal disease. They show significantly higher yields and better differentiation toward these tissues than MSCs derived from adipose.

Hydroxyapatite and Calcified Elastin Induce Osteoblast-like Differentiation in Rat Aortic Smooth Muscle Cells

PubMed Central

Lei, Yang; Sinha, Aditi; Nosoudi, Nasim; Grover, Ankit; Vyavahare, Naren

2014-01-01

Vascular calcification can be categorized into two different types. Intimal calcification related to atherosclerosis and elastin-specific medial arterial calcification (MAC). Osteoblast-like differentiation of vascular smooth muscle cells (VSMCs) has been shown in both types; however, how this relates to initiation of vascular calcification is unclear. We hypothesize that the initial deposition of hydroxyapatite-like mineral in MAC occurs on degraded elastin first and that causes osteogenic transformation of VSMCs. To test this, rat aortic smooth muscle cells (RASMCs) were cultured on hydroxyapatite crystals and calcified aortic elastin. Using RT-PCR and specific protein assays, we demonstrate that RASMCs lose their smooth muscle lineage markers like alpha smooth muscle actin (SMA) and myosin heavy chain (MHC) and undergo chondrogenic/osteogenic transformation. This is indicated by an increase in the expression of typical chondrogenic proteins such as aggrecan, collagen type II alpha 1(Col2a1) and bone proteins such as runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN). Furthermore, when calcified conditions are removed, cells return to their original phenotype. Our data supports the hypothesis that elastin degradation and calcification precedes VSMCs' osteoblast-like differentiation. PMID:24447384

Effects of in vitro endochondral priming and pre-vascularisation of human MSC cellular aggregates in vivo.

PubMed

Freeman, Fiona E; Allen, Ashley B; Stevens, Hazel Y; Guldberg, Robert E; McNamara, Laoise M

2015-11-05

During endochondral ossification, both the production of a cartilage template and the subsequent vascularisation of that template are essential precursors to bone tissue formation. Recent studies have found the application of both chondrogenic and vascular priming of mesenchymal stem cells (MSCs) enhanced the mineralisation potential of MSCs in vitro whilst also allowing for immature vessel formation. However, the in vivo viability, vascularisation and mineralisation potential of MSC aggregates that have been pre-conditioned in vitro by a combination of chondrogenic and vascular priming, has yet to be established. In this study, we test the hypothesis that a tissue regeneration approach that incorporates both chondrogenic priming of MSCs, to first form a cartilage template, and subsequent pre-vascularisation of the cartilage constructs, by co-culture with human umbilical vein endothelial cells (HUVECs) in vitro, will improve vessel infiltration and thus mineral formation once implanted in vivo. Human MSCs were chondrogenically primed for 21 days, after which they were co-cultured with MSCs and HUVECs and cultured in endothelial growth medium for another 21 days. These aggregates were then implanted subcutaneously in nude rats for 4 weeks. We used a combination of bioluminescent imaging, microcomputed tomography, histology (Masson's trichrome and Alizarin Red) and immunohistochemistry (CD31, CD146, and α-smooth actin) to assess the vascularisation and mineralisation potential of these MSC aggregates in vivo. Pre-vascularised cartilaginous aggregates were found to have mature endogenous vessels (indicated by α-smooth muscle actin walls and erythrocytes) after 4 weeks subcutaneous implantation, and also viable human MSCs (detected by bioluminescent imaging) 21 days after subcutaneous implantation. In contrast, aggregates that were not pre-vascularised had no vessels within the aggregate interior and human MSCs did not remain viable beyond 14 days. Interestingly, the pre-vascularised cartilaginous aggregates were also the only group to have mineralised nodules within the cellular aggregates, whereas mineralisation occurred in the alginate surrounding the aggregates for all other groups. Taken together these results indicate that a combined chondrogenic priming and pre-vascularisation approach for in vitro culture of MSC aggregates shows enhanced vessel formation and increased mineralisation within the cellular aggregate when implanted subcutaneously in vivo.

PLGA-Based Nanoparticles: a Safe and Suitable Delivery Platform for Osteoarticular Pathologies.

PubMed

Riffault, Mathieu; Six, Jean-Luc; Netter, Patrick; Gillet, Pierre; Grossin, Laurent

2015-12-01

Despite the promising applications of PLGA based particles, studies examining the fate and consequences of these particles after intra-articular administration in the joint are scanty. This study was carried out to evaluate the neutrality of the unloaded delivery system on different articular cell types. To facilitate tracking, we have thus developed a fluorescent core of particles, combined to a hyaluronate shell for cell recognition. Fluorescence pictures were taken at time intervals to assess the internalization and the corresponding inflammatory response was monitored by RT-qPCR and biochemical measurements. After NPs pre-treatment, mesenchymal stem cells (MSCs) were cultured into chondrogenic, adipogenic or osteogenic differentiation media, to investigate if NPs exposure interferes with differentiation ability. Finally, intra-articular injections were performed in healthy rat knees and joint's structure analysed by histological studies. Particles were detected in cytoplasm 8 h after exposure. Internalization led to a slight and reversible increase of inflammatory markers, but lower than in inflammatory conditions. We have confirmed particles exposure minimal neutrality on MSCs pluripotency. Histological exams of joint after intra-articular injections do not demonstrate any side effects of NPs. Our findings suggest that such a delivery platform is well tolerated locally and could be used to deliver active molecules to the joint.

Combating Posttraumatic Osteoarthritis via Enhanced Intra-Articular Recruitment of Stem Cells and Targeting Delivery of a Chondrogenic Agent

DTIC Science & Technology

2016-08-01

the animals’ intestinal tract. With this new protocol in protocol in place, as well as increased breeding of transgenic (GFP + ) animals by our vendor...bone marrow cells from a transgenic rat (Lewis background) bearing the GFP transgene . Experimentation (ACL rupture) is now underway with the GFP...a reduction in the supply of transgenic (green fluorescent protein-positive) animals from a vendor. After resolving those issues, we experienced

Jellyfish collagen scaffolds for cartilage tissue engineering.

PubMed

Hoyer, Birgit; Bernhardt, Anne; Lode, Anja; Heinemann, Sascha; Sewing, Judith; Klinger, Matthias; Notbohm, Holger; Gelinsky, Michael

2014-02-01

Porous scaffolds were engineered from refibrillized collagen of the jellyfish Rhopilema esculentum for potential application in cartilage regeneration. The influence of collagen concentration, salinity and temperature on fibril formation was evaluated by turbidity measurements and quantification of fibrillized collagen. The formation of collagen fibrils with a typical banding pattern was confirmed by atomic force microscopy and transmission electron microscopy analysis. Porous scaffolds from jellyfish collagen, refibrillized under optimized conditions, were fabricated by freeze-drying and subsequent chemical cross-linking. Scaffolds possessed an open porosity of 98.2%. The samples were stable under cyclic compression and displayed an elastic behavior. Cytotoxicity tests with human mesenchymal stem cells (hMSCs) did not reveal any cytotoxic effects of the material. Chondrogenic markers SOX9, collagen II and aggrecan were upregulated in direct cultures of hMSCs upon chondrogenic stimulation. The formation of typical extracellular matrix components was further confirmed by quantification of sulfated glycosaminoglycans. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

L-carnitine significantly decreased aging of rat adipose tissue-derived mesenchymal stem cells.

PubMed

Mobarak, Halimeh; Fathi, Ezzatollah; Farahzadi, Raheleh; Zarghami, Nosratollah; Javanmardi, Sara

2017-03-01

Mesenchymal stem cells are undifferentiated cells that have the ability to divide continuously and tissue regeneration potential during the transplantation. Aging and loss of cell survival, is one of the main problems in cell therapy. Since the production of free radicals in the aging process is effective, the use of antioxidant compounds can help in scavenging free radicals and prevent the aging of cells. The aim of this study is evaluate the effects of L-carnitine (LC) on proliferation and aging of rat adipose tissue-derived mesenchymal stem cells (rADSC). rADSCs were isolated from inguinal region of 5 male Rattus rats. Oil red-O, alizarin red-S and toluidine blue staining were performed to evaluate the adipogenic, osteogenic and chondrogenic differentiation of rADSCs, respectively. Flow cytometric analysis was done for investigating the cell surface markers. The methyl thiazol tetrazolium (MTT) method was used to determine the cell proliferation of rADSCs following exposure to different concentrations of LC. rADSCs aging was evaluated by beta-galactosidase staining. The results showed significant proliferation of rADSCs 48 h after treatment with concentrations of 0.2 mM LC. In addition, in the presence of 0.2 mM LC, rADSCs appeared to be growing faster than control group and 0.2 mM LC supplementation could significantly decrease the population doubling time and aging of rADSCs. It seems that LC would be a good antioxidant to improve lifespan of rADSCs due to the decrease in aging.

A composite scaffold of MSC affinity peptide-modified demineralized bone matrix particles and chitosan hydrogel for cartilage regeneration

NASA Astrophysics Data System (ADS)

Meng, Qingyang; Man, Zhentao; Dai, Linghui; Huang, Hongjie; Zhang, Xin; Hu, Xiaoqing; Shao, Zhenxing; Zhu, Jingxian; Zhang, Jiying; Fu, Xin; Duan, Xiaoning; Ao, Yingfang

2015-12-01

Articular cartilage injury is still a significant challenge because of the poor intrinsic healing potential of cartilage. Stem cell-based tissue engineering is a promising technique for cartilage repair. As cartilage defects are usually irregular in clinical settings, scaffolds with moldability that can fill any shape of cartilage defects and closely integrate with the host cartilage are desirable. In this study, we constructed a composite scaffold combining mesenchymal stem cells (MSCs) E7 affinity peptide-modified demineralized bone matrix (DBM) particles and chitosan (CS) hydrogel for cartilage engineering. This solid-supported composite scaffold exhibited appropriate porosity, which provided a 3D microenvironment that supports cell adhesion and proliferation. Cell proliferation and DNA content analysis indicated that the DBM-E7/CS scaffold promoted better rat bone marrow-derived MSCs (BMMSCs) survival than the CS or DBM/CS groups. Meanwhile, the DBM-E7/CS scaffold increased matrix production and improved chondrogenic differentiation ability of BMMSCs in vitro. Furthermore, after implantation in vivo for four weeks, compared to those in control groups, the regenerated issue in the DBM-E7/CS group exhibited translucent and superior cartilage-like structures, as indicated by gross observation, histological examination, and assessment of matrix staining. Overall, the functional composite scaffold of DBM-E7/CS is a promising option for repairing irregularly shaped cartilage defects.

Isolation of mesenchymal stromal/stem cells from cryopreserved umbilical cord blood cells.

PubMed

Fujii, Sumie; Miura, Yasuo; Iwasa, Masaki; Yoshioka, Satoshi; Fujishiro, Aya; Sugino, Noriko; Kaneko, Hitomi; Nakagawa, Yoko; Hirai, Hideyo; Takaori-Kondo, Akifumi; Ichinohe, Tatsuo; Maekawa, Taira

2017-07-05

Umbilical cord blood (UCB) has advantages over other tissues because it can be obtained without an invasive procedure and complex processing. We explored the availability of cryopreserved UCB cells as a source of mesenchymal stromal/stem cells (MSCs). MSCs were successfully isolated from six of 30 UCB units (median volume, 34.0 mL; median nucleated cell number, 4.4×10 8 ) that were processed and cryopreserved using CP-1/human serum albumin. This isolation rate was lower than that (57%) from non-cryopreserved UCB cells. The number of nucleated cells before and after hydroxyethyl starch separation, UCB unit volume, and cell viability after thawing did not significantly differ between UCB units from which MSCs were successfully isolated and those from which they were not. When CryoSure-DEX40 was used as a cryoprotectant, MSCs were isolated from two of ten UCB units. Logistic regression analysis demonstrated that the cryopreservation method was not significantly associated with the success of MSC isolation. The isolated MSCs had a similar morphology and surface marker expression profile as bone marrow-derived MSCs and were able to differentiate into osteogenic, adipogenic, and chondrogenic cells. In summary, MSCs can be isolated from cryopreserved UCB cells. However, the cryopreservation process reduces the isolation rate; therefore, freshly donated UCB cells are preferable for the isolation of MSCs.

Effects of mechanical loading on human mesenchymal stem cells for cartilage tissue engineering.

PubMed

Choi, Jane Ru; Yong, Kar Wey; Choi, Jean Yu

2018-03-01

Today, articular cartilage damage is a major health problem, affecting people of all ages. The existing conventional articular cartilage repair techniques, such as autologous chondrocyte implantation (ACI), microfracture, and mosaicplasty, have many shortcomings which negatively affect their clinical outcomes. Therefore, it is essential to develop an alternative and efficient articular repair technique that can address those shortcomings. Cartilage tissue engineering, which aims to create a tissue-engineered cartilage derived from human mesenchymal stem cells (MSCs), shows great promise for improving articular cartilage defect therapy. However, the use of tissue-engineered cartilage for the clinical therapy of articular cartilage defect still remains challenging. Despite the importance of mechanical loading to create a functional cartilage has been well demonstrated, the specific type of mechanical loading and its optimal loading regime is still under investigation. This review summarizes the most recent advances in the effects of mechanical loading on human MSCs. First, the existing conventional articular repair techniques and their shortcomings are highlighted. The important parameters for the evaluation of the tissue-engineered cartilage, including chondrogenic and hypertrophic differentiation of human MSCs are briefly discussed. The influence of mechanical loading on human MSCs is subsequently reviewed and the possible mechanotransduction signaling is highlighted. The development of non-hypertrophic chondrogenesis in response to the changing mechanical microenvironment will aid in the establishment of a tissue-engineered cartilage for efficient articular cartilage repair. © 2017 Wiley Periodicals, Inc.

Mesenchymal stem cells for cartilage repair in osteoarthritis

PubMed Central

2012-01-01

Osteoarthritis (OA) is a degenerative disease of the connective tissue and progresses with age in the older population or develops in young athletes following sports-related injury. The articular cartilage is especially vulnerable to damage and has poor potential for regeneration because of the absence of vasculature within the tissue. Normal load-bearing capacity and biomechanical properties of thinning cartilage are severely compromised during the course of disease progression. Although surgical and pharmaceutical interventions are currently available for treating OA, restoration of normal cartilage function has been difficult to achieve. Since the tissue is composed primarily of chondrocytes distributed in a specialized extracellular matrix bed, bone marrow stromal cells (BMSCs), also known as bone marrow-derived 'mesenchymal stem cells' or 'mesenchymal stromal cells', with inherent chondrogenic differentiation potential appear to be ideally suited for therapeutic use in cartilage regeneration. BMSCs can be easily isolated and massively expanded in culture in an undifferentiated state for therapeutic use. Owing to their potential to modulate local microenvironment via anti-inflammatory and immunosuppressive functions, BMSCs have an additional advantage for allogeneic application. Moreover, by secreting various bioactive soluble factors, BMSCs can protect the cartilage from further tissue destruction and facilitate regeneration of the remaining progenitor cells in situ. This review broadly describes the advances made during the last several years in BMSCs and their therapeutic potential for repairing cartilage damage in OA. PMID:22776206

Bioreactor mechanically guided 3D mesenchymal stem cell chondrogenesis using a biocompatible novel thermo-reversible methylcellulose-based hydrogel

PubMed Central

Cochis, A.; Grad, S.; Stoddart, M. J.; Farè, S.; Altomare, L.; Azzimonti, B.; Alini, M.; Rimondini, L.

2017-01-01

Autologous chondrocyte implantation for cartilage repair represents a challenge because strongly limited by chondrocytes’ poor expansion capacity in vitro. Mesenchymal stem cells (MSCs) can differentiate into chondrocytes, while mechanical loading has been proposed as alternative strategy to induce chondrogenesis excluding the use of exogenous factors. Moreover, MSC supporting material selection is fundamental to allow for an active interaction with cells. Here, we tested a novel thermo-reversible hydrogel composed of 8% w/v methylcellulose (MC) in a 0.05 M Na2SO4 solution. MC hydrogel was obtained by dispersion technique and its thermo-reversibility, mechanical properties, degradation and swelling were investigated, demonstrating a solution-gelation transition between 34 and 37 °C and a low bulk degradation (<20%) after 1 month. The lack of any hydrogel-derived immunoreaction was demonstrated in vivo by mice subcutaneous implantation. To induce in vitro chondrogenesis, MSCs were seeded into MC solution retained within a porous polyurethane (PU) matrix. PU-MC composites were subjected to a combination of compression and shear forces for 21 days in a custom made bioreactor. Mechanical stimulation led to a significant increase in chondrogenic gene expression, while histological analysis detected sulphated glycosaminoglycans and collagen II only in loaded specimens, confirming MC hydrogel suitability to support load induced MSCs chondrogenesis. PMID:28332587

Chondrogenesis and integration of mesenchymal stem cells within an in vitro cartilage defect repair model.

PubMed

Vinardell, T; Thorpe, S D; Buckley, C T; Kelly, D J

2009-12-01

Integration of repair tissue is a key indicator of the long-term success of cell-based therapies for cartilage repair. The objective of this study was to compare the in vitro chondrogenic differentiation and integration of agarose hydrogels seeded with either chondrocytes or bone marrow-derived mesenchymal stem cells (MSCs) in defects created in cartilage explants. Chondrocytes and MSCs were isolated from porcine donors, suspended in 2% agarose and then injected into cylindrical defects within the explants. These constructs were maintained in a chemically defined medium supplemented with 10 ng/mL of TGF-beta3. Cartilage integration was assessed by histology and mechanical push-out tests. After 6 weeks in culture, chondrocyte-seeded constructs demonstrated a higher integration strength (64.4 +/- 8.3 kPa) compared to MSC-seeded constructs (22.7 +/- 5.9 kPa). Glycosaminoglycan (GAG) (1.27 +/- 0.3 vs. 0.19 +/- 0.03 kPa) and collagen (0.31 +/- 0.08 vs. 0.09 +/- 0.01 kPa) accumulation in chondrocyte-seeded constructs was greater than that measured in the MSC-seeded group. The GAG, collagen, and DNA content of both chondrocyte- and MSC-seeded hydrogels cultured in cartilage explants was significantly lower than control constructs cultured in free swelling conditions. The results of this study suggest that the explant model may constitute a more rigorous in vitro test to assess MSC therapies for cartilage defect repair.

3D bioprinting mesenchymal stem cell-laden construct with core-shell nanospheres for cartilage tissue engineering

NASA Astrophysics Data System (ADS)

Zhu, Wei; Cui, Haitao; Boualam, Benchaa; Masood, Fahed; Flynn, Erin; Rao, Raj D.; Zhang, Zhi-Yong; Zhang, Lijie Grace

2018-05-01

Cartilage tissue is prone to degradation and has little capacity for self-healing due to its avascularity. Tissue engineering, which provides artificial scaffolds to repair injured tissues, is a novel and promising strategy for cartilage repair. 3D bioprinting offers even greater potential for repairing degenerative tissue by simultaneously integrating living cells, biomaterials, and biological cues to provide a customized scaffold. With regard to cell selection, mesenchymal stem cells (MSCs) hold great capacity for differentiating into a variety of cell types, including chondrocytes, and could therefore be utilized as a cartilage cell source in 3D bioprinting. In the present study, we utilize a tabletop stereolithography-based 3D bioprinter for a novel cell-laden cartilage tissue construct fabrication. Printable resin is composed of 10% gelatin methacrylate (GelMA) base, various concentrations of polyethylene glycol diacrylate (PEGDA), biocompatible photoinitiator, and transforming growth factor beta 1 (TGF-β1) embedded nanospheres fabricated via a core-shell electrospraying technique. We find that the addition of PEGDA into GelMA hydrogel greatly improves the printing resolution. Compressive testing shows that modulus of the bioprinted scaffolds proportionally increases with the concentrations of PEGDA, while swelling ratio decreases with the increase of PEGDA concentration. Confocal microscopy images illustrate that the cells and nanospheres are evenly distributed throughout the entire bioprinted construct. Cells grown on 5%/10% (PEGDA/GelMA) hydrogel present the highest cell viability and proliferation rate. The TGF-β1 embedded in nanospheres can keep a sustained release up to 21 d and improve chondrogenic differentiation of encapsulated MSCs. The cell-laden bioprinted cartilage constructs with TGF-β1-containing nanospheres is a promising strategy for cartilage regeneration.

3D bioprinting mesenchymal stem cell-laden construct with core-shell nanospheres for cartilage tissue engineering.

PubMed

Zhu, Wei; Cui, Haitao; Boualam, Benchaa; Masood, Fahed; Flynn, Erin; Rao, Raj D; Zhang, Zhi-Yong; Zhang, Lijie Grace

2018-05-04

Cartilage tissue is prone to degradation and has little capacity for self-healing due to its avascularity. Tissue engineering, which provides artificial scaffolds to repair injured tissues, is a novel and promising strategy for cartilage repair. 3D bioprinting offers even greater potential for repairing degenerative tissue by simultaneously integrating living cells, biomaterials, and biological cues to provide a customized scaffold. With regard to cell selection, mesenchymal stem cells (MSCs) hold great capacity for differentiating into a variety of cell types, including chondrocytes, and could therefore be utilized as a cartilage cell source in 3D bioprinting. In the present study, we utilize a tabletop stereolithography-based 3D bioprinter for a novel cell-laden cartilage tissue construct fabrication. Printable resin is composed of 10% gelatin methacrylate (GelMA) base, various concentrations of polyethylene glycol diacrylate (PEGDA), biocompatible photoinitiator, and transforming growth factor beta 1 (TGF-β1) embedded nanospheres fabricated via a core-shell electrospraying technique. We find that the addition of PEGDA into GelMA hydrogel greatly improves the printing resolution. Compressive testing shows that modulus of the bioprinted scaffolds proportionally increases with the concentrations of PEGDA, while swelling ratio decreases with the increase of PEGDA concentration. Confocal microscopy images illustrate that the cells and nanospheres are evenly distributed throughout the entire bioprinted construct. Cells grown on 5%/10% (PEGDA/GelMA) hydrogel present the highest cell viability and proliferation rate. The TGF-β1 embedded in nanospheres can keep a sustained release up to 21 d and improve chondrogenic differentiation of encapsulated MSCs. The cell-laden bioprinted cartilage constructs with TGF-β1-containing nanospheres is a promising strategy for cartilage regeneration.

Silk-fibrin/hyaluronic acid composite gels for nucleus pulposus tissue regeneration.

PubMed

Park, Sang-Hyug; Cho, Hongsik; Gil, Eun Seok; Mandal, Biman B; Min, Byoung-Hyun; Kaplan, David L

2011-12-01

Scaffold designs are critical for in vitro culture of tissue-engineered cartilage in three-dimensional environments to enhance cellular differentiation for tissue engineering and regenerative medicine. In the present study we demonstrated silk and fibrin/hyaluronic acid (HA) composite gels as scaffolds for nucleus pulposus (NP) cartilage formation, providing both biochemical support for NP outcomes as well as fostering the retention of size of the scaffold during culture due to the combined features of the two proteins. Passage two (P2) human chondrocytes cultured in 10% serum were encapsulated within silk-fibrin/HA gels. Five study groups with fibrin/HA gel culture (F/H) along with varying silk concentrations (2% silk gel only, fibrin/HA gel culture with 1% silk [F/H+1S], 1.5% silk [F/H+1.5S], and 2% silk [F/H+2S]) were cultured in serum-free chondrogenic defined media (CDM) for 4 weeks. Histological examination with alcian blue showed a defined chondrogenic area at 1 week in all groups that widened homogenously until 4 weeks. In particular, chondrogenic differentiation observed in the F/H+1.5S had no reduction in size throughout the culture period. The results of biochemical and molecular biological evaluations supported observations made during histological examination. Mechanical strength measurements showed that the silk mixed gels provided stronger mechanical properties for NP tissue than fibrin/HA composite gels in CDM. This effect could potentially be useful in the study of in vitro NP tissue engineering as well as for clinical implications for NP tissue regeneration.

Silk-Fibrin/Hyaluronic Acid Composite Gels for Nucleus Pulposus Tissue Regeneration

PubMed Central

Park, Sang-Hyug; Cho, Hongsik; Gil, Eun Seok; Mandal, Biman B.; Min, Byoung-Hyun

2011-01-01

Scaffold designs are critical for in vitro culture of tissue-engineered cartilage in three-dimensional environments to enhance cellular differentiation for tissue engineering and regenerative medicine. In the present study we demonstrated silk and fibrin/hyaluronic acid (HA) composite gels as scaffolds for nucleus pulposus (NP) cartilage formation, providing both biochemical support for NP outcomes as well as fostering the retention of size of the scaffold during culture due to the combined features of the two proteins. Passage two (P2) human chondrocytes cultured in 10% serum were encapsulated within silk-fibrin/HA gels. Five study groups with fibrin/HA gel culture (F/H) along with varying silk concentrations (2% silk gel only, fibrin/HA gel culture with 1% silk [F/H+1S], 1.5% silk [F/H+1.5S], and 2% silk [F/H+2S]) were cultured in serum-free chondrogenic defined media (CDM) for 4 weeks. Histological examination with alcian blue showed a defined chondrogenic area at 1 week in all groups that widened homogenously until 4 weeks. In particular, chondrogenic differentiation observed in the F/H+1.5S had no reduction in size throughout the culture period. The results of biochemical and molecular biological evaluations supported observations made during histological examination. Mechanical strength measurements showed that the silk mixed gels provided stronger mechanical properties for NP tissue than fibrin/HA composite gels in CDM. This effect could potentially be useful in the study of in vitro NP tissue engineering as well as for clinical implications for NP tissue regeneration. PMID:21736446

Skeletal Cell Differentiation Is Enhanced by Atmospheric Dielectric Barrier Discharge Plasma Treatment

PubMed Central

Zhang, Jun; Kurpad, Deepa S.; Fridman, Gregory; Fridman, Alexander; Freeman, Theresa A.

2013-01-01

Enhancing chondrogenic and osteogenic differentiation is of paramount importance in providing effective regenerative therapies and improving the rate of fracture healing. This study investigated the potential of non-thermal atmospheric dielectric barrier discharge plasma (NT-plasma) to enhance chondrocyte and osteoblast proliferation and differentiation. Although the exact mechanism by which NT-plasma interacts with cells is undefined, it is known that during treatment the atmosphere is ionized generating extracellular reactive oxygen and nitrogen species (ROS and RNS) and an electric field. Appropriate NT-plasma conditions were determined using lactate-dehydrogenase release, flow cytometric live/dead assay, flow cytometric cell cycle analysis, and Western blots to evaluate DNA damage and mitochondrial integrity. We observed that specific NT-plasma conditions were required to prevent cell death, and that loss of pre-osteoblastic cell viability was dependent on intracellular ROS and RNS production. To further investigate the involvement of intracellular ROS, fluorescent intracellular dyes Mitosox (superoxide) and dihydrorhodamine (peroxide) were used to assess onset and duration after NT-plasma treatment. Both intracellular superoxide and peroxide were found to increase immediately post NT-plasma treatment. These increases were sustained for one hour but returned to control levels by 24 hr. Using the same treatment conditions, osteogenic differentiation by NT-plasma was assessed and compared to peroxide or osteogenic media containing β-glycerolphosphate. Although both NT-plasma and peroxide induced differentiation-specific gene expression, neither was as effective as the osteogenic media. However, treatment of cells with NT-plasma after 24 hr in osteogenic or chondrogenic media significantly enhanced differentiation as compared to differentiation media alone. The results of this study show that NT-plasma can selectively initiate and amplify ROS signaling to enhance differentiation, and suggest this technology could be used to enhance bone fusion and improve healing after skeletal injury. PMID:24349203

Skeletal cell differentiation is enhanced by atmospheric dielectric barrier discharge plasma treatment.

PubMed

Steinbeck, Marla J; Chernets, Natalie; Zhang, Jun; Kurpad, Deepa S; Fridman, Gregory; Fridman, Alexander; Freeman, Theresa A

2013-01-01

Enhancing chondrogenic and osteogenic differentiation is of paramount importance in providing effective regenerative therapies and improving the rate of fracture healing. This study investigated the potential of non-thermal atmospheric dielectric barrier discharge plasma (NT-plasma) to enhance chondrocyte and osteoblast proliferation and differentiation. Although the exact mechanism by which NT-plasma interacts with cells is undefined, it is known that during treatment the atmosphere is ionized generating extracellular reactive oxygen and nitrogen species (ROS and RNS) and an electric field. Appropriate NT-plasma conditions were determined using lactate-dehydrogenase release, flow cytometric live/dead assay, flow cytometric cell cycle analysis, and Western blots to evaluate DNA damage and mitochondrial integrity. We observed that specific NT-plasma conditions were required to prevent cell death, and that loss of pre-osteoblastic cell viability was dependent on intracellular ROS and RNS production. To further investigate the involvement of intracellular ROS, fluorescent intracellular dyes Mitosox (superoxide) and dihydrorhodamine (peroxide) were used to assess onset and duration after NT-plasma treatment. Both intracellular superoxide and peroxide were found to increase immediately post NT-plasma treatment. These increases were sustained for one hour but returned to control levels by 24 hr. Using the same treatment conditions, osteogenic differentiation by NT-plasma was assessed and compared to peroxide or osteogenic media containing β-glycerolphosphate. Although both NT-plasma and peroxide induced differentiation-specific gene expression, neither was as effective as the osteogenic media. However, treatment of cells with NT-plasma after 24 hr in osteogenic or chondrogenic media significantly enhanced differentiation as compared to differentiation media alone. The results of this study show that NT-plasma can selectively initiate and amplify ROS signaling to enhance differentiation, and suggest this technology could be used to enhance bone fusion and improve healing after skeletal injury.

Relative impact of uniaxial alignment vs. form-induced stress on differentiation of human adipose derived stem cells

PubMed Central

Huang, Samuel; Li, Julie Yi-Shuan; Chien, Shu; Zhang, Kang; Chen, Shaochen

2013-01-01

ADSCs are a great cell source for tissue engineering and regenerative medicine. However, the development of methods to appropriately manipulate these cells in vitro remains a challenge. Here the proliferation and differentiation of ADSCs on microfabricated surfaces with varying geometries were investigated. To create the patterned substrates, a maskless biofabrication method was developed based on dynamic optical projection stereolithography. Proliferation and early differentiation of ADSCs were compared across three distinct multicellular patterns, namely stripes (ST), symmetric fork (SF), and asymmetric fork (AF). The ST pattern was designed for uniaxial cell alignment while the SF and AF pattern were designed with altered cell directionality to different extents. The SF and AF patterns generated similar levels of regional peak stress, which were both significantly higher than those within the ST pattern. No significant difference in ADSC proliferation was observed among the three patterns. In comparison to the ST pattern, higher peak stress levels of the SF and AF patterns were associated with up-regulation of the chondrogenic and osteogenic markers SOX9 and RUNX2. Interestingly, uniaxial cell alignment in the ST pattern seemed to increase the expression of SM22α and smooth muscle α-actin, suggesting an early smooth muscle lineage progression. These results indicate that geometric cues that promote uniaxial alignment might be more potent for myogenesis than those with increased peak stress. Overall, the use of these patterned geometric cues for modulating cell alignment and form-induced stress can serve as a powerful and versatile technique towards controlling differentiation in ADSCs. PMID:24060419

Generation of scaffoldless hyaline cartilaginous tissue from human iPSCs.

PubMed

Yamashita, Akihiro; Morioka, Miho; Yahara, Yasuhito; Okada, Minoru; Kobayashi, Tomohito; Kuriyama, Shinichi; Matsuda, Shuichi; Tsumaki, Noriyuki

2015-03-10

Defects in articular cartilage ultimately result in loss of joint function. Repairing cartilage defects requires cell sources. We developed an approach to generate scaffoldless hyaline cartilage from human induced pluripotent stem cells (hiPSCs). We initially generated an hiPSC line that specifically expressed GFP in cartilage when teratoma was formed. We optimized the culture conditions and found BMP2, transforming growth factor β1 (TGF-β1), and GDF5 critical for GFP expression and thus chondrogenic differentiation of the hiPSCs. The subsequent use of scaffoldless suspension culture contributed to purification, producing homogenous cartilaginous particles. Subcutaneous transplantation of the hiPSC-derived particles generated hyaline cartilage that expressed type II collagen, but not type I collagen, in immunodeficiency mice. Transplantation of the particles into joint surface defects in immunodeficiency rats and immunosuppressed mini-pigs indicated that neocartilage survived and had potential for integration into native cartilage. The immunodeficiency mice and rats suffered from neither tumors nor ectopic tissue formation. The hiPSC-derived cartilaginous particles constitute a viable cell source for regenerating cartilage defects. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Generation of Scaffoldless Hyaline Cartilaginous Tissue from Human iPSCs

PubMed Central

Yamashita, Akihiro; Morioka, Miho; Yahara, Yasuhito; Okada, Minoru; Kobayashi, Tomohito; Kuriyama, Shinichi; Matsuda, Shuichi; Tsumaki, Noriyuki

2015-01-01

Summary Defects in articular cartilage ultimately result in loss of joint function. Repairing cartilage defects requires cell sources. We developed an approach to generate scaffoldless hyaline cartilage from human induced pluripotent stem cells (hiPSCs). We initially generated an hiPSC line that specifically expressed GFP in cartilage when teratoma was formed. We optimized the culture conditions and found BMP2, transforming growth factor β1 (TGF-β1), and GDF5 critical for GFP expression and thus chondrogenic differentiation of the hiPSCs. The subsequent use of scaffoldless suspension culture contributed to purification, producing homogenous cartilaginous particles. Subcutaneous transplantation of the hiPSC-derived particles generated hyaline cartilage that expressed type II collagen, but not type I collagen, in immunodeficiency mice. Transplantation of the particles into joint surface defects in immunodeficiency rats and immunosuppressed mini-pigs indicated that neocartilage survived and had potential for integration into native cartilage. The immunodeficiency mice and rats suffered from neither tumors nor ectopic tissue formation. The hiPSC-derived cartilaginous particles constitute a viable cell source for regenerating cartilage defects. PMID:25733017

Water-based polyurethane 3D printed scaffolds with controlled release function for customized cartilage tissue engineering.

PubMed

Hung, Kun-Che; Tseng, Ching-Shiow; Dai, Lien-Guo; Hsu, Shan-hui

2016-03-01

Conventional 3D printing may not readily incorporate bioactive ingredients for controlled release because the process often involves the use of heat, organic solvent, or crosslinkers that reduce the bioactivity of the ingredients. Water-based 3D printing materials with controlled bioactivity for customized cartilage tissue engineering is developed in this study. The printing ink contains the water dispersion of synthetic biodegradable polyurethane (PU) elastic nanoparticles, hyaluronan, and bioactive ingredients TGFβ3 or a small molecule drug Y27632 to replace TGFβ3. Compliant scaffolds are printed from the ink at low temperature. These scaffolds promote the self-aggregation of mesenchymal stem cells (MSCs) and, with timely release of the bioactive ingredients, induce the chondrogenic differentiation of MSCs and produce matrix for cartilage repair. Moreover, the growth factor-free controlled release design may prevent cartilage hypertrophy. Rabbit knee implantation supports the potential of the novel 3D printing scaffolds in cartilage regeneration. We consider that the 3D printing composite scaffolds with controlled release bioactivity may have potential in customized tissue engineering. Copyright © 2016 Elsevier Ltd. All rights reserved.

3D printing of novel osteochondral scaffolds with graded microstructure

NASA Astrophysics Data System (ADS)

Nowicki, Margaret A.; Castro, Nathan J.; Plesniak, Michael W.; Zhang, Lijie Grace

2016-10-01

Osteochondral tissue has a complex graded structure where biological, physiological, and mechanical properties vary significantly over the full thickness spanning from the subchondral bone region beneath the joint surface to the hyaline cartilage region at the joint surface. This presents a significant challenge for tissue-engineered structures addressing osteochondral defects. Fused deposition modeling (FDM) 3D bioprinters present a unique solution to this problem. The objective of this study is to use FDM-based 3D bioprinting and nanocrystalline hydroxyapatite for improved bone marrow human mesenchymal stem cell (hMSC) adhesion, growth, and osteochondral differentiation. FDM printing parameters can be tuned through computer aided design and computer numerical control software to manipulate scaffold geometries in ways that are beneficial to mechanical performance without hindering cellular behavior. Additionally, the ability to fine-tune 3D printed scaffolds increases further through our investment casting procedure which facilitates the inclusion of nanoparticles with biochemical factors to further elicit desired hMSC differentiation. For this study, FDM was used to print investment-casting molds innovatively designed with varied pore distribution over the full thickness of the scaffold. The mechanical and biological impacts of the varied pore distributions were compared and evaluated to determine the benefits of this physical manipulation. The results indicate that both mechanical properties and cell performance improve in the graded pore structures when compared to homogeneously distributed porous and non-porous structures. Differentiation results indicated successful osteogenic and chondrogenic manipulation in engineered scaffolds.

Proteomic Definitions of Mesenchymal Stem Cells

PubMed Central

Maurer, Martin H.

2011-01-01

Mesenchymal stem cells (MSCs) are pluripotent cells isolated from the bone marrow and various other organs. They are able to proliferate and self-renew, as well as to give rise to progeny of at least the osteogenic, chondrogenic, and adipogenic lineages. Despite this functional definition, MSCs can also be defined by their expression of a distinct set of cell surface markers. In the current paper, studies investigating the proteome of human MSCs are reviewed with the aim to identify common protein markers of MSCs. The proteomic analysis of MSCs revealed a distinct set of proteins representing the basic molecular inventory, including proteins for (i) cell surface markers, (ii) the responsiveness to growth factors, (iii) the reuse of developmental signaling cascades in adult stem cells, (iv) the interaction with molecules of the extracellular matrix, (v) the expression of genes regulating transcription and translation, (vi) the control of the cell number, and (vii) the protection against cellular stress. PMID:21437194

Current Therapeutic Strategies for Stem Cell-Based Cartilage Regeneration

PubMed Central

Nam, Yoojun; Lee, Jennifer

2018-01-01

The process of cartilage destruction in the diarthrodial joint is progressive and irreversible. This destruction is extremely difficult to manage and frustrates researchers, clinicians, and patients. Patients often take medication to control their pain. Surgery is usually performed when pain becomes uncontrollable or joint function completely fails. There is an unmet clinical need for a regenerative strategy to treat cartilage defect without surgery due to the lack of a suitable regenerative strategy. Clinicians and scientists have tried to address this using stem cells, which have a regenerative potential in various tissues. Cartilage may be an ideal target for stem cell treatment because it has a notoriously poor regenerative potential. In this review, we describe past, present, and future strategies to regenerate cartilage in patients. Specifically, this review compares a surgical regenerative technique (microfracture) and cell therapy, cell therapy with and without a scaffold, and therapy with nonaggregated and aggregated cells. We also review the chondrogenic potential of cells according to their origin, including autologous chondrocytes, mesenchymal stem cells, and induced pluripotent stem cells. PMID:29765426

Rapid Selection of Mesenchymal Stem and Progenitor Cells in Primary Prostate Stromal Cultures

PubMed Central

Brennen, W. Nathaniel; Kisteman, L. Nelleke; Isaacs, John T.

2016-01-01

BACKGROUND Carcinoma-associated fibroblasts (CAFs) are a dominant component of the tumor microenvironment with pro-tumorigenic properties. Despite this knowledge, their physiologic origins remain poorly understood. Mesenchymal stem cells (MSCs) can be recruited from the bone marrow to areas of tissue damage and inflammation, including prostate cancer. MSCs can generate and have many overlapping properties with CAFs in preclinical models. METHODS Multiparameter flow cytometry and multipotent differentiation assays used to define MSCs in primary prostate stromal cultures derived from young (>25 yrs) organ donors and prostate cancer patients compared with bone marrow-derived stromal cultures. Population doubling times, population doublings, cell size, and differentiation potential determined under multiple culture conditions, including normoxia, hypoxia, and a variety of media. TGF-β measured by ELISA. RESULTS MSCs and stromal progenitors are not only present in normal and malignant prostate tissue, but are quickly selected for in primary stromal cultures derived from these tissues; becoming the dominant population within just a few passages. Growth potential inversely associated with TGF-β concentrations. All conditions generated populations with an average cell diameter >15 μm. All cultures tested had the ability to undergo osteogenic and chondrogenic differentiation, but unlike bone marrow-derived MSCs, primary stromal cultures derived from normal prostate tissue lack adipogenic differentiation potential. In contrast, a subset of stromal cultures derived from prostate cancer patients retain the ability to differentiate into adipocytes; a property that is significantly suppressed under hypoxic conditions in both bone marrow- and prostate-derived MSCs. CONCLUSIONS Primary prostate stromal cultures are highly enriched in cells with an MSC or stromal progenitor phenotype. The use of primary cultures such as these to study CAFs raises interesting implications when considering their overlapping properties. The lack of adipogenesis in stromal cultures derived from normal prostates suggests they have a lineage-restricted progenitor phenotype. The retention of adipogenic differentiation in cultures from a subset of prostate cancer patients suggests the active recruitment of less committed progenitors or MSCs from the bone marrow as a function of disease progression. This recruitment can potentially be exploited for prognostic purposes or a cell-based platform for the systemic delivery of cytotoxic agents to sites of prostate cancer. PMID:26732992

Rapid selection of mesenchymal stem and progenitor cells in primary prostate stromal cultures.

PubMed

Brennen, W Nathaniel; Kisteman, L Nelleke; Isaacs, John T

2016-05-01

Carcinoma-associated fibroblasts (CAFs) are a dominant component of the tumor microenvironment with pro-tumorigenic properties. Despite this knowledge, their physiologic origins remain poorly understood. Mesenchymal stem cells (MSCs) can be recruited from the bone marrow to areas of tissue damage and inflammation, including prostate cancer. MSCs can generate and have many overlapping properties with CAFs in preclinical models. Multiparameter flow cytometry and multipotent differentiation assays used to define MSCs in primary prostate stromal cultures derived from young (<25 yrs) organ donors and prostate cancer patients compared with bone marrow-derived stromal cultures. Population doubling times, population doublings, cell size, and differentiation potential determined under multiple culture conditions, including normoxia, hypoxia, and a variety of media. TGF-β measured by ELISA. MSCs and stromal progenitors are not only present in normal and malignant prostate tissue, but are quickly selected for in primary stromal cultures derived from these tissues; becoming the dominant population within just a few passages. Growth potential inversely associated with TGF-β concentrations. All conditions generated populations with an average cell diameter >15 µm. All cultures tested had the ability to undergo osteogenic and chondrogenic differentiation, but unlike bone marrow-derived MSCs, primary stromal cultures derived from normal prostate tissue lack adipogenic differentiation potential. In contrast, a subset of stromal cultures derived from prostate cancer patients retain the ability to differentiate into adipocytes; a property that is significantly suppressed under hypoxic conditions in both bone marrow- and prostate-derived MSCs. Primary prostate stromal cultures are highly enriched in cells with an MSC or stromal progenitor phenotype. The use of primary cultures such as these to study CAFs raises interesting implications when considering their overlapping properties. The lack of adipogenesis in stromal cultures derived from normal prostates suggests they have a lineage-restricted progenitor phenotype. The retention of adipogenic differentiation in cultures from a subset of prostate cancer patients suggests the active recruitment of less committed progenitors or MSCs from the bone marrow as a function of disease progression. This recruitment can potentially be exploited for prognostic purposes or a cell-based platform for the systemic delivery of cytotoxic agents to sites of prostate cancer. © 2016 Wiley Periodicals, Inc.

Effects of hydrostatic pressure and transforming growth factor-beta 3 on adult human mesenchymal stem cell chondrogenesis in vitro.

PubMed

Miyanishi, Keita; Trindade, Michael C D; Lindsey, Derek P; Beaupré, Gary S; Carter, Dennis R; Goodman, Stuart B; Schurman, David J; Smith, R Lane

2006-06-01

This study examined the effects of intermittent hydrostatic pressure (IHP) and transforming growth factor-beta 3 on chondrogenesis of adult human mesenchymal stem cells (hMSCs) in vitro. Chondrogenic gene expression was determined by quantifying mRNA signal levels for SOX9, a transcription factor critical for cartilage development and the cartilage matrix proteins, aggrecan and type II collagen. Extracellular matrix production was determined by weight and histology. IHP was applied to hMSCs in pellet culture at a level of 10 MPa and a frequency of 1 Hz for 4 h per day for periods of 3, 7, and 14 days. hMSCs responded to addition of TGF-beta 3 (10 ng/mL) with a greater than 10-fold increase (p < 0.01) in mRNA levels for each, SOX9, type II collagen, and aggrecan during a 14-day culture period. Applying IHP in the presence of TGF-beta 3 further increased the mRNA levels for these proteins by 1.9-, 3.3-, and 1.6-fold, respectively, by day 14. Chondrogenic mRNA levels were increased with just exposure to IHP. Extracellular matrix deposition of type II collagen and aggrecan increased in the pellets as a function of treatment conditions and time of culture. This study demonstrated adjunctive effects of IHP on TGF-beta 3-induced chondrogenesis and suggests that mechanical loading can facilitate articular cartilage tissue engineering.

Matrix-associated implantation of predifferentiated mesenchymal stem cells versus articular chondrocytes: in vivo results of cartilage repair after 1 year.

PubMed

Marquass, Bastian; Schulz, Ronny; Hepp, Pierre; Zscharnack, Matthias; Aigner, Thomas; Schmidt, Stefanie; Stein, Frank; Richter, Robert; Osterhoff, Georg; Aust, Gabriele; Josten, Christoph; Bader, Augustinus

2011-07-01

The use of predifferentiated mesenchymal stem cells (MSC) leads to better histological results compared with undifferentiated MSC in sheep. This raises the need for a longer term follow-up study and comparison with a clinically established method. We hypothesized that chondrogenic in vitro predifferentiation of autologous MSC embedded in a collagen I hydrogel leads to better structural repair of a chronic osteochondral defect in an ovine stifle joint after 1 year. We further hypothesized that resulting histological results would be comparable with those of chondrocyte-seeded matrix-associated autologous chondrocyte transplantation (MACT). Controlled laboratory study. Predifferentiation period of ovine MSC within collagen gel in vitro was defined by assessment of several cellular and molecular biological parameters. For the animal study, 2 osteochondral lesions (7-mm diameter) were created at the medial femoral condyles of the hind legs in 9 sheep. Implantation of MSC gels was performed 6 weeks after defect creation. Thirty-six defects were divided into 4 treatment groups: (1) chondrogenically predifferentiated MSC gels (pre-MSC gels), (2) undifferentiated MSC gels (un-MSC gels), (3) MACT gels, and (4) untreated controls (UC). Histological, immunohistochemical, and radiological evaluations followed after 12 months. After 12 months in vivo, pre-MSC gels showed significantly better histological outcome compared with un-MSC gels and UC. Compared with MACT gels, the overall scores were higher for O'Driscoll and International Cartilage Repair Society (ICRS). The repair tissue of the pre-MSC group showed immunohistochemical detection of interzonal collagen type II staining. Radiological evaluation supported superior bonding of pre-MSC gels to perilesional native cartilage. Compared with previous work by our group, no degradation of the repair tissue between 6 and 12 months in vivo, particularly in pre-MSC gels, was observed. Repair of chronic osteochondral defects with collagen hydrogels composed of chondrogenically predifferentiated MSC shows no signs of degradation after 1 year in vivo. In addition, pre-MSC gels lead to partially superior histological results compared with articular chondrocytes. The results suggest an encouraging method for future treatment of focal osteochondral defects without donor site morbidity by harvesting articular chondrocytes.

In vitro mesenchymal trilineage differentiation and extracellular matrix production by adipose and bone marrow derived adult equine multipotent stromal cells on a collagen scaffold.

PubMed

Xie, Lin; Zhang, Nan; Marsano, Anna; Vunjak-Novakovic, Gordana; Zhang, Yanru; Lopez, Mandi J

2013-12-01

Directed differentiation of adult multipotent stromal cells (MSC) is critical for effective treatment strategies. This study was designed to evaluate the capability of equine MSC from bone marrow (BMSC) and adipose tissue (ASC) on a type I collagen (COLI) scaffold to undergo chondrogenic, osteogenic and adipogenic differentiation and form extracellular matrix (ECM) in vitro. Following determination of surface antigen expression, MSC were loaded into scaffolds in a perfusion bioreactor and loading efficiency was quantified. Cell-scaffold constructs were assessed after loading and 7, 14 and 21 days of culture in stromal or induction medium. Cell number was determined with DNA content, cell viability and spatial uniformity with confocal laser microscopy and cell phenotype and matrix production with light and scanning electron microscopy and mRNA levels. The MSC were positive for CD29 (>90 %), CD44 (>99 %), and CD105 (>60 %). Loading efficiencies were >70 %. The ASC and BMSC cell numbers on scaffolds were affected by culture in induction medium differently. Viable cells remained uniformly distributed in scaffolds for up to 21 days and could be directed to differentiate or to maintain an MSC phenotype. Micro- and ultrastructure showed lineage-specific cell and ECM changes. Lineage-specific mRNA levels differed between ASC and BMSC with induction and changed with time. Based on these results, equine ASC and BMSC differentiate into chondrogenic, osteogenic and adipogenic lineages and form ECM similarly on COLI scaffolds. The collected data supports the potential for equine MSC-COLI constructs to support diverse equine tissue formation for controlled biological studies.

Fully Dedifferentiated Chondrocytes Expanded in Specific Mesenchymal Stem Cell Growth Medium with FGF2 Obtains Mesenchymal Stem Cell Phenotype In Vitro but Retains Chondrocyte Phenotype In Vivo

PubMed Central

Lee, Jungsun; Lee, Jin-Yeon; Chae, Byung-Chul; Jang, Jeongho

2017-01-01

Given recent progress in regenerative medicine, we need a means to expand chondrocytes in quantity without losing their regenerative capability. Although many reports have shown that growth factor supplementation can have beneficial effects, the use of growth factor–supplemented basal media has widespread effect on the characteristics of chondrocytes. Chondrocytes were in vitro cultured in the 2 most widely used chondrocyte growth media, conventional chondrocyte culture medium and mesenchymal stem cell (MSC) culture medium, both with and without fibroblast growth factor-2 (FGF2) supplementation. Their expansion rates, expressions of extracellular matrix–related factors, senescence, and differentiation potentials were examined in vitro and in vivo. Our results revealed that chondrocytes quickly dedifferentiated during expansion in all tested media, as assessed by the loss of type II collagen expression. The 2 basal media (chondrocyte culture medium vs. MSC culture medium) were associated with distinct differences in cell senescence. Consistent with the literature, FGF2 was associated with accelerated dedifferentiation during expansion culture and superior redifferentiation upon induction. However, chondrocytes expanded in FGF2-containing conventional chondrocyte culture medium showed MSC-like features, as indicated by their ability to direct ectopic bone formation and cartilage formation. In contrast, chondrocytes cultured in FGF2-supplemented MSC culture medium showed potent chondrogenesis and almost no bone formation. The present findings show that the chosen basal medium can exert profound effects on the characteristics and activity of in vitro–expanded chondrocytes and indicate that right growth factor/medium combination can help chondrocytes retain a high-level chondrogenic potential without undergoing hypertrophic transition. PMID:29251111

Non-invasive in vivo molecular imaging of intra-articularly transplanted immortalized bone marrow stem cells for osteoarthritis treatment.

PubMed

Peng, Bou-Yue; Chiou, Chi-Sheng; Dubey, Navneet Kumar; Yu, Sung-Hsun; Deng, Yue-Hua; Tsai, Feng-Chou; Chiang, Han-Sun; Shieh, Ying-Hua; Chen, Wei-Hong; Deng, Win-Ping

2017-11-14

Pathophysiology of osteoarthritis (OA) is characterized by progressive loss of articular cartilage in the knee-joints. To impart regenerative ability in lowly metabolizing chondrocytes, the bone marrow stem cells (BMSCs) has recently been recognized as a superior alternative treatment for OA. However, study of primary BMSCs-mediated chondrogenesis is difficult due to progressive cellular aging and replicative senescence. To obtain a therapeutic cell population for OA, BMSCs were immortalized by human papilloma virus (HPV)-16 E6/E7 along with mCherry luciferase (mCL), a gene marker for non-invasive imaging, and designated as iBMSCs-mCL. Next, their cell morphology, population doubling time (PDT) and colony forming ability (CFU) were evaluated. Furthermore, pluripotency and immunophenotypic markers were investigated. To deduce therapeutic ability, iBMSCs-mCL were intra-articularly injected into right knee of anterior cruciate ligament transaction (ACLT)-OA mice model and tracked through non-invasive bioluminescence imaging. Cell morphology of iBMSCs-mCL was similar to parental BMSCs. PDT and CFU ability of iBMSCs-mCLs were significantly increased. Pluripotency and immunophenotypic markers were highly expressed in iBMSC-mCL. Long-term survival and tri-lineage differentiation particularly chondrogenic potential of iBMSCs-mCL were also demonstrated in vitro and then in vivo which was monitored through non-invasive imaging. Intensive bioluminescent signals in iBMSCs-mCL administered knee-joint indicated a marked in vivo survival and proliferation of iBMSCs-mCL. Immunohistochemical staining for type II collagen (IHC of Col II) and alcian blue & safranin o staining of proteoglycans also corroborated cartilage regeneration by iBMSCs-mCL. Conclusively, iBMSCs-mCL maintains stemness and in vivo cartilage regeneration potential suggesting a promising avenue for development of OA therapeutics.

Osmolyte Type and the Osmolarity Level Affect Chondrogenesis of Mesenchymal Stem Cells.

PubMed

Ahmadyan, Sorour; Kabiri, Mahboubeh; Hanaee-Ahvaz, Hana; Farazmand, Ali

2018-06-01

The inductive effects of increased osmolarity on chondrogenesis are well approved. However, the effects of the osmolyte agent invoked to induce hyperosmolarity are largely neglected. Herein, we scrutinized how hyperosmotic conditions acquired by addition of different osmolytes would impact chondrogenesis. We briefly assessed whether such conditions would differentially affect hypertrophy and angiogenesis during MSC chondrogenesis. Chondrogenic and hypertrophic marker expression along with VEGF secretion during adipose-derived (AD)-MSC chondrogenesis under three osmolarity levels (350, 450, and 550 mOsm) using three different osmolytes (NaCl, sorbitol, and PEG) were assessed. MTT assay, qRT-PCR, immunocytochemistry, Alcian Blue staining, ELISA, and ALP assays proved osmolyte-type dependent effects of hyperosmolarity on chondrogenesis, hypertrophy, and angiogenesis. At same osmolarity level, PEG had least cytotoxic/cytostatic effect and most prohibitive effects on angiogenesis. As expected, all hyperosmolar conditions led to enhanced chondrogenesis with slightly varying degrees. PEG and sorbitol had higher chondro-promotive and hypertrophy-suppressive effects compared to NaCl, while NaCl had exacerbated hypertrophy. We observed that TonEBP was involved in osmoadaptation of all treatments in varying degrees. Of importance, we highlighted differential effects of hyperosmolarity obtained by different osmolytes on the efficacy of chondrogenesis and more remarkably on the induction/suppression of cartilage pathologic markers. Our study underlies the need for a more vigilant exploitation of physicobiochemical inducers in order to maximize chondrogenesis while restraining unwanted hypertrophy and angiogenesis.

Wnt and FGF signals interact to coordinate growth with cell fate specification during limb development.

PubMed

ten Berge, Derk; Brugmann, Samantha A; Helms, Jill A; Nusse, Roel

2008-10-01

A fundamental question in developmental biology is how does an undifferentiated field of cells acquire spatial pattern and undergo coordinated differentiation? The development of the vertebrate limb is an important paradigm for understanding these processes. The skeletal and connective tissues of the developing limb all derive from a population of multipotent progenitor cells located in its distal tip. During limb outgrowth, these progenitors segregate into a chondrogenic lineage, located in the center of the limb bud, and soft connective tissue lineages located in its periphery. We report that the interplay of two families of signaling proteins, fibroblast growth factors (FGFs) and Wnts, coordinate the growth of the multipotent progenitor cells with their simultaneous segregation into these lineages. FGF and Wnt signals act together to synergistically promote proliferation while maintaining the cells in an undifferentiated, multipotent state, but act separately to determine cell lineage specification. Withdrawal of both signals results in cell cycle withdrawal and chondrogenic differentiation. Continued exposure to Wnt, however, maintains proliferation and re-specifies the cells towards the soft connective tissue lineages. We have identified target genes that are synergistically regulated by Wnts and FGFs, and show how these factors actively suppress differentiation and promote growth. Finally, we show how the spatial restriction of Wnt and FGF signals to the limb ectoderm, and to a specialized region of it, the apical ectodermal ridge, controls the distribution of cell behaviors within the growing limb, and guides the proper spatial organization of the differentiating tissues.

Lysyl Oxidase-like-2 (LOXL2) Is a Major Isoform in Chondrocytes and Is Critically Required for Differentiation*

PubMed Central

Iftikhar, Mussadiq; Hurtado, Paola; Bais, Manish V.; Wigner, Nate; Stephens, Danielle N.; Gerstenfeld, Louis C.; Trackman, Philip C.

2011-01-01

The lysyl oxidase family is made up of five members: lysyl oxidase (LOX) and lysyl oxidase-like 1–4 (LOXL1-LOXL4). All members share conserved C-terminal catalytic domains that provide for lysyl oxidase or lysyl oxidase-like enzyme activity; and more divergent propeptide regions. LOX family enzyme activities catalyze the final enzymatic conversion required for the formation of normal biosynthetic collagen and elastin cross-links. The importance of lysyl oxidase enzyme activity to normal bone development has long been appreciated, but regulation and roles for specific LOX isoforms in bone formation in vivo is largely unexplored. Fracture healing recapitulates aspects of endochondral bone development. The present study first investigated the expression of all LOX isoforms in fracture healing. A remarkable coincidence of LOXL2 expression with the chondrogenic phase of fracture healing was found, prompting more detailed analyses of LOXL2 expression in normal growth plates, and LOXL2 expression and function in developing ATDC5 chondrogenic cells. Data show that LOXL2 is expressed by pre-hypertrophic and hypertrophic chondrocytes in vivo, and that LOXL2 expression is regulated in vitro as a function of chondrocyte differentiation. Moreover, LOXL2 knockdown studies in vitro show that LOXL2 expression is required for ATDC5 chondrocyte cell line differentiation through regulation of SNAIL and SOX9, important transcription factors that control chondrocyte differentiation. Taken together, data provide evidence that LOXL2, like LOX, is a multifunctional protein. LOXL2 promotes chondrocyte differentiation by mechanisms that are likely to include roles as both a regulator and an effector of chondrocyte differentiation. PMID:21071451

Large-scale expansion of Wharton's jelly-derived mesenchymal stem cells on gelatin microbeads, with retention of self-renewal and multipotency characteristics and the capacity for enhancing skin wound healing.

PubMed

Zhao, Guifang; Liu, Feilin; Lan, Shaowei; Li, Pengdong; Wang, Li; Kou, Junna; Qi, Xiaojuan; Fan, Ruirui; Hao, Deshun; Wu, Chunling; Bai, Tingting; Li, Yulin; Liu, Jin Yu

2015-03-19

Successful stem cell therapy relies on large-scale generation of stem cells and their maintenance in a proliferative multipotent state. This study aimed to establish a three-dimension culture system for large-scale generation of hWJ-MSC and investigated the self-renewal activity, genomic stability and multi-lineage differentiation potential of such hWJ-MSC in enhancing skin wound healing. hWJ-MSC were seeded on gelatin microbeads and cultured in spinning bottles (3D). Cell proliferation, karyotype analysis, surface marker expression, multipotent differentiation (adipogenic, chondrogenic, and osteogenic potentials), and expression of core transcription factors (OCT4, SOX2, NANOG, and C-MYC), as well as their efficacy in accelerating skin wound healing, were investigated and compared with those of hWJ-MSC derived from plate cultres (2D), using in vivo and in vitro experiments. hWJ-MSC attached to and proliferated on gelatin microbeads in 3D cultures reaching a maximum of 1.1-1.30×10(7) cells on 0.5 g of microbeads by days 8-14; in contrast, hWJ-MSC derived from 2D cultures reached a maximum of 6.5 -11.5×10(5) cells per well in a 24-well plate by days 6-10. hWJ-MSC derived by 3D culture incorporated significantly more EdU (P<0.05) and had a significantly higher proliferation index (P<0.05) than those derived from 2D culture. Immunofluorescence staining, real-time PCR, flow cytometry analysis, and multipotency assays showed that hWJ-MSC derived from 3D culture retained MSC surface markers and multipotency potential similar to 2D culture-derived cells. 3D culture-derived hWJ-MSC also retained the expression of core transcription factors at levels comparable to their 2D culture counterparts. Direct injection of hWJ-MSC derived from 3D or 2D cultures into animals exhibited similar efficacy in enhancing skin wound healing. Thus, hWJ-MSC can be expanded markedly in gelatin microbeads, while retaining MSC surface marker expression, multipotent differential potential, and expression of core transcription factors. These cells also efficiently enhanced skin wound healing in vivo, in a manner comparable to that of hWJ-MSC obtained from 2D culture.

Mesenchymal Stromal Cells for Sphincter Regeneration: Role of Laminin Isoforms upon Myogenic Differentiation

PubMed Central

Seeger, Tanja; Hart, Melanie; Patarroyo, Manuel; Rolauffs, Bernd; Aicher, Wilhelm K.; Klein, Gerd

2015-01-01

Multipotent mesenchymal stromal cells (MSCs) are well known for their tri-lineage potential and ability to differentiate in vitro into osteogenic, chondrogenic or adipogenic lineages. By selecting appropriate conditions MSCs can also be differentiated in vitro into the myogenic lineage and are therefore a promising option for cell-based regeneration of muscle tissue such as an aged or damaged sphincter muscle. For the differentiation into the myogenic lineage there is still a need to evaluate the effects of extracellular matrix proteins such as laminins (LM) which are crucial for different stem cell types and for normal muscle function. The laminin family consists of 16 functionally different isoforms with LM-211 being the most abundant isoform of adult muscle tissues. In the sphincter tissue a strong expression of the isoforms LM-211/221, LM-411/421 and LM-511/521 can be detected in the different cell layers. Bone marrow-derived MSCs in culture, however, mainly express the isoforms LM-411 and LM-511, but not LM-211. Even after myogenic differentiation, LM-211 can hardly be detected. All laminin isoforms tested (LM-211, LM-411, LM-511 and LM-521) showed a significant inhibition of the proliferation of undifferentiated MSCs but, with the exception of LM-521, they had no influence on the proliferation of MSCs cultivated in myogenic medium. The strongest cellular adhesion of MSCs was to LM-511 and LM-521, whereas LM-211 was only a weakly-adhesive substrate for MSCs. Myogenic differentiation of MSCs even reduced the interaction with LM-211, but it did not affect the interaction with LM-511 and LM-521. Since during normal myogenesis the latter two isoforms are the major laminins surrounding developing myogenic progenitors, α5 chain-containing laminins are recommended for further improvements of myogenic differentiation protocols of MSCs into smooth muscle cells. PMID:26406476

Engineering spheroids potentiating cell-cell and cell-ECM interactions by self-assembly of stem cell microlayer.

PubMed

Lee, Yu Bin; Kim, Eun Mi; Byun, Hayeon; Chang, Hyung-Kwan; Jeong, Kwanghee; Aman, Zachary M; Choi, Yu Suk; Park, Jungyul; Shin, Heungsoo

2018-05-01

Numerous methods have been reported for the fabrication of 3D multi-cellular spheroids and their use in stem cell culture. Current methods typically relying on the self-assembly of trypsinized, suspended stem cells, however, show limitations with respect to cell viability, throughput, and accurate recapitulation of the natural microenvironment. In this study, we developed a new system for engineering cell spheroids by self-assembly of micro-scale monolayer of stem cells. We prepared synthetic hydrogels with the surface of chemically formed micropatterns (squares/circles with width/diameter of 200 μm) on which mesenchymal stem cells isolated from human nasal turbinate tissue (hTMSCs) were selectively attached and formed a monolayer. The hydrogel is capable of thermally controlled expansion. As the temperature was decreased from 37 to 4 °C, the cell layer detached rapidly (<10 min) and assembled to form spheroids with consistent size (∼100 μm) and high viability (>90%). Spheroidization was significantly delayed and occurred with reduced efficiency on circle patterns compared to square patterns. Multi-physics mapping supported that delamination of the micro-scale monolayer may be affected by stress concentrated at the corners of the square pattern. In contrast, stress was distributed symmetrically along the boundary of the circle pattern. In addition, treatment of the micro-scale monolayer with a ROCK inhibitor significantly retarded spheroidization, highlighting the importance of contraction mediated by actin stress fibers for the stable generation of spheroidal stem cell structures. Spheroids prepared from the assembly of monolayers showed higher expression, both on the mRNA and protein levels, of ECM proteins (fibronectin and laminin) and stemness markers (Oct4, Sox2, and Nanog) compared to spheroids prepared from low-attachment plates, in which trypsinized single cells are assembled. The hTMSC spheroids also presented enhanced expression levels of markers related to tri-lineage (osteogenic, chondrogenic and adipogenic) differentiation. The changes in microcellular environments and functionalities were double-confirmed by using adipose derived mesenchymal stem cells (ADSCs). This spheroid engineering technique may have versatile applications in regenerative medicine for functionally improved 3D culture and therapeutic cell delivery. Copyright © 2018 Elsevier Ltd. All rights reserved.

Deciphering Mechanical Regulation of Chondrogenesis in Fibrin–Polyurethane Composite Scaffolds Enriched with Human Mesenchymal Stem Cells: A Dual Computational and Experimental Approach

PubMed Central

Stoddart, Martin; Lezuo, Patrick; Forkmann, Christoph; Wimmmer, Markus A.; Alini, Mauro; Van Oosterwyck, Hans

2014-01-01

Fibrin–polyurethane composite scaffolds support chondrogenesis of human mesenchymal stem cells (hMSCs) derived from bone marrow and due to their robust mechanical properties allow mechanical loading in dynamic bioreactors, which has been shown to increase the chondrogenic differentiation of MSCs through the transforming growth factor beta pathway. The aim of this study was to use the finite element method, mechanical testing, and dynamic in vitro cell culture experiments on hMSC-enriched fibrin–polyurethane composite scaffolds to quantitatively decipher the mechanoregulation of chondrogenesis within these constructs. The study identified compressive principal strains as the key regulator of chondrogenesis in the constructs. Although dynamic uniaxial compression did not induce chondrogenesis, multiaxial loading by combined application of dynamic compression and interfacial shear induced significant chondrogenesis at locations where all the three principal strains were compressive and had a minimum magnitude of 10%. In contrast, no direct correlation was identified between the level of pore fluid velocity and chondrogenesis. Due to the high permeability of the constructs, the pore fluid pressures could not be increased sufficiently by mechanical loading, and instead, chondrogenesis was induced by triaxial compressive deformations of the matrix with a minimum magnitude of 10%. Thus, it can be concluded that dynamic triaxial compressive deformations of the matrix is sufficient to induce chondrogenesis in a threshold-dependent manner, even where the pore fluid pressure is negligible. PMID:24199606

ROCK inhibitor prevents the dedifferentiation of human articular chondrocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matsumoto, Emi; Furumatsu, Takayuki, E-mail: matino@md.okayama-u.ac.jp; Kanazawa, Tomoko

2012-03-30

Highlights: Black-Right-Pointing-Pointer ROCK inhibitor stimulates chondrogenic gene expression of articular chondrocytes. Black-Right-Pointing-Pointer ROCK inhibitor prevents the dedifferentiation of monolayer-cultured chondrocytes. Black-Right-Pointing-Pointer ROCK inhibitor enhances the redifferentiation of cultured chondrocytes. Black-Right-Pointing-Pointer ROCK inhibitor is useful for preparation of un-dedifferentiated chondrocytes. Black-Right-Pointing-Pointer ROCK inhibitor may be a useful reagent for chondrocyte-based regeneration therapy. -- Abstract: Chondrocytes lose their chondrocytic phenotypes in vitro. The Rho family GTPase ROCK, involved in organizing the actin cytoskeleton, modulates the differentiation status of chondrocytic cells. However, the optimum method to prepare a large number of un-dedifferentiated chondrocytes is still unclear. In this study, we investigated the effectmore » of ROCK inhibitor (ROCKi) on the chondrogenic property of monolayer-cultured articular chondrocytes. Human articular chondrocytes were subcultured in the presence or absence of ROCKi (Y-27632). The expression of chondrocytic marker genes such as SOX9 and COL2A1 was assessed by quantitative real-time PCR analysis. Cellular morphology and viability were evaluated. Chondrogenic redifferentiation potential was examined by a pellet culture procedure. The expression level of SOX9 and COL2A1 was higher in ROCKi-treated chondrocytes than in untreated cells. Chondrocyte morphology varied from a spreading form to a round shape in a ROCKi-dependent manner. In addition, ROCKi treatment stimulated the proliferation of chondrocytes. The deposition of safranin O-stained proteoglycans and type II collagen was highly detected in chondrogenic pellets derived from ROCKi-pretreated chondrocytes. Our results suggest that ROCKi prevents the dedifferentiation of monolayer-cultured chondrocytes, and may be a useful reagent to maintain chondrocytic phenotypes in vitro for chondrocyte-based regeneration therapy.« less

Transient chondrogenic phase in the intramembranous pathway during normal skeletal development.

PubMed

Nah, H D; Pacifici, M; Gerstenfeld, L C; Adams, S L; Kirsch, T

2000-03-01

Calvarial and facial bones form by intramembranous ossification, in which bone cells arise directly from mesenchyme without an intermediate cartilage anlage. However, a number of studies have reported the emergence of chondrocytes from in vitro calvarial cell or organ cultures and the expression of type II collagen, a cartilage-characteristic marker, in developing calvarial bones. Based on these findings we hypothesized that a covert chondrogenic phase may be an integral part of the normal intramembranous pathway. To test this hypothesis, we analyzed the temporal and spatial expression patterns of cartilage characteristic genes in normal membranous bones from chick embryos at various developmental stages (days 12, 15 and 19). Northern and RNAse protection analyses revealed that embryonic frontal bones expressed not only the type I collagen gene but also a subset of cartilage characteristic genes, types IIA and XI collagen and aggrecan, thus resembling a phenotype of prechondrogenic-condensing mesenchyme. The expression of cartilage-characteristic genes decreased with the progression of bone maturation. Immunohistochemical analyses of developing embryonic chick heads indicated that type II collagen and aggrecan were produced by alkaline phosphatase activity positive cells engaged in early stages of osteogenic differentiation, such as cells in preosteogenic-condensing mesenchyme, the cambium layer of periosteum, the advancing osteogenic front, and osteoid bone. Type IIB and X collagen messenger RNAs (mRNA), markers for mature chondrocytes, were also detected at low levels in calvarial bone but not until late embryonic stages (day 19), indicating that some calvarial cells may undergo overt chondrogenesis. On the basis of our findings, we propose that the normal intramembranous pathway in chicks includes a previously unrecognized transient chondrogenic phase similar to prechondrogenic mesenchyme, and that the cells in this phase retain chondrogenic potential that can be expressed in specific in vitro and in vivo microenvironments.

Cartilage Repair Using Composites of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells and Hyaluronic Acid Hydrogel in a Minipig Model.

PubMed

Ha, Chul-Won; Park, Yong-Beom; Chung, Jun-Young; Park, Yong-Geun

2015-09-01

The cartilage regeneration potential of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) with a hyaluronic acid (HA) hydrogel composite has shown remarkable results in rat and rabbit models. The purpose of the present study was to confirm the consistent regenerative potential in a pig model using three different cell lines. A full-thickness chondral injury was intentionally created in the trochlear groove of each knee in 6 minipigs. Three weeks later, an osteochondral defect, 5 mm wide by 10 mm deep, was created, followed by an 8-mm-wide and 5-mm-deep reaming. A mixture (1.5 ml) of hUCB-MSCs (0.5×10(7) cells per milliliter) and 4% HA hydrogel composite was then transplanted into the defect on the right knee. Each cell line was used in two minipigs. The osteochondral defect created in the same manner on the left knee was untreated to act as the control. At 12 weeks postoperatively, the pigs were sacrificed, and the degree of subsequent cartilage regeneration was evaluated by gross and histological analysis. The transplanted knee resulted in superior and more complete hyaline cartilage regeneration compared with the control knee. The cellular characteristics (e.g., cellular proliferation and chondrogenic differentiation capacity) of the hUCB-MSCs influenced the degree of cartilage regeneration potential. This evidence of consistent cartilage regeneration using composites of hUCB-MSCs and HA hydrogel in a large animal model could be a stepping stone to a human clinical trial in the future. To date, several studies have investigated the chondrogenic potential of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs); however, the preclinical studies are still limited in numbers with various results. In parallel, in the past several years, the cartilage regeneration potential of hUCB-MSCs with a hyaluronic acid (HA) hydrogel composite have been investigated and remarkable results in rat and rabbit models have been attained. (These experimental results are currently in preparation for publication.) Before applying the cartilage regeneration technique in a human clinical trial, it seemed necessary to confirm the consistent result in a larger animal model. At 12 weeks postoperatively, the minipigs were sacrificed, and the degree of subsequent cartilage regeneration was evaluated by gross and histological analysis. The transplanted knee resulted in superior and more complete hyaline cartilage regeneration compared with the control knee. This evidence of consistent cartilage regeneration with composites of hUCB-MSCs and HA hydrogel in a large animal model could be a stepping stone to a human clinical trial in the future. ©AlphaMed Press.

Cartilage Repair Using Composites of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells and Hyaluronic Acid Hydrogel in a Minipig Model

PubMed Central

Ha, Chul-Won; Chung, Jun-Young; Park, Yong-Geun

2015-01-01

The cartilage regeneration potential of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) with a hyaluronic acid (HA) hydrogel composite has shown remarkable results in rat and rabbit models. The purpose of the present study was to confirm the consistent regenerative potential in a pig model using three different cell lines. A full-thickness chondral injury was intentionally created in the trochlear groove of each knee in 6 minipigs. Three weeks later, an osteochondral defect, 5 mm wide by 10 mm deep, was created, followed by an 8-mm-wide and 5-mm-deep reaming. A mixture (1.5 ml) of hUCB-MSCs (0.5 × 107 cells per milliliter) and 4% HA hydrogel composite was then transplanted into the defect on the right knee. Each cell line was used in two minipigs. The osteochondral defect created in the same manner on the left knee was untreated to act as the control. At 12 weeks postoperatively, the pigs were sacrificed, and the degree of subsequent cartilage regeneration was evaluated by gross and histological analysis. The transplanted knee resulted in superior and more complete hyaline cartilage regeneration compared with the control knee. The cellular characteristics (e.g., cellular proliferation and chondrogenic differentiation capacity) of the hUCB-MSCs influenced the degree of cartilage regeneration potential. This evidence of consistent cartilage regeneration using composites of hUCB-MSCs and HA hydrogel in a large animal model could be a stepping stone to a human clinical trial in the future. Significance To date, several studies have investigated the chondrogenic potential of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs); however, the preclinical studies are still limited in numbers with various results. In parallel, in the past several years, the cartilage regeneration potential of hUCB-MSCs with a hyaluronic acid (HA) hydrogel composite have been investigated and remarkable results in rat and rabbit models have been attained. (These experimental results are currently in preparation for publication.) Before applying the cartilage regeneration technique in a human clinical trial, it seemed necessary to confirm the consistent result in a larger animal model. At 12 weeks postoperatively, the minipigs were sacrificed, and the degree of subsequent cartilage regeneration was evaluated by gross and histological analysis. The transplanted knee resulted in superior and more complete hyaline cartilage regeneration compared with the control knee. This evidence of consistent cartilage regeneration with composites of hUCB-MSCs and HA hydrogel in a large animal model could be a stepping stone to a human clinical trial in the future. PMID:26240434

A comparative assessment of cartilage and joint fat pad as a potential source of cells for autologous therapy development in knee osteoarthritis.

PubMed

English, A; Jones, E A; Corscadden, D; Henshaw, K; Chapman, T; Emery, P; McGonagle, D

2007-11-01

The utility of autologous chondrocytes for cartilage repair strategies in older subjects with osteoarthritis (OA) may be limited by both age-related and disease-associated decline in chondrogenesis. The aim of this work was to assess OA Hoffa's fat pad as an alternative source of autologous chondroprogenitor cells and to compare it with OA chondrocytes derived from different areas of cartilage. Cartilage and fat pad tissue digests were obtained from 26 subjects with knee OA and compared with normal bone marrow (BM) mesenchymal stem cells (MSCs) with respect to their in vitro colony-forming potential, growth kinetics, multipotentiality and clonogenicity. Flow cytometry was used to investigate their MSC marker phenotype. Expanded cultures derived from eroded areas of cartilage were slightly more chondrogenic than those derived from macroscopically normal cartilage or chondro-osteophytes; however, all cartilage-derived cultures failed to maintain their chondrogenic potency following extended expansion. In contrast, OA fat pads contained highly clonogenic and multipotential cells with stable chondrogenic potency in vitro, even after 16 population doublings. Standard colony-forming assays failed to reflect the observed functional differences between the studied tissues whereas flow cytometry revealed higher levels of a putative MSC marker low-affinity growth factor receptor (LNGFR) on culture expanded fat pad-derived, but not cartilage-derived, MSCs. In contrast to OA cartilage from three different sites, OA Hoffa's fat pad contains clonogenic cells that meet the criteria for MSCs and produce multipotential cultures that maintain their chondrogenesis long term. These findings have broad implications for future strategies aimed at cartilage repair in OA.

Regulation of decellularized tissue remodeling via scaffold-mediated lentiviral delivery in anatomically-shaped osteochondral constructs.

PubMed

Rowland, Christopher R; Glass, Katherine A; Ettyreddy, Adarsh R; Gloss, Catherine C; Matthews, Jared R L; Huynh, Nguyen P T; Guilak, Farshid

2018-05-30

Cartilage-derived matrix (CDM) has emerged as a promising scaffold material for tissue engineering of cartilage and bone due to its native chondroinductive capacity and its ability to support endochondral ossification. Because it consists of native tissue, CDM can undergo cellular remodeling, which can promote integration with host tissue and enables it to be degraded and replaced by neotissue over time. However, enzymatic degradation of decellularized tissues can occur unpredictably and may not allow sufficient time for mechanically competent tissue to form, especially in the harsh inflammatory environment of a diseased joint. The goal of the current study was to engineer cartilage and bone constructs with the ability to inhibit aberrant inflammatory processes caused by the cytokine interleukin-1 (IL-1), through scaffold-mediated delivery of lentiviral particles containing a doxycycline-inducible IL-1 receptor antagonist (IL-1Ra) transgene on anatomically-shaped CDM constructs. Additionally, scaffold-mediated lentiviral gene delivery was used to facilitate spatial organization of simultaneous chondrogenic and osteogenic differentiation via site-specific transduction of a single mesenchymal stem cell (MSC) population to overexpress either chondrogenic, transforming growth factor-beta 3 (TGF-β3), or osteogenic, bone morphogenetic protein-2 (BMP-2), transgenes. Controlled induction of IL-1Ra expression protected CDM hemispheres from inflammation-mediated degradation, and supported robust bone and cartilage tissue formation even in the presence of IL-1. In the absence of inflammatory stimuli, controlled cellular remodeling was exploited as a mechanism for fusing concentric CDM hemispheres overexpressing BMP-2 and TGF-β3 into a single bi-layered osteochondral construct. Our findings demonstrate that site-specific delivery of inducible and tunable transgenes confers spatial and temporal control over both CDM scaffold remodeling and neotissue composition. Furthermore, these constructs provide a microphysiological in vitro joint organoid model with site-specific, tunable, and inducible protein delivery systems for examining the spatiotemporal response to pro-anabolic and/or inflammatory signaling across the osteochondral interface. Copyright © 2018 Elsevier Ltd. All rights reserved.

Nfatc1 Is a Functional Transcriptional Factor Mediating Nell-1-Induced Runx3 Upregulation in Chondrocytes.

PubMed

Li, Chenshuang; Zheng, Zhong; Zhang, Xinli; Asatrian, Greg; Chen, Eric; Song, Richard; Culiat, Cymbeline; Ting, Kang; Soo, Chia

2018-01-06

Neural EGFL like 1 (Nell-1) is essential for chondrogenic differentiation, maturation, and regeneration. Our previous studies have demonstrated that Nell-1's pro-chondrogenic activities are predominantly reliant upon runt-related transcription factor 3 (Runx3)-mediated Indian hedgehog (Ihh) signaling. Here, we identify the nuclear factor of activated T-cells 1 (Nfatc1) as the key transcriptional factor mediating the Nell-1 → Runx3 signal transduction in chondrocytes. Using chromatin immunoprecipitation assay, we were able to determine that Nfatc1 binds to the -833--810 region of the Runx3 -promoter in response to Nell-1 treatment. By revealing the Nell-1 → Nfatc1 → Runx3 → Ihh cascade, we demonstrate the involvement of Nfatc1, a nuclear factor of activated T-cells, in chondrogenesis, while providing innovative insights into developing a novel therapeutic strategy for cartilage regeneration and other chondrogenesis-related conditions.

Chondrogenesis of infrapatellar fat pad derived adipose stem cells in 3D printed chitosan scaffold.

PubMed

Ye, Ken; Felimban, Raed; Traianedes, Kathy; Moulton, Simon E; Wallace, Gordon G; Chung, Johnson; Quigley, Anita; Choong, Peter F M; Myers, Damian E

2014-01-01

Infrapatellar fat pad adipose stem cells (IPFP-ASCs) have been shown to harbor chondrogenic potential. When combined with 3D polymeric structures, the stem cells provide a source of stem cells to engineer 3D tissues for cartilage repair. In this study, we have shown human IPFP-ASCs seeded onto 3D printed chitosan scaffolds can undergo chondrogenesis using TGFβ3 and BMP6. By week 4, a pearlescent, cartilage-like matrix had formed that penetrated the top layers of the chitosan scaffold forming a 'cap' on the scaffold. Chondrocytic morphology showed typical cells encased in extracellular matrix which stained positively with toluidine blue. Immunohistochemistry demonstrated positive staining for collagen type II and cartilage proteoglycans, as well as collagen type I. Real time PCR analysis showed up-regulation of collagen type II, aggrecan and SOX9 genes when IPFP-ASCs were stimulated by TGFβ3 and BMP6. Thus, IPFP-ASCs can successfully undergo chondrogenesis using TGFβ3 and BMP6 and the cartilage-like tissue that forms on the surface of 3D-printed chitosan scaffold may prove useful as an osteochondral graft.

Acute Phosphate Restriction Leads to Impaired Fracture Healing and Resistance to BMP-2

PubMed Central

Wigner, Nathan A; Luderer, Hilary F; Cox, Megan K; Sooy, Karen; Gerstenfeld, Louis C; Demay, Marie B

2010-01-01

Hypophosphatemia leads to rickets and osteomalacia, the latter of which results in decreased biomechanical integrity of bones, accompanied by poor fracture healing. Impaired phosphate-dependent apoptosis of hypertrophic chondrocytes is the molecular basis for rickets. However, the underlying pathophysiology of impaired fracture healing has not been characterized previously. To address the role of phosphate in fracture repair, mice were placed on a phosphate-restricted diet 2 days prior to or 3 days after induction of a mid-diaphyseal femoral fracture to assess the effects of phosphate deficiency on the initial recruitment of mesenchymal stem cells and their subsequent differentiation. Histologic and micro-computed tomographic (µCT) analyses demonstrated that both phosphate restriction models dramatically impaired fracture healing primarily owing to a defect in differentiation along the chondrogenic lineage. Based on Sox9 and Sox5 mRNA levels, neither the initial recruitment of cells to the callus nor their lineage commitment was effected by hypophosphatemia. However, differentiation of these cells was impaired in association with impaired bone morphogenetic protein (BMP) signaling. In vivo ectopic bone-formation assays and in vitro investigations in ST2 stromal cells confirmed that phosphate restriction leads to BMP-2 resistance. Marrow ablation studies demonstrate that hypophosphatemia has different effects on injury-induced intramembranous bone formation compared with endochondral bone formation. Thus phosphate plays an important role in the skeleton that extends beyond mineralized matrix formation and growth plate maturation and is critical for endochondral bone repair. © 2010 American Society for Bone and Mineral Research. PMID:19839770

Effective genetic modification and differentiation of hMSCs upon controlled release of rAAV vectors using alginate/poloxamer composite systems.

PubMed

Díaz-Rodríguez, P; Rey-Rico, A; Madry, H; Landin, M; Cucchiarini, M

2015-12-30

Viral vectors are common tools in gene therapy to deliver foreign therapeutic sequences in a specific target population via their natural cellular entry mechanisms. Incorporating such vectors in implantable systems may provide strong alternatives to conventional gene transfer procedures. The goal of the present study was to generate different hydrogel structures based on alginate (AlgPH155) and poloxamer PF127 as new systems to encapsulate and release recombinant adeno-associated viral (rAAV) vectors. Inclusion of rAAV in such polymeric capsules revealed an influence of the hydrogel composition and crosslinking temperature upon the vector release profiles, with alginate (AlgPH155) structures showing the fastest release profiles early on while over time vector release was more effective from AlgPH155+PF127 [H] capsules crosslinked at a high temperature (50°C). Systems prepared at room temperature (AlgPH155+PF127 [C]) allowed instead to achieve a more controlled release profile. When tested for their ability to target human mesenchymal stem cells, the different systems led to high transduction efficiencies over time and to gene expression levels in the range of those achieved upon direct vector application, especially when using AlgPH155+PF127 [H]. No detrimental effects were reported on either cell viability or on the potential for chondrogenic differentiation. Inclusion of PF127 in the capsules was also capable of delaying undesirable hypertrophic cell differentiation. These findings are of promising value for the further development of viral vector controlled release strategies. Copyright © 2015 Elsevier B.V. All rights reserved.

An overview of cartilage tissue engineering.

PubMed

Kim, H W; Han, C D

2000-12-01

Articular cartilage regeneration refers to the formation of new tissue that is indistinguishable from the native articular cartilage with respect to zonal organization, biochemical composition, and mechanical properties. Due to a limited capacity to repair cartilage, scar tissue frequently has a poorly organized structure and lacks the functional characteristics of normal cartilage. The degree of success to date achieved using a purely cell- or biological-based approach has been modest. Potentially the development of a hybrid strategy, whereby, chondrocytes or chondrogenic stem cells are combined with a matrix, making cartilage in vitro, which is then subsequently transplanted, offers a route towards a new successful treatment modality. The success of this approach depends upon the material being biocompatible, processable into a suitable three-dimensional structure and eventually biodegradable without harmful effects. In addition, the material should have a sufficient porosity to facilitate high cell loading and tissue ingrowth, and it should be able to support cell proliferation, differentiation, and function. The cell-polymer-bioreactor system provides a basis for studying the structural and functional properties of the cartilaginous matrix during its development, because tissue concentrations of glycosaminoglycan and collagen can be modulated by altering the conditions of tissue cultivation.

Retinoic acid, hemin and hexamethylen bisacetamide interference with "in vitro" differentiation of chick embryo chondrocytes.

PubMed

Manduca, P; Abelmoschi, M L

1992-01-01

We have investigated the effect of all-trans Retinoic acid, and of substances (Hemine and Hexamethylene bisacetamide) which interfere with "in vitro" differentiation of mesenchyme derived cell lineages on the expression of specific markers of hyperthrophy in "in vitro" differentiating chick embryo chondrocytes. (Castagnola P., et al., 1986). Continuous treatment of chondrogenic cells in conditions allowing differentiation "in vitro" with Retinoic acid resulted in persistence of type I collagen synthesis and in lack of type X collagen and Ch 21 protein expression. Hemin treated cells secreted a reduced amount of type X collagen. HMBA treatment inhibited type X collagen expression and caused reduction of the ratio between type II collagen and Ch 21 synthesized. The data indicate an independent regulation of these markers during chondrocyte differentiation.

Stem cell-based tissue-engineering for treatment of meniscal tears in the avascular zone.

PubMed

Zellner, Johannes; Hierl, Katja; Mueller, Michael; Pfeifer, Christian; Berner, Arne; Dienstknecht, Thomas; Krutsch, Werner; Geis, Sebastian; Gehmert, Sebastian; Kujat, Richard; Dendorfer, Sebastian; Prantl, Lukas; Nerlich, Michael; Angele, Peter

2013-10-01

Meniscal tears in the avascular zone have a poor self-healing potential, however partial meniscectomy predisposes the knee for early osteoarthritis. Tissue engineering with mesenchymal stem cells and a hyaluronan collagen based scaffold is a promising approach to repair meniscal tears in the avascular zone. 4 mm longitudinal meniscal tears in the avascular zone of lateral menisci of New Zealand White Rabbits were performed. The defect was left empty, sutured with a 5-0 suture or filled with a hyaluronan/collagen composite matrix without cells, with platelet rich plasma or with autologous mesenchymal stem cells. Matrices with stem cells were in part precultured in chondrogenic medium for 14 days prior to the implantation. Menisci were harvested at 6 and 12 weeks. The developed repair tissue was analyzed macroscopically, histologically and biomechanically. Untreated defects, defects treated with suture alone, with cell-free or with platelet rich plasma seeded implants showed a muted fibrous healing response. The implantation of stem cell-matrix constructs initiated fibrocartilage-like repair tissue, with better integration and biomechanical properties in the precultured stem cell-matrix group. A hyaluronan-collagen based composite scaffold seeded with mesenchymal stem cells is more effective in the repair avascular meniscal tear with stable meniscus-like tissue and to restore the native meniscus. Copyright © 2013 Wiley Periodicals, Inc., a Wiley Company.

Ajuga decumbens stimulates mesenchymal stem cell differentiation and regenerates cartilage in a rabbit osteoarthritis model

PubMed Central

Sawada, Yoko; Sugimoto, Atsushi; Osaki, Tomohiro; Okamoto, Yoshiharu

2018-01-01

In a previous study by our group, Ajuga decumbens extract (ADE) was demonstrated to decrease the number of osteoclasts in subchondral bone and to have a synergistic effect with glucosamine in improving cartilaginous injuries in a rabbit model of osteoarthritis. In the present study, a concentrate of the useful fraction of ADE, termed extra ADE (EADE), which includes higher concentrations of the active component 20-hydroxyecdysone, was evaluated for its efficacy to accelerate the healing of experimental cartilage injury. Cartilage injuries were surgically induced in rabbits by creating three holes; one in the articular cartilage of the medial trochlea and two in the trochlear sulcus of the distal femur. The rabbits were divided into the following four groups (n=3 in each): Control, ADE (0.5 g/kg), low dosage EADE (0.05 g/kg; low EADE) and high dosage EADE (0.5 g/kg; high EADE). ADE and EADE were dissolved in tap water and each dosage was orally administered every day for 3 weeks. At the end of the experimental period, histological analysis indicated that the cartilage matrix was regenerated in the low and high EADE groups. On counting of cells in the histological specimens, it was determined that the mean number of osteoclasts per 100 osteoblasts in subchondral bone was lower in the high EADE group compared with the control group. Furthermore, the results indicated that treatment with EADE (1–100 µg/ml) stimulated chondrogenic differentiation of mesenchymal stem cells and induced proteoglycan production to a greater extent than the control in vitro. EADE treatment (10 and 100 µg/ml) was also observed to significantly attenuate interleukin-1β-induced prostaglandin E2 production in chondrocytes (P<0.05). In summary, the results of the present study suggest that EADE may have greater curative effects on bone injury compared with the currently used therapeutic ADE. PMID:29725360

Different osteochondral potential of clonal cell lines derived from adult human trabecular bone.

PubMed

Osyczka, Anna M; Nöth, Ulrich; Danielson, Keith G; Tuan, Rocky S

2002-06-01

Cells derived from human trabecular bones have been shown to have multipotential differentiation ability along osteogenic, chondrogenic, and adipogenic lineages. In this study, we have derived two clonal sublines of human trabecular bone cells by means of stable transduction with human papilloma virus E6/E7 genes. Our results showed that these clonal sublines differ in their osteochondral potential, but are equally adipogenic, indicative of the heterogeneous nature of the parental cell population. The availability of these cell lines should be useful for the analysis of the mechanisms regulating the differentiation of adult mesenchymal progenitor cells.

Biofabrication of soft tissue templates for engineering the bone-ligament interface.

PubMed

Harris, Ella; Liu, Yurong; Cunniffe, Grainne; Morrissey, David; Carroll, Simon; Mulhall, Kevin; Kelly, Daniel J

2017-10-01

Regenerating damaged tissue interfaces remains a significant clinical challenge, requiring recapitulation of the structure, composition, and function of the native enthesis. In the ligament-to-bone interface, this region transitions from ligament to fibrocartilage, to calcified cartilage and then to bone. This gradation in tissue types facilitates the transfer of load between soft and hard structures while minimizing stress concentrations at the interface. Previous attempts to engineer the ligament-bone interface have utilized various scaffold materials with an array of various cell types and/or biological cues. The primary goal of this study was to engineer a multiphased construct mimicking the ligament-bone interface by driving differentiation of a single population of mesenchymal stem cells (MSCs), seeded within blended fibrin-alginate hydrogels, down an endochondral, fibrocartilaginous, or ligamentous pathway through spatial presentation of growth factors along the length of the construct within a custom-developed, dual-chamber culture system. MSCs within these engineered constructs demonstrated spatially distinct regions of differentiation, adopting either a cartilaginous or ligamentous phenotype depending on their local environment. Furthermore, there was also evidence of spatially defined progression toward an endochondral phenotype when chondrogenically primed MSCs within this construct were additionally exposed to hypertrophic cues. The study demonstrates the feasibility of engineering spatially complex soft tissues within a single MSC laden hydrogel through the defined presentation of biochemical cues. This novel approach represents a new strategy for engineering the ligament-bone interface. Biotechnol. Bioeng. 2017;114: 2400-2411. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Molecular Study of a Hoxa2 Gain-of-Function in Chondrogenesis: A Model of Idiopathic Proportionate Short Stature

PubMed Central

Deprez, Pierre M. L.; Nichane, Miloud G.; Lengelé, Benoît G.; Rezsöhazy, René; Nyssen-Behets, Catherine

2013-01-01

In a previous study using transgenic mice ectopically expressing Hoxa2 during chondrogenesis, we associated the animal phenotype to human idiopathic proportionate short stature. Our analysis showed that this overall size reduction was correlated with a negative influence of Hoxa2 at the first step of endochondral ossification. However, the molecular pathways leading to such phenotype are still unknown. Using protein immunodetection and histological techniques comparing transgenic mice to controls, we show here that the persistent expression of Hoxa2 in chondrogenic territories provokes a general down-regulation of the main factors controlling the differentiation cascade, such as Bapx1, Bmp7, Bmpr1a, Ihh, Msx1, Pax9, Sox6, Sox9 and Wnt5a. These data confirm the impairment of chondrogenic differentiation by Hoxa2 overexpression. They also show a selective effect of Hoxa2 on endochondral ossification processes since Gdf5 and Gdf10, and Bmp4 or PthrP were up-regulated and unmodified, respectively. Since Hoxa2 deregulation in mice induces a proportionate short stature phenotype mimicking human idiopathic conditions, our results give an insight into understanding proportionate short stature pathogenesis by highlighting molecular factors whose combined deregulation may be involved in such a disease. PMID:24129174

Secretion of immunoregulatory cytokines by mesenchymal stem cells

PubMed Central

Kyurkchiev, Dobroslav; Bochev, Ivan; Ivanova-Todorova, Ekaterina; Mourdjeva, Milena; Oreshkova, Tsvetelina; Belemezova, Kalina; Kyurkchiev, Stanimir

2014-01-01

According to the minimal criteria of the International Society of Cellular Therapy, mesenchymal stem cells (MSCs) are a population of undifferentiated cells defined by their ability to adhere to plastic surfaces when cultured under standard conditions, express a certain panel of phenotypic markers and can differentiate into osteogenic, chondrogenic and adipogenic lineages when cultured in specific inducing media. In parallel with their major role as undifferentiated cell reserves, MSCs have immunomodulatory functions which are exerted by direct cell-to-cell contacts, secretion of cytokines and/or by a combination of both mechanisms. There are no convincing data about a principal difference in the profile of cytokines secreted by MSCs isolated from different tissue sources, although some papers report some quantitative but not qualitative differences in cytokine secretion. The present review focuses on the basic cytokines secreted by MSCs as described in the literature by which the MSCs exert immunodulatory effects. It should be pointed out that MSCs themselves are objects of cytokine regulation. Hypothetical mechanisms by which the MSCs exert their immunoregulatory effects are also discussed in this review. These mechanisms may either influence the target immune cells directly or indirectly by affecting the activities of predominantly dendritic cells. Chemokines are also discussed as participants in this process by recruiting cells of the immune systems and thus making them targets of immunosuppression. This review aims to present and discuss the published data and the personal experience of the authors regarding cytokines secreted by MSCs and their effects on the cells of the immune system. PMID:25426252

Importance of Sox2 in maintenance of cell proliferation and multipotency of mesenchymal stem cells in low-density culture.

PubMed

Yoon, D S; Kim, Y H; Jung, H S; Paik, S; Lee, J W

2011-10-01

This study has aimed to repopulate 'primitive' cells from late-passage mesenchymal stem cells (MSCs) of poor multipotentiality and low cell proliferation rate, by simply altering plating density. Effects of low density culture compared t high density culture on late-passage bone marrow (BM)-derived MSCs and pluripotency markers of multipotentiality were investigated. Cell proliferation, gene expression, RNA interference and differentiation potential were assayed. We repopulated 'primitive' cells by replating late-passage MSCs at low density (17 cells/cm(2) ) regardless of donor age. Repopulated MSCs from low-density culture were smaller cells with spindle shaped morphology compared to MSCs from high-density culture. The latter had enhanced colony-forming ability, proliferation rate, and adipogenic and chondrogenic potential. Strong expression of osteogenic-related genes (Cbfa1, Dlx5, alkaline phosphatase and type Ι collagen) in late-passage MSCs was reduced by replating at low density, whereas expression of three pluripotency markers (Sox2, Nanog and Oct-4), Osterix and Msx2 reverted to levels of early-passage MSCs. Knockdown of Sox2 and Msx2 but not Nanog, using RNA interference, showed significant decrease in colony-forming ability. Specifically, knockdown of Sox2 significantly inhibited multipotentiality and cell proliferation. Our data suggest that plating density should be considered to be a critical factor for enrichment of 'primitive' cells from heterogeneous BM and that replicative senescence and multipotentiality of MSCs during in vitro expansion may be predominantly regulated through Sox2. © 2011 Blackwell Publishing Ltd.

Single-Stage Cartilage Repair Using Platelet-Rich Fibrin Scaffolds With Autologous Cartilaginous Grafts.

PubMed

Wong, Chin-Chean; Chen, Chih-Hwa; Chan, Wing P; Chiu, Li-Hsuan; Ho, Wei-Pin; Hsieh, Fon-Jou; Chen, You-Tzung; Yang, Tsung-Lin

2017-11-01

To avoid complicated procedures requiring in vitro chondrocyte expansion for cartilage repair, the development of a culture-free, 1-stage approach combining platelet-rich fibrin (PRF) and autologous cartilage grafts may be the solution. To develop a feasible 1-step procedure to combine PRF and autologous cartilage grafts for articular chondral defects. Controlled laboratory study Methods: The chemotactic effects of PRF on chondrocytes harvested from the primary culture of rabbit cartilage were evaluated in vitro and ex vivo. The rabbit chondrocytes were cultured with different concentrations of PRF media and evaluated for their cell proliferation, chondrogenic gene expression, cell viability, and extracellular matrix synthesis abilities. For the in vivo study, the chondral defects were created on established animal models of rabbits. The gross anatomy, histology, and objective scores were evaluated to validate the treatment results. PRF improved the chemotaxis, proliferation, and viability of the cultured chondrocytes. The gene expression of the chondrogenic markers, including type II collagen and aggrecan, revealed that PRF induced the chondrogenic differentiation of cultured chondrocytes. PRF increased the formation and deposition of the cartilaginous matrix produced by cultured chondrocytes. The efficacy of PRF on cell viability was comparable with that of fetal bovine serum. In animal disease models, morphologic, histological, and objectively quantitative evaluation demonstrated that PRF combined with cartilage granules was feasible in facilitating chondral repair. PRF enhances the migration, proliferation, viability, and differentiation of chondrocytes, thus showing an appealing capacity for cartilage repair. The data altogether provide evidence to confirm the feasibility of 1-stage, culture-free method of combining PRF and autologous cartilage graft for repairing articular chondral defects. The single-stage, culture-free method of combining PRF and autologous cartilage is useful for repairing articular chondral defects. These advantages benefit clinical translation by simplifying and potentiating the efficacy of autologous cartilage transplantation.

The Importance of Connexin Hemichannels During Chondroprogenitor Cell Differentiation in Hydrogel Versus Microtissue Culture Models

PubMed Central

Schrobback, Karsten; Klein, Travis Jacob

2015-01-01

Appropriate selection of scaffold architecture is a key challenge in cartilage tissue engineering. Gap junction-mediated intercellular contacts play important roles in precartilage condensation of mesenchymal cells. However, scaffold architecture could potentially restrict cell–cell communication and differentiation. This is particularly important when choosing the appropriate culture platform as well as scaffold-based strategy for clinical translation, that is, hydrogel or microtissues, for investigating differentiation of chondroprogenitor cells in cartilage tissue engineering. We, therefore, studied the influence of gap junction-mediated cell–cell communication on chondrogenesis of bone marrow-derived mesenchymal stromal cells (BM-MSCs) and articular chondrocytes. Expanded human chondrocytes and BM-MSCs were either (re-) differentiated in micromass cell pellets or encapsulated as isolated cells in alginate hydrogels. Samples were treated with and without the gap junction inhibitor 18-α glycyrrhetinic acid (18αGCA). DNA and glycosaminoglycan (GAG) content and gene expression levels (collagen I/II/X, aggrecan, and connexin 43) were quantified at various time points. Protein localization was determined using immunofluorescence, and adenosine-5′-triphosphate (ATP) was measured in conditioned media. While GAG/DNA was higher in alginate compared with pellets for chondrocytes, there were no differences in chondrogenic gene expression between culture models. Gap junction blocking reduced collagen II and extracellular ATP in all chondrocyte cultures and in BM-MSC hydrogels. However, differentiation capacity was not abolished completely by 18αGCA. Connexin 43 levels were high throughout chondrocyte cultures and peaked only later during BM-MSC differentiation, consistent with the delayed response of BM-MSCs to 18αGCA. Alginate hydrogels and microtissues are equally suited culture platforms for the chondrogenic (re-)differentiation of expanded human articular chondrocytes and BM-MSCs. Therefore, reducing direct cell–cell contacts does not affect in vitro chondrogenesis. However, blocking gap junctions compromises cell differentiation, pointing to a prominent role for hemichannel function in this process. Therefore, scaffold design strategies that promote an increasing distance between single chondroprogenitor cells do not restrict their differentiation potential in tissue-engineered constructs. PMID:25693425

The importance of connexin hemichannels during chondroprogenitor cell differentiation in hydrogel versus microtissue culture models.

PubMed

Schrobback, Karsten; Klein, Travis Jacob; Woodfield, Tim B F

2015-06-01

Appropriate selection of scaffold architecture is a key challenge in cartilage tissue engineering. Gap junction-mediated intercellular contacts play important roles in precartilage condensation of mesenchymal cells. However, scaffold architecture could potentially restrict cell-cell communication and differentiation. This is particularly important when choosing the appropriate culture platform as well as scaffold-based strategy for clinical translation, that is, hydrogel or microtissues, for investigating differentiation of chondroprogenitor cells in cartilage tissue engineering. We, therefore, studied the influence of gap junction-mediated cell-cell communication on chondrogenesis of bone marrow-derived mesenchymal stromal cells (BM-MSCs) and articular chondrocytes. Expanded human chondrocytes and BM-MSCs were either (re-) differentiated in micromass cell pellets or encapsulated as isolated cells in alginate hydrogels. Samples were treated with and without the gap junction inhibitor 18-α glycyrrhetinic acid (18αGCA). DNA and glycosaminoglycan (GAG) content and gene expression levels (collagen I/II/X, aggrecan, and connexin 43) were quantified at various time points. Protein localization was determined using immunofluorescence, and adenosine-5'-triphosphate (ATP) was measured in conditioned media. While GAG/DNA was higher in alginate compared with pellets for chondrocytes, there were no differences in chondrogenic gene expression between culture models. Gap junction blocking reduced collagen II and extracellular ATP in all chondrocyte cultures and in BM-MSC hydrogels. However, differentiation capacity was not abolished completely by 18αGCA. Connexin 43 levels were high throughout chondrocyte cultures and peaked only later during BM-MSC differentiation, consistent with the delayed response of BM-MSCs to 18αGCA. Alginate hydrogels and microtissues are equally suited culture platforms for the chondrogenic (re-)differentiation of expanded human articular chondrocytes and BM-MSCs. Therefore, reducing direct cell-cell contacts does not affect in vitro chondrogenesis. However, blocking gap junctions compromises cell differentiation, pointing to a prominent role for hemichannel function in this process. Therefore, scaffold design strategies that promote an increasing distance between single chondroprogenitor cells do not restrict their differentiation potential in tissue-engineered constructs.

Human Urine-Derived Stem Cells: Potential for Cell-Based Therapy of Cartilage Defects

PubMed Central

Chen, Long; Li, Lang; Xing, Fei; Peng, Jing; Peng, Kun; Wang, Yuanzheng

2018-01-01

Stem cell therapy is considered an optimistic approach to replace current treatments for cartilage defects. Recently, human urine-derived stem cells (hUSCs), which are isolated from the urine, are studied as a promising candidate for many tissue engineering therapies due to their multipotency and sufficient proliferation activities. However, it has not yet been reported whether hUSCs can be employed in cartilage defects. In this study, we revealed that induced hUSCs expressed chondrogenic-related proteins, including aggrecan and collagen II, and their gene expression levels were upregulated in vitro. Moreover, we combined hUSCs with hyaluronic acid (HA) and injected hUSCs-HA into a rabbit knee joint with cartilage defect. Twelve weeks after the injection, the histologic analyses (HE, toluidine blue, and Masson trichrome staining), immunohistochemistry (aggrecan and collagen II), and histologic grade of the sample indicated that hUSCs-HA could stimulate much more neocartilage formation compared with hUSCs alone, pure HA, and saline, which only induced the modest cartilage regeneration. In this study, we demonstrated that hUSCs could be a potential cell source for stem cell therapies to treat cartilage-related defects in the future. PMID:29765413

Fabrication of human hair keratin/jellyfish collagen/eggshell-derived hydroxyapatite osteoinductive biocomposite scaffolds for bone tissue engineering: From waste to regenerative medicine products.

PubMed

Arslan, Yavuz Emre; Sezgin Arslan, Tugba; Derkus, Burak; Emregul, Emel; Emregul, Kaan C

2017-06-01

In the present study, we aimed at fabricating an osteoinductive biocomposite scaffold using keratin obtained from human hair, jellyfish collagen and eggshell-derived nano-sized spherical hydroxyapatite (nHA) for bone tissue engineering applications. Keratin, collagen and nHA were characterized with the modified Lowry method, free-sulfhydryl groups and hydroxyproline content analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR) and thermal gravimetric analysis (TGA) which confirmed the success of the extraction and/or isolation processes. Human adipose mesenchymal stem cells (hAMSCs) were isolated and the cell surface markers were characterized via flow cytometry analysis in addition to multilineage differentiation capacity. The undifferentiated hAMSCs were highly positive for CD29, CD44, CD73, CD90 and CD105, but were not seen to express hematopoietic cell surface markers such as CD14, CD34 and CD45. The cells were successfully directed towards osteogenic, chondrogenic and adipogenic lineages in vitro. The microarchitecture of the scaffolds and cell attachment were evaluated using scanning electron microscopy (SEM). The cell viability on the scaffolds was assessed by the MTT assay which revealed no evidence of cytotoxicity. The osteogenic differentiation of hAMSCs on the scaffolds was determined histologically using alizarin red S, osteopontin and osteonectin stainings. Early osteogenic differentiation markers of hAMSCs were significantly expressed on the collagen-keratin-nHA scaffolds. In conclusion, it is believed that collagen-keratin-nHA osteoinductive biocomposite scaffolds have the potential of being used in bone tissue engineering. Copyright © 2017 Elsevier B.V. All rights reserved.

CARTILAGE OLIGOMERIC MATRIX PROTEIN ENHANCES MATRIX ASSEMBLY DURING CHONDROGENESIS OF HUMAN MESENCHYMAL STEM CELLS

PubMed Central

Haleem-Smith, Hana; Calderon, Raul; Song, Yingjie; Tuan, Rocky S.; Chen, Faye H.

2011-01-01

Cartilage oligomeric matrix protein/thrombospondin-5 (COMP/TSP5) is an abundant cartilage extracellular matrix (ECM) protein that interacts with major cartilage ECM components, including aggrecan and collagens. To test our hypothesis that COMP/TSP5 functions in the assembly of the ECM during cartilage morphogenesis, we have employed mesenchymal stem cell (MSC) chondrogenesis in vitro as a model to examine the effects of COMP over-expression on neo-cartilage formation. Human bone marrow-derived MSCs were transfected with either full-length COMP cDNA or control plasmid, followed by chondrogenic induction in three-dimensional pellet or alginate-hydrogel culture. MSC chondrogenesis and ECM production was estimated based on quantitation of sulfated glycosaminoglycan (sGAG) accumulation, immunohistochemistry of the presence and distribution of cartilage ECM proteins, and real-time RT-PCR analyis of mRNA expression of cartilage markers. Our results showed that COMP over-expression resulted in increased total sGAG content during the early phase of MSC chondrogenesis, and increased immuno-detectable levels of aggrecan and collagen type II in the ECM of COMP-transfected pellet and alginate cultures, indicating more abundant cartilaginous matrix. COMP transfection did not significantly increase the transcript levels of the early chondrogenic marker, Sox9, or aggrecan, suggesting that enhancement of MSC cartilage ECM was effected at post-transcriptional levels. These findings strongly suggest that COMP functions in mesenchymal chondrogenesis by enhancing cartilage ECM organization and assembly. The action of COMP is most likely mediated not via direct changes in cartilage matrix gene expression but via interactions of COMP with other cartilage ECM proteins, such as aggrecan and collagens, that result in enhanced assembly and retention. PMID:22095699

Mesenchymal Stem/Progenitor Cells Derived from Articular Cartilage, Synovial Membrane and Synovial Fluid for Cartilage Regeneration: Current Status and Future Perspectives.

PubMed

Huang, Yi-Zhou; Xie, Hui-Qi; Silini, Antonietta; Parolini, Ornella; Zhang, Yi; Deng, Li; Huang, Yong-Can

2017-10-01

Large articular cartilage defects remain an immense challenge in the field of regenerative medicine because of their poor intrinsic repair capacity. Currently, the available medical interventions can relieve clinical symptoms to some extent, but fail to repair the cartilaginous injuries with authentic hyaline cartilage. There has been a surge of interest in developing cell-based therapies, focused particularly on the use of mesenchymal stem/progenitor cells with or without scaffolds. Mesenchymal stem/progenitor cells are promising graft cells for tissue regeneration, but the most suitable source of cells for cartilage repair remains controversial. The tissue origin of mesenchymal stem/progenitor cells notably influences the biological properties and therapeutic potential. It is well known that mesenchymal stem/progenitor cells derived from synovial joint tissues exhibit superior chondrogenic ability compared with those derived from non-joint tissues; thus, these cell populations are considered ideal sources for cartilage regeneration. In addition to the progress in research and promising preclinical results, many important research questions must be answered before widespread success in cartilage regeneration is achieved. This review outlines the biology of stem/progenitor cells derived from the articular cartilage, the synovial membrane, and the synovial fluid, including their tissue distribution, function and biological characteristics. Furthermore, preclinical and clinical trials focusing on their applications for cartilage regeneration are summarized, and future research perspectives are discussed.

Sonic hedgehog is required for survival of both myogenic and chondrogenic somitic lineages.

PubMed

Teillet, M; Watanabe, Y; Jeffs, P; Duprez, D; Lapointe, F; Le Douarin, N M

1998-06-01

In vertebrates, the medial moieties of the somites give rise to the vertebrae and epaxial muscles, which develop in close relationship with the axial organs, neural tube and notochord. The lateral moieties contribute to the ribs and to limb and body wall muscles (hypaxial muscles) after a phase of lateral and ventral migration. Surgical ablation of the neural tube and notochord in the chick embryo during segmentation and early differentiation of the somites (day 2 of incubation) does not affect primary development of the hypaxial muscles, but leads to a complete absence of epaxial muscles, vertebrae and ribs, due to cell death in the somites. Here we demonstrate that cell death, which occurs within 24 hours of excision of the axial organs, affects both myogenic and chondrogenic cell lineages defined, respectively, by the expression of MyoD and Pax-1 genes. In contrast, Pax-3 transcripts, normally present in cells giving rise to hypaxial muscles, are preserved in the excised embryos. Backgrafting either the ventral neural tube or the notochord allows survival of MyoD- and Pax-1-expressing cells. Similarly, Sonic hedgehog-producing cells grafted in place of axial organs also rescue MyoD- and Pax-1-expressing cells from death and allow epaxial muscles, ribs and vertebrae to undergo organogenesis. These results demonstrate that the ventral neural tube and the notochord promote the survival of both myogenic and chondrogenic cell lineages in the somites and that this action is mediated by Sonic hedgehog.

Human acellular cartilage matrix powders as a biological scaffold for cartilage tissue engineering with synovium-derived mesenchymal stem cells.

PubMed

Chang, Chih-Hung; Chen, Chia-Chun; Liao, Cheng-Hao; Lin, Feng-Huei; Hsu, Yuan-Ming; Fang, Hsu-Wei

2014-07-01

In our previous study, we found that cartilage fragments from osteoarthritic knee promoted chondrogenesis of mesenchymal stem cells. In this study, we further transformed the cartilage tissues into acellular cartilage matrix (ACM) and explored the feasibility of using ACM as a biological scaffold. Nonworn parts of cartilage tissues were obtained during total knee arthroplasty (TKA) surgery and were successfully fabricated into ACM powders. The ACM powders and human synovium-derived mesenchymal stem cells (SMSCs) were mixed into collagen gel for in vitro culture. Histological results showed a synergistic effect of ACM powders and chondrogenic growth factors in the formation of engineered cartilage. The findings of real-time polymerase chain reaction (PCR) suggested that ACM powders had the potential of promoting type II collagen gene expression in the growth factors-absent environment. Moreover, with growth factors induction, the ACM powders could reduce the hypertrophy in chondrogenesis of SMSCs. In summary, ACM powders could serve as a functional scaffold that benefited the chondrogenesis of SMSCs for cartilage tissue engineering. © 2013 Wiley Periodicals, Inc.

Isolation and biological characteristic evaluation of a novel type of cartilage stem/progenitor cell derived from Small‑tailed Han sheep embryos.

PubMed

Ma, Caiyun; Lu, Tengfei; Wen, Hebao; Zheng, Yanjie; Han, Xiao; Ji, Xongda; Guan, Weijun

2018-07-01

Cartilage stem/progenitor cells (CSPCs) are a novel stem cell population and function as promising therapeutic candidates for cell‑based cartilage repair. Until now, numerous existing research materials have been obtained from humans, horses, cows and other mammals, but rarely from sheep. In the present study, CSPCs with potential applications in repairing tissue damage and cell‑based therapy were isolated from 45‑day‑old Small‑tailed Han Sheep embryos, and examined at the cellular and molecular level. The expression level of characteristic surface markers of the fetal sheep CSPCs were also evaluated by immunofluorescence, reverse transcription‑polymerase chain reaction analysis and flow cytometric assays. Biological growth curves were drawn in accordance with cell numbers. Additionally, karyotype analysis showed no marked differences in the in vitro cultured CSPCs and they were genetically stable among different passages. The CSPCs were also capable of adipogenic, osteogenic and chondrogenic lineage progression under the appropriate induction medium in vitro. Together, these findings provide a theoretical basis and experimental evidence for cellular transplant therapy in tissue engineering.

Biphasic influence of dexamethasone exposure on embryonic vertebrate skeleton development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheng, Xin; Chen, Jian-long; Ma, Zheng-lai

Dexamethasone (Dex) has anti-inflammatory and immunomodulatory properties against many conditions. There is a potential teratogenic risk, however, for pregnant women receiving Dex treatment. It has been claimed that Dex exposure during pregnancy could affect osteogenesis in the developing embryo, which still remains highly controversial. In this study, we employed chick embryos to investigate the effects of Dex exposure on skeletal development using combined in vivo and in vitro approach. First, we demonstrated that Dex (10{sup −8}–10{sup −6} μmol/egg) exposure resulted in a shortening of the developing long bones of chick embryos, and it accelerated the deposition of calcium salts. Secondly,more » histological analysis of chick embryo phalanxes exhibited Dex exposure inhibited the proliferation of chondrocytes, increased apoptosis of chondrocytes and osteocytes, and led to atypical arranged hypertrophic chondrocytes. The expression of genes related to skeletogenesis was also analyzed by semi-quantitative RT-PCR. The expression of ALP, Col1a2 and Col2a1 was decreased in the Dex treated phalanxes. A detectable increase was observed in Runx-2 and Mmp-13 expression. We next examined how Dex affected the different stages of skeletogenesis in vitro. Utilizing limb bud mesenchyme micromass cultures, we determined that Dex exposure exerted no effect on apoptosis but impaired chondrogenic cell proliferation. Interestingly, low dose of Dex moderately prompted nodule formation as revealed by alcian blue staining, but higher doses of Dex significantly inhibited similar chondrogenic differentiation. Dex exposure did not induce apoptosis when the chondrogenic precursors were still at the mesenchymal stage, however, cell viability was suppressed when the mesenchyme differentiated into chondrocytes. Alizarin red staining revealed that the capacity to form mineralized bone nodules was correspondingly enhanced as Dex concentrations increased. The mRNA level of Sox-9 was slightly increased in mesenchymal cell mass treated by low concentration of Dex. Mmp-13 expression was obviously up-regulated by Dex in both mesenchymal cells and primary chondrocyte cultures. And Col10a1 expression was also increased by Dex exposure in chondrocyte. In summary, we have revealed that different concentrations of Dex exposure during early gestation could exert a biphasic effect on vertebrate skeletal development. - Highlights: • Chick embryos occurred shortening of the long bone following Dex exposure. • Dex suppressed chondrocytes proliferation and promoted apoptosis. • Dex exposure decreased ALP production and up-regulated Runx-2 and Mmp-13. • Dex exhibited biphasic effects on chondrogenic proliferation and nodule formation. • The hypertrophy and ossification were accelerated by Dex both in vivo and in vitro.« less