Electrospun biomimetic scaffold of hydroxyapatite/chitosan supports enhanced osteogenic differentiation of mMSCs

NASA Astrophysics Data System (ADS)

Peng, Hongju; Yin, Zi; Liu, Huanhuan; Chen, Xiao; Feng, Bei; Yuan, Huihua; Su, Bo; Ouyang, Hongwei; Zhang, Yanzhong

2012-12-01

Engaging functional biomaterial scaffolds to regulate stem cell differentiation has drawn a great deal of attention in the tissue engineering and regenerative medicine community. In this study, biomimetic composite nanofibrous scaffolds of hydroxyapatite/chitosan (HAp/CTS) were prepared to investigate their capacity for inducing murine mesenchymal stem cells (mMSCs) to differentiate into the osteogenic lineage, in the absence and presence of an osteogenic supplementation (i.e., ascorbic acid, β-glycerol phosphate, and dexamethasone), respectively. Using electrospun chitosan (CTS) nanofibrous scaffolds as the control, cell morphology, growth, specific osteogenic genes expression, and quantified proteins secretion on the HAp/CTS scaffolds were sequentially examined and assessed. It appeared that the HAp/CTS scaffolds supported better attachment and proliferation of the mMSCs. Most noteworthy was that in the absence of the osteogenic supplementation, expression of osteogenic genes including collagen I (Col I), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OCN) were significantly upregulated in mMSCs cultured on the HAp/CTS nanofibrous scaffolds. Also increased secretion of the osteogenesis protein markers of alkaline phosphatase and collagen confirmed that the HAp/CTS nanofibrous scaffold markedly promoted the osteogenic commitment in the mMSCs. Moreover, the presence of osteogenic supplementation proved an enhanced efficacy of mMSC osteogenesis on the HAp/CTS nanofibrous scaffolds. Collectively, this study demonstrated that the biomimetic nanofibrous HAp/CTS scaffolds could support and enhance the adhesion, proliferation, and particularly osteogenic differentiation of the mMSCs. It also substantiated the potential of using biomimetic nanofibrous scaffolds of HAp/CTS for functional bone repair and regeneration applications.

Vitamin C treatment promotes mesenchymal stem cell sheet formation and tissue regeneration by elevating telomerase activity.

PubMed

Wei, Fulan; Qu, Cunye; Song, Tieli; Ding, Gang; Fan, Zhipeng; Liu, Dayong; Liu, Yi; Zhang, Chunmei; Shi, Songtao; Wang, Songlin

2012-09-01

Cell sheet engineering has been developed as an alternative approach to improve mesenchymal stem cell-mediated tissue regeneration. In this study, we found that vitamin C (Vc) was capable of inducing telomerase activity in periodontal ligament stem cells (PDLSCs), leading to the up-regulated expression of extracellular matrix type I collagen, fibronectin, and integrin β1, stem cell markers Oct4, Sox2, and Nanog as well as osteogenic markers RUNX2, ALP, OCN. Under Vc treatment, PDLSCs can form cell sheet structures because of increased cell matrix production. Interestingly, PDLSC sheets demonstrated a significant improvement in tissue regeneration compared with untreated control dissociated PDLSCs and offered an effective treatment for periodontal defects in a swine model. In addition, bone marrow mesenchymal stem cell sheets and umbilical cord mesenchymal stem cell sheets were also well constructed using this method. The development of Vc-mediated mesenchymal stem cell sheets may provide an easy and practical approach for cell-based tissue regeneration. Copyright © 2011 Wiley Periodicals, Inc.

Potential dental pulp revascularization and odonto-/osteogenic capacity of a novel transplant combined with dental pulp stem cells and platelet-rich fibrin.

PubMed

Chen, Yong-Jin; Zhao, Yin-Hua; Zhao, Ya-Juan; Liu, Nan-Xia; Lv, Xin; Li, Qiang; Chen, Fa-Ming; Zhang, Min

2015-08-01

Our aim is to investigate the cytobiological effects of autologous platelet-rich fibrin (PRF) on dental pulp stem cells (DPSCs) and to explore the ectopic and orthotopic possibilities of dental pulp revascularization and pulp-dentin complex regeneration along the root canal cavities of the tooth by using a novel tissue-engineered transplant composed of cell-sheet fragments of DPSCs and PRF granules. Canine DPSCs were isolated and characterized by assaying their colony-forming ability and by determining their cell surface markers and osteogenic/adipogenic differentiation potential. The biological effects of autologous PRF on DPSCs, including cell proliferation, alkaline phosphatase (ALP) activity and odonto-/osteogenic gene expression, were then investigated and quantified. A novel transplant consisting of cell-sheet fragments of DPSCs and PRF granules was adopted to regenerate pulp-dentin-like tissues in the root canal, both subcutaneously in nude mice and in the roots of canines. PRF promoted the proliferation of DPSCs in a dose- and time-dependent manner and induced the differentiation of DPSCs to odonto-/osteoblastic fates by increasing the expression of the Alp, Dspp, Dmp1 and Bsp genes. Transplantation of the DPSC/PRF construct led both to a favorable regeneration of homogeneous and compact pulp-like tissues with abundantly distributed blood capillaries and to the deposition of regenerated dentin along the intracanal walls at 8 weeks post-operation. Thus, the application of DPSC/PRF tissue constructs might serve as a potential therapy in regenerative endodontics for pulp revitalization or revascularization.

Silk ionomers for encapsulation and differentiation of human MSCs

PubMed Central

Calabrese, Rossella; Kaplan, David L.

2012-01-01

The response of human bone marrow derived human mesenchymal stem cells (hMSCs) encapsulated in silk ionomer hydrogels was studied. Silk aqueous solutions with silk-poly-L-lysine or silk-poly-L-glutamate were formed into hydrogels via ultrasonication in situ with different net charges. hMSCs were encapsulated within the hydrogels and the impact of matrix charge was assessed over weeks in osteogenic, adipogenic and maintenance growth media. These modified silk charged polymers supported cell viability and proliferative potential, and the hMSCs were able to differentiate toward osteogenic or adipogenic lineages in the corresponding differentiation media. The silk/silk-poly-L-lysine hydrogels exhibited a positive effect on selective osteogenesis of hMSCs, inducing differentiation toward an osteogenic lineage even in the absence of osteogenic supplements, while also inhibiting adipogenesis. In contrast, silk/silk fibroin-poly-L-glutamate hydrogels supported both osteogenic and adipogenic differentiation of hMSCs when cultured under induction conditions. The results demonstrate the potential utility of silk-based ionomers in gel formats for hMSCs encapsulation and for directing hMSCs long term functional differentiation toward specific lineages. PMID:22824008

[Proliferation and osteogenic differentiation of mesenchymal stem cells in hydrogels of human blood plasma].

PubMed

Linero, Itali M; Doncel, Adriana; Chaparro, Orlando

2014-01-01

The use of mesenchymal stem cells in clinical practice has increased considerably in the last decade because they play a supporting role in the processes of tissue repair and regeneration, becoming the main tool of cell therapy for the treatment of diseases functionally affecting bone and cartilage tissue . To evaluate in vitro the proliferative and osteogenic differentiation ability of mesenchymal stem cells derived from human adipose tissue in a blood plasma hydrogel. Mesenchymal stem cells were obtained from human adipose tissue explants and characterized by flow cytometry. Their multipotentiality was demonstrated by their ability to differentiate to adipogenic and osteogenic lineages. Cell proliferation and osteogenic differentiation ability of the cells cultured in blood plasma hydrogels were also evaluated. Mesenchymal stem cells derived from human adipose tissue growing in human blood plasma hydrogels showed a pattern of proliferation similar to that of the cells cultured in monolayer and also maintained their ability to differentiate to osteogenic lineage. Human blood plasma hydrogels are a suitable support for proliferation and osteogenic differentiation of mesenchymal stem cells derived from human adipose tissue and provides a substrate that is autologous, biocompatible, reabsorbable, easy to use, potentially injectable and economic, which could be used as a successful strategy for the management and clinical application of cell therapy in regenerative medicine.

Synergistic effect of scaffold composition and dynamic culturing environment in multilayered systems for bone tissue engineering.

PubMed

Rodrigues, Márcia T; Martins, Albino; Dias, Isabel R; Viegas, Carlos A; Neves, Nuno M; Gomes, Manuela E; Reis, Rui L

2012-11-01

Bone extracellular matrix (ECM) is composed of mineralized collagen fibrils which support biological apatite nucleation that participates in bone outstanding properties. Understanding and mimicking bone morphological and physiological parameters at a biological scale is a major challenge in tissue engineering scaffolding. Using emergent (nano)technologies scaffold designing may be critically improved, enabling highly functional tissue substitutes for bone applications. This study aims to develop novel biodegradable composite scaffolds of tricalcium phosphate (TCPs) and electrospun nanofibers of poly(ϵ-caprolactone) (PCL), combining TCPs osteoconductivity with PCL biocompatibility and elasticity, mimicking bone structure and composition. We hypothesized that scaffolds with such structure/composition would stimulate the proliferation and differentiation of bone marrow stromal cells (BMSCs) towards the osteogenic phenotype. Composite scaffolds, developed by electrospining using consecutive stacked layers of PCL and TCPs, were characterized by FTIR spectroscopy, X-Ray diffraction and scanning electronic microscopy. Cellular behavior was assessed in goat BMSCs seeded onto composite scaffolds and cultured in static or dynamic conditions, using basal or osteogenic media during 7, 14 or 21 days. Cellular proliferation was quantified and osteogenic differentiation confirmed by alkaline phosphatase activity, alizarin red staining and immunocytochemistry for osteocalcin and collagen I. Results suggest that PCL-TCP scaffolds provide a 3D support for gBMSCs proliferation and osteogenic differentiation with production of ECM. TCPs positively stimulate the osteogenic process, especially under dynamic conditions, where PCL-TCP scaffolds are sufficient to promote osteogenic differentiation even in basal medium conditions. The enhancement of the osteogenic potential in dynamic conditions evidences the synergistic effect of scaffold composition and dynamic stimulation in gBMSCs osteogenic differentiation. Copyright © 2012 John Wiley & Sons, Ltd.

In vitro studies on human periodontal ligament stem cell sheets enhanced by enamel matrix derivative.

PubMed

Wang, Zhongshan; Feng, Zhihong; Wu, Guofeng; Bai, Shizhu; Dong, Yan; Zhao, Yimin

2016-05-01

Numerous preclinical and clinical studies have focused on the periodontal regenerative functions of enamel matrix derivative (EMD), a heat-treated preparation derived from enamel matrix proteins (EMPs) of developing porcine teeth. In this study, periodontal ligament (PDL) stem cells (PDLSCs) were isolated, and the effects of EMD on the extracorporeal induction process and the characteristics of PDLSC sheets were investigated for their potential as a more effective stem-cell therapy. EMD-enhanced cell sheets could be induced by complete medium supplemented with 50 μg/mL vitamin C and 100 μg/mL EMD. The EMD-enhanced cell sheets appeared thicker and more compact than the normal PDLSC sheets, demonstrated more layers of cells (3-7 layers), secreted richer extracellular matrix (ECM), showed varying degrees of increases in mRNA expression of periodontal tissue-specific genes (COL I, POSTN), calcification-related genes (RUNX2, OPN, OCN) and a cementum tissue-specific gene (CAP), and possessed a better mineralization ability in terms of osteogenic differentiation in vitro. These EMD-enhanced cell sheets may represent a potential option for stem-cell therapy for PDL regeneration. Copyright © 2016 Elsevier B.V. All rights reserved.

Transplantation of periodontal ligament cell sheets expressing human β-defensin-3 promotes anti-inflammation in a canine model of periodontitis

PubMed Central

Zhu, Minwen; Miao, Bo; Zhu, Jianhua; Wang, Haiyan; Zhou, Zengtong

2017-01-01

Periodontitis is a chronic oral inflammatory disease caused by microorganisms. Human β-defensin-3 (HBD-3) is an endogenous antimicrobial peptide that inhibits a broad spectrum of microorganisms. Cell sheet technology has been widely applied in tissue and organ reconstructions. In the current study, it was aimed to investigate the anti-inflammatory effect of periodontal tissue engineered by HBD-3 gene-modified periodontal ligament cell (PDLC) sheets, and to identify a suitable method of promoting the regeneration of periodontal tissues. Western blot analysis and antimicrobial tests were used to confirm the expression of HBD-3. The effect of the cell sheets on anti-inflammatory activity and bone remodeling in a dog model of periodontitis was demonstrated by immunohistochemistry. The results demonstrated that the transfected PDLCs stably expressed HBD-3. Periodontal pathogens were susceptible to the antimicrobial activity of the cell sheets. In addition, the cell sheets relieved the bone resorption caused by inflammation in the in vivo model. HBD-3 may potentially be applied in the treatment of periodontitis and may function as osteogenic promoter via its anti-inflammatory effect. PMID:28944821

Transplantation of periodontal ligament cell sheets expressing human β‑defensin‑3 promotes anti‑inflammation in a canine model of periodontitis.

PubMed

Zhu, Minwen; Miao, Bo; Zhu, Jianhua; Wang, Haiyan; Zhou, Zengtong

2017-11-01

Periodontitis is a chronic oral inflammatory disease caused by microorganisms. Human β‑defensin‑3 (HBD‑3) is an endogenous antimicrobial peptide that inhibits a broad spectrum of microorganisms. Cell sheet technology has been widely applied in tissue and organ reconstructions. In the current study, it was aimed to investigate the anti‑inflammatory effect of periodontal tissue engineered by HBD‑3 gene‑modified periodontal ligament cell (PDLC) sheets, and to identify a suitable method of promoting the regeneration of periodontal tissues. Western blot analysis and antimicrobial tests were used to confirm the expression of HBD‑3. The effect of the cell sheets on anti‑inflammatory activity and bone remodeling in a dog model of periodontitis was demonstrated by immunohistochemistry. The results demonstrated that the transfected PDLCs stably expressed HBD‑3. Periodontal pathogens were susceptible to the antimicrobial activity of the cell sheets. In addition, the cell sheets relieved the bone resorption caused by inflammation in the in vivo model. HBD‑3 may potentially be applied in the treatment of periodontitis and may function as osteogenic promoter via its anti‑inflammatory effect.

The effect of mesenchymal stem cell sheets on structural allograft healing of critical-sized femoral defects in mice

PubMed Central

Long, Teng; Zhu, Zhenan; Awad, Hani A.; Schwarz, Edward M.; Hilton, Matthew J.; Dong, Yufeng

2014-01-01

Structural bone allografts are widely used in the clinic to treat critical sized bone defects, despite lacking the osteoinductive characteristics of live autografts. To address this, we generated revitalized structural allografts wrapped with mesenchymal stem/progenitor cell (MSC) sheets, which were produced by expanding primary syngenic bone marrow derived cells on temperature-responsive plates, as a tissue engineered periosteum. In vitro assays demonstrated maintenance of the MSC phenotype in the sheets, suggesting that short-term culturing of MSC sheets is not detrimental. To test their efficacy in vivo, allografts wrapped with MSC sheets were transplanted into 4-mm murine femoral defects and compared to allografts with direct seeding of MSCs and allografts without cells. Evaluations consisted of x-ray plain radiography, 3D microCT, histology, and biomechanical testing at 4- and 6-weeks post-surgery. Our findings demonstrate that MSC sheets induce prolonged cartilage formation at the graft-host junction and enhanced bone callus formation, as well as graft-host osteointegration. Moreover, a large periosteal callus was observed spanning the allografts with MSC sheets, which partially mimics live autograft healing. Finally, biomechanical testing showed a significant increase in the structural and functional properties of MSC sheet grafted femurs. Taken together, MSC sheets exhibit enhanced osteogenicity during critical sized bone defect repair, demonstrating the feasibility of this tissue engineering solution for massive allograft healing. PMID:24393269

Lipopolysaccharide induces proliferation and osteogenic differentiation of adipose-derived mesenchymal stromal cells in vitro via TLR4 activation

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

Herzmann, Nicole; Salamon, Achim; Fiedler, Tomas

Multipotent mesenchymal stromal cells (MSC) are capable of multi-lineage differentiation and support regenerative processes. In bacterial infections, resident MSC can come intocontact with and need to react to bacterial components. Lipopolysaccharide (LPS), a typical structure of Gram-negative bacteria, increases the proliferation and osteogenic differentiation of MSC. LPS is usually recognized by the toll-like receptor (TLR) 4 and induces pro-inflammatory reactions in numerous cell types. In this study, we quantified the protein expression of TLR4 and CD14 on adipose-derived MSC (adMSC) in osteogenic differentiation and investigated the effect of TLR4 activation by LPS on NF-κB activation, proliferation and osteogenic differentiation ofmore » adMSC. We found that TLR4 is expressed on adMSC whereas CD14 is not, and that osteogenic differentiation induced an increase of the amount of TLR4 protein whereas LPS stimulation did not. Moreover, we could show that NF-κB activation via TLR4 occurs upon LPS treatment. Furthermore, we were able to show that competitive inhibition of TLR4 completely abolished the stimulatory effect of LPS on the proliferation and osteogenic differentiation of adMSC. In addition, the inhibition of TLR4 leads to the complete absence of osteogenic differentiation of adMSC, even when osteogenically stimulated. Thus, we conclude that LPS induces proliferation and osteogenic differentiation of adMSC in vitro through the activation of TLR4 and that the TLR4 receptor seems to play a role during osteogenic differentiation of adMSC.« less

Comparative facile methods for preparing graphene oxide-hydroxyapatite for bone tissue engineering.

PubMed

Raucci, M G; Giugliano, D; Longo, A; Zeppetelli, S; Carotenuto, G; Ambrosio, L

2017-08-01

Motivated by the success of using graphene oxide (GO) as a nanofiller of composites, there is a drive to search for this new kind of carbon material as a bioactive component in ceramic materials. In the present study, biomineralized GO was prepared by two different approaches, represented by in situ sol-gel synthesis and biomimetic treatment. It was found that in the biocomposites obtained by the sol-gel approach, the spindle-like hydroxyapatite nanoparticles, with a diameter of ca. 5 ± 0.37 nm and a length of ca. 70 ± 2.5 nm, were presented randomly and strongly on the surface. The oxygen-containing functional groups, such as hydroxyl and carbonyl, present on the basal plane and edges of the GO sheets, play an important role in anchoring calcium ions, as demonstrated by FT-IR and TEM investigations. A different result was obtained for biocomposites after biomimetic treatment: an amorphous calcium phosphate on GO sheet was observed after 5 days of treatment. These different approaches resulted in a diverse effect on the proliferation and differentiation of osteogenic mesenchymal stem cells. In fact, in biocomposites prepared by the sol-gel approach the expression of an early marker of osteogenic differentiation, ALP, increases with the amount of GO in the first days of cell culture. Meanwhile, biomimetic materials sustain cell viability and proliferation, even if the expression of alkaline phosphatase activity in a basal medium is delayed. These findings may provide new prospects for utilizing GO-based hydroxyapatite biocomposites in bone repair, bone augmentation and coating of biomedical implants and broaden the application of GO sheets in biological areas. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Relevance of fiber integrated gelatin-nanohydroxyapatite composite scaffold for bone tissue regeneration

NASA Astrophysics Data System (ADS)

Halima Shamaz, Bibi; Anitha, A.; Vijayamohan, Manju; Kuttappan, Shruthy; Nair, Shantikumar; Nair, Manitha B.

2015-10-01

Porous nanohydroxyapatite (nanoHA) is a promising bone substitute, but it is brittle, which limits its utility for load bearing applications. To address this issue, herein, biodegradable electrospun microfibrous sheets of poly(L-lactic acid)-(PLLA)-polyvinyl alcohol (PVA) were incorporated into a gelatin-nanoHA matrix which was investigated for its mechanical properties, the physical integration of the fibers with the matrix, cell infiltration, osteogenic differentiation and bone regeneration. The inclusion of sacrificial fibers like PVA along with PLLA and leaching resulted in improved cellular infiltration towards the center of the scaffold. Furthermore, the treatment of PLLA fibers with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide enhanced their hydrophilicity, ensuring firm anchorage between the fibers and the gelatin-HA matrix. The incorporation of PLLA microfibers within the gelatin-nanoHA matrix reduced the brittleness of the scaffolds, the effect being proportional to the number of layers of fibrous sheets in the matrix. The proliferation and osteogenic differentiation of human adipose-derived mesenchymal stem cells was augmented on the fibrous scaffolds in comparison to those scaffolds devoid of fibers. Finally, the scaffold could promote cell infiltration, together with bone regeneration, upon implantation in a rabbit femoral cortical defect within 4 weeks. The bone regeneration potential was significantly higher when compared to commercially available HA (Surgiwear™). Thus, this biomimetic, porous, 3D composite scaffold could be offered as a promising candidate for bone regeneration in orthopedics.

The effect of mesenchymal stem cell sheets on structural allograft healing of critical sized femoral defects in mice.

PubMed

Long, Teng; Zhu, Zhenan; Awad, Hani A; Schwarz, Edward M; Hilton, Matthew J; Dong, Yufeng

2014-03-01

Structural bone allografts are widely used in the clinic to treat critical sized bone defects, despite lacking the osteoinductive characteristics of live autografts. To address this, we generated revitalized structural allografts wrapped with mesenchymal stem/progenitor cell (MSC) sheets, which were produced by expanding primary syngenic bone marrow derived cells on temperature-responsive plates, as a tissue-engineered periosteum. In vitro assays demonstrated maintenance of the MSC phenotype in the sheets, suggesting that short-term culturing of MSC sheets is not detrimental. To test their efficacy in vivo, allografts wrapped with MSC sheets were transplanted into 4-mm murine femoral defects and compared to allografts with direct seeding of MSCs and allografts without cells. Evaluations consisted of X-ray plain radiography, 3D microCT, histology, and biomechanical testing at 4- and 6-weeks post-surgery. Our findings demonstrate that MSC sheets induce prolonged cartilage formation at the graft-host junction and enhanced bone callus formation, as well as graft-host osteointegration. Moreover, a large periosteal callus was observed spanning the allografts with MSC sheets, which partially mimics live autograft healing. Finally, biomechanical testing showed a significant increase in the structural and functional properties of MSC sheet grafted femurs. Taken together, MSC sheets exhibit enhanced osteogenicity during critical sized bone defect repair, demonstrating the feasibility of this tissue engineering solution for massive allograft healing. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

Human decellularized bone scaffolds from aged donors show improved osteoinductive capacity compared to young donor bone.

PubMed

Smith, Christopher A; Board, Tim N; Rooney, Paul; Eagle, Mark J; Richardson, Stephen M; Hoyland, Judith A

2017-01-01

To improve the safe use of allograft bone, decellularization techniques may be utilized to produce acellular scaffolds. Such scaffolds should retain their innate biological and biomechanical capacity and support mesenchymal stem cell (MSC) osteogenic differentiation. However, as allograft bone is derived from a wide age-range, this study aimed to determine whether donor age impacts on the ability an osteoinductive, acellular scaffold produced from human bone to promote the osteogenic differentiation of bone marrow MSCs (BM-MSC). BM-MSCs from young and old donors were seeded on acellular bone cubes from young and old donors undergoing osteoarthritis related hip surgery. All combinations resulted in increased osteogenic gene expression, and alkaline phosphatase (ALP) enzyme activity, however BM-MSCs cultured on old donor bone displayed the largest increases. BM-MSCs cultured in old donor bone conditioned media also displayed higher osteogenic gene expression and ALP activity than those exposed to young donor bone conditioned media. ELISA and Luminex analysis of conditioned media demonstrated similar levels of bioactive factors between age groups; however, IGF binding protein 1 (IGFBP1) concentration was significantly higher in young donor samples. Additionally, structural analysis of old donor bone indicated an increased porosity compared to young donor bone. These results demonstrate the ability of a decellularized scaffold produced from young and old donors to support osteogenic differentiation of cells from young and old donors. Significantly, the older donor bone produced greater osteogenic differentiation which may be related to reduced IGFBP1 bioavailability and increased porosity, potentially explaining the excellent clinical results seen with the use of allograft from aged donors.

In vivo differentiation of human periodontal ligament cells leads to formation of dental hard tissue.

PubMed

Wolf, M; Lossdörfer, S; Abuduwali, N; Meyer, R; Kebir, S; Götz, W; Jäger, A

2013-11-01

Following trauma, periodontal disease, or orthodontic tooth movement, residual periodontal ligament (PDL) cells at the defect site are considered mandatory for successful regeneration of the injured structures. Recent developments in tissue engineering focus, as one pillar, on the transplantation of PDL cells to support periodontal regeneration processes. Here, we examined the ability of osteogenically predifferentiated PDL cells to undergo further osteoblastic or cementoblastic differentiation and to mineralize their extracellular matrix when transplanted in an in vivo microenvironment. Using collagen sponges as carriers, osteogenically predifferentiated human PDL cells were transplanted subcutaneously into six immunocompromised CD-1® nude mice. Following explantation after 28 days, osteogenic and cementogenic marker protein expression was visualized immunohistochemically. After 28 days, transplanted PDL cells revealed both cellular, cytoplasmatic and extracellular immunoreactivity for the chosen markers alkaline phosphatase, osteopontin, PTH-receptor 1, and osteocalcin. Specific osteogenic and cementoblastic differentiation was demonstrated by RUNX2 and CEMP1 immunoreactivity. Early stages of mineralization were demonstrated by calcium and phosphate staining. Our results reinforce the previously published reports of PDL cell mineralization in vivo and further demonstrate the successful induction of specific osteogenic and cementogenic differentiation of transplanted human PDL cells in vivo. These findings reveal promising possibilities for supporting periodontal remodeling and regeneration processes with PDL cells being potential target cells with which to influence the process of orthodontically induced root resorption.

Synergistic effects of BMP-2, BMP-6 or BMP-7 with human plasma fibronectin onto hydroxyapatite coatings: A comparative study.

PubMed

Brigaud, Isabelle; Agniel, Rémy; Leroy-Dudal, Johanne; Kellouche, Sabrina; Ponche, Arnaud; Bouceba, Tahar; Mihailescu, Natalia; Sopronyi, Mihai; Viguier, Eric; Ristoscu, Carmen; Sima, Felix; Mihailescu, Ion N; Carreira, Ana Claudia O; Sogayar, Mari Cleide; Gallet, Olivier; Anselme, Karine

2017-06-01

Design of new osteoinductive biomaterials to reproduce an optimized physiological environment capable of recruiting stem cells and instructing their fate towards the osteoblastic lineage has become a priority in orthopaedic surgery. This work aims at evaluating the bioactivity of BMP combined with human plasma fibronectin (FN/BMP) delivered in solution or coated onto titanium-hydroxyapatite (TiHA) surfaces. Herein, we focus on the comparison of in vitro osteogenic efficacy in mouse C2C12 pre-osteoblasts of three BMP members, namely: BMP-2, BMP-6 and BMP-7. In parallel, we evaluated the molecular binding strength between each BMP with FN using the Surface Plasmon Resonance (SPR) technology. The affinity of BMPs for FN was found totally different and dependent on BMP type. Indeed, the combination of FN with BMP-2 on TiHA surfaces potentiates the burst of gene-mediated osteogenic induction, while it prolongs the osteogenic activity of BMP-6 and surprisingly annihilates the BMP-7 one. These results correlate with FN/BMP affinity for TiHA, since BMP-6>BMP-2>BMP-7. In addition, by analyzing the osteogenic activity in the peri-implant environment, we showed that osteoinductive paracrine effects were significantly decreased upon (FN/BMP-6), as opposed to (FN/BMP-2) coatings. Altogether, our results support the use of FN/BMP-6 to develop a biomimetic microenvironment capable to induce osteogenic activity under physiological conditions, with minimum paracrine signalization. The originality of our paper relies on the first direct comparison of the in vitro osteogenic potential of three osteogenic BMPs (BMP-2, -6 and -7) combined with native human plasma fibronectin delivered in solution or coated by laser transfer onto titanium hydroxyapatite surfaces. We confirm that BMP association with fibronectin enhances the osteogenic activity of BMP-2, -6 and -7, but with essential discrepancies, depending on the BMP member, and in agreement with the affinity of BMPs for fibronectin. Moreover, we bring elements to explain the origin of the BMP-2 medical life-threatening side-effects by analyzing in vitro paracrine effects. Finally, this work supports the alternative use of FN/BMP-6 to induce osteogenic activity under physiological conditions, with minimum side effects. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Biomimetic Nucleation of Hydroxyapatite Crystals Mediated by Antheraea pernyi Silk Sericin Promotes Osteogenic Differentiation of Human Bone Marrow Derived Mesenchymal Stem Cells

PubMed Central

2015-01-01

Biomacromolecules have been used as templates to grow hydroxyapatite crystals (HAps) by biomineralization to fabricate mineralized materials for potential application in bone tissue engineering. Silk sericin is a protein with features desirable as a biomaterial, such as increased hydrophilicity and biodegradation. Mineralization of the silk sericin from Antheraea pernyi (A. pernyi) silkworm has rarely been reported. Here, for the first time, nucleation of HAps on A. pernyi silk sericin (AS) was attempted through a wet precipitation method and consequently the cell viability and osteogenic differentiation of BMSCs on mineralized AS were investigated. It was found that AS mediated the nucleation of HAps in the form of nanoneedles while self-assembling into β-sheet conformation, leading to the formation of a biomineralized protein based biomaterial. The cell viability assay of BMSCs showed that the mineralization of AS stimulated cell adhesion and proliferation, showing that the resultant AS biomaterial is biocompatible. The differentiation assay confirmed that the mineralized AS significantly promoted the osteogenic differentiation of BMSCs when compared to nonmineralized AS as well as other types of sericin (B. mori sericin), suggesting that the resultant mineralized AS biomaterial has potential in promoting bone formation. This result represented the first work proving the osteogenic differentiation of BMSCs directed by silk sericin. Therefore, the biomineralization of A. pernyi silk sericin coupled with seeding BMSCs on the resultant mineralized biomaterials is a useful strategy to develop the potential application of this unexplored silk sericin in the field of bone tissue engineering. This study lays the foundation for the use of A. pernyi silk sericin as a potential scaffold for tissue engineering. PMID:24666022

Biomimetic nucleation of hydroxyapatite crystals mediated by Antheraea pernyi silk sericin promotes osteogenic differentiation of human bone marrow derived mesenchymal stem cells.

PubMed

Yang, Mingying; Shuai, Yajun; Zhang, Can; Chen, Yuyin; Zhu, Liangjun; Mao, Chuanbin; OuYang, Hongwei

2014-04-14

Biomacromolecules have been used as templates to grow hydroxyapatite crystals (HAps) by biomineralization to fabricate mineralized materials for potential application in bone tissue engineering. Silk sericin is a protein with features desirable as a biomaterial, such as increased hydrophilicity and biodegradation. Mineralization of the silk sericin from Antheraea pernyi (A. pernyi) silkworm has rarely been reported. Here, for the first time, nucleation of HAps on A. pernyi silk sericin (AS) was attempted through a wet precipitation method and consequently the cell viability and osteogenic differentiation of BMSCs on mineralized AS were investigated. It was found that AS mediated the nucleation of HAps in the form of nanoneedles while self-assembling into β-sheet conformation, leading to the formation of a biomineralized protein based biomaterial. The cell viability assay of BMSCs showed that the mineralization of AS stimulated cell adhesion and proliferation, showing that the resultant AS biomaterial is biocompatible. The differentiation assay confirmed that the mineralized AS significantly promoted the osteogenic differentiation of BMSCs when compared to nonmineralized AS as well as other types of sericin (B. mori sericin), suggesting that the resultant mineralized AS biomaterial has potential in promoting bone formation. This result represented the first work proving the osteogenic differentiation of BMSCs directed by silk sericin. Therefore, the biomineralization of A. pernyi silk sericin coupled with seeding BMSCs on the resultant mineralized biomaterials is a useful strategy to develop the potential application of this unexplored silk sericin in the field of bone tissue engineering. This study lays the foundation for the use of A. pernyi silk sericin as a potential scaffold for tissue engineering.

Human decellularized bone scaffolds from aged donors show improved osteoinductive capacity compared to young donor bone

PubMed Central

Smith, Christopher A.; Board, Tim N.; Rooney, Paul; Eagle, Mark J.; Richardson, Stephen M.

2017-01-01

To improve the safe use of allograft bone, decellularization techniques may be utilized to produce acellular scaffolds. Such scaffolds should retain their innate biological and biomechanical capacity and support mesenchymal stem cell (MSC) osteogenic differentiation. However, as allograft bone is derived from a wide age-range, this study aimed to determine whether donor age impacts on the ability an osteoinductive, acellular scaffold produced from human bone to promote the osteogenic differentiation of bone marrow MSCs (BM-MSC). BM-MSCs from young and old donors were seeded on acellular bone cubes from young and old donors undergoing osteoarthritis related hip surgery. All combinations resulted in increased osteogenic gene expression, and alkaline phosphatase (ALP) enzyme activity, however BM-MSCs cultured on old donor bone displayed the largest increases. BM-MSCs cultured in old donor bone conditioned media also displayed higher osteogenic gene expression and ALP activity than those exposed to young donor bone conditioned media. ELISA and Luminex analysis of conditioned media demonstrated similar levels of bioactive factors between age groups; however, IGF binding protein 1 (IGFBP1) concentration was significantly higher in young donor samples. Additionally, structural analysis of old donor bone indicated an increased porosity compared to young donor bone. These results demonstrate the ability of a decellularized scaffold produced from young and old donors to support osteogenic differentiation of cells from young and old donors. Significantly, the older donor bone produced greater osteogenic differentiation which may be related to reduced IGFBP1 bioavailability and increased porosity, potentially explaining the excellent clinical results seen with the use of allograft from aged donors. PMID:28505164

Influence of cell culture media conditions on the osteogenic differentiation of cord blood-derived mesenchymal stem cells.

PubMed

Hildebrandt, Cornelia; Büth, Heiko; Thielecke, Hagen

2009-01-01

In this study the critical parameters directing osteogenic differentiation of umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) were investigated, key factors and conditions identified and improved protocols for a more cell-type adapted differentiation developed. Today only little information about the specific conditions directing osteogenic development is available and current protocols for cultivation and differentiation of UCB-MSCs are based mainly on experience with bone marrow-derived MSCs (BM-MSCs) without further adaptation. Thus, protocols for improved osteoinduction are of particular interest. The goal of this study was to investigate the influence of three different culture media (A) alpha MEM, 15% FBS, (B) DMEM, 15% FBS and (C) MSCGM, 10% SingleQuot growth supplement on the osteogenic differentiation of UCB-MSCs. Moreover, a systematic analysis of two concentrations of dexamethasone (10(-8)M/10(-7)M) in combination with or without BMP-2 (10(-7)M) was carried out by detecting the expression of alkaline phosphatase (ALP), collagen-1 and the mineralization of ECM. We found that MSCGM, 10% SingleQuot had a supportive effect on the osteogenic differentiation of UCB-MSCs. In case of treatment with 10(-8)M dexamethasone, mineralization occurred in combination with BMP-2 exclusively, while a concentration of 10(-7)M dexamethasone led to a high amount of mineralized ECM and the expression of collagen-1 independent of BMP-2 addition. According to this data dexamethasone is the leading osteoinductive factor, but BMP-2 seems to have supportive properties in UCB-MSCs. In conclusion, MSCGM supplemented with 10% SingleQuot and 10(-7)M dexamethasone was the condition identified to be best for inducing the osteogenic differentiation of UCB-MSCs.

Gradients in pore size enhance the osteogenic differentiation of human mesenchymal stromal cells in three-dimensional scaffolds

NASA Astrophysics Data System (ADS)

di Luca, Andrea; Ostrowska, Barbara; Lorenzo-Moldero, Ivan; Lepedda, Antonio; Swieszkowski, Wojcech; van Blitterswijk, Clemens; Moroni, Lorenzo

2016-03-01

Small fractures in bone tissue can heal by themselves, but in case of larger defects current therapies are not completely successful due to several drawbacks. A possible strategy relies on the combination of additive manufactured polymeric scaffolds and human mesenchymal stromal cells (hMSCs). The architecture of bone tissue is characterized by a structural gradient. Long bones display a structural gradient in the radial direction, while flat bones in the axial direction. Such gradient presents a variation in bone density from the cancellous bone to the cortical bone. Therefore, scaffolds presenting a gradient in porosity could be ideal candidates to improve bone tissue regeneration. In this study, we present a construct with a discrete gradient in pore size and characterize its ability to further support the osteogenic differentiation of hMSCs. Furthermore, we studied the behaviour of hMSCs within the different compartments of the gradient scaffolds, showing a correlation between osteogenic differentiation and ECM mineralization, and pore dimensions. Alkaline phosphatase activity and calcium content increased with increasing pore dimensions. Our results indicate that designing structural porosity gradients may be an appealing strategy to support gradual osteogenic differentiation of adult stem cells.

Strontium attenuates rhBMP-2-induced osteogenic differentiation via formation of Sr-rhBMP-2 complex and suppression of Smad-dependent signaling pathway.

PubMed

Zhang, Wenjing; Tian, Yu; He, Hongyan; Chen, Rui; Ma, Yifan; Guo, Han; Yuan, Yuan; Liu, Changsheng

2016-03-01

Strontium (Sr(2+)) has pronounced effects on stimulating bone formation and inhibiting bone resorption in bone regeneration. In this current study, the effect and the underlying mechanism involved of Sr(2+) on the biological activity of bone morphogenetic protein-2 (BMP-2) were studied in detail with pluripotent skeletal muscle myogenic progenitor C2C12 model cell line. The results indicated that Sr(2+) could bind recombinant human BMP-2 (rhBMP-2) rapidly, even in the presence of Ca(2+) and Mg(2+), and inhibited rhBMP-2-induced osteogenic differentiation in vitro and osteogenetic efficiency in vivo. Further studies demonstrated that Sr(2+) treatment undermined the binding capacity of rhBMP-2 with its receptor BMPRIA and thus attenuated Smad 1/5/8 phosphorylation without affecting their dephosphorylation in C2C12 cells. Furthermore, circular dichroism spectroscopy, fluorescence spectroscopy and X-ray photoelectron spectroscopy all revealed that the inhibitory effect of Sr(2+) on the rhBMP-2 osteogenic activity was associated with the formation of Sr-rhBMP-2 complex and ensuing enhancement of β-sheet structure. Our work suggests the activity of rhBMP-2 to induce osteogenic differentiation was decreased by directly interaction with free Sr ions in solution, which should provide guide and assist for development of BMP-2-based materials for bone regeneration. Due to easy denaturation and ensuing the reduced activity of rhBMP-2, preserving/enhancing the capacity of rhBMP-2 to induce osteogenic differentiation is of critical importance in developing the protein-based therapy. Cations as effective elements influence the conformation and thereby the bioactivity of protein. Strontium (Sr(2+)), stimulating bone formation and inhibiting bone resorption, has been incorporated into biomaterials/scaffold to improve the bioactivity for bone-regeneration applications. However, Sr(2+)-induced changes in the conformation and bioactivity of BMP-2 have never been investigated. In this study, the formation of Sr-rhBMP-2 complex inhibited the osteogenic differentiation in vitro and osteogenetic efficiency in vivo through the inhibition of BMP/Smad signaling pathway, providing guidance for development of Sr-containing BMP-2-based bone scaffold/matrice and other Sr-dopped protein therapy. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Induction of Osteogenic Differentiation of Adipose Derived Stem Cells by Microstructured Nitinol Actuator-Mediated Mechanical Stress

PubMed Central

Strauß, Sarah; Dudziak, Sonja; Hagemann, Ronny; Barcikowski, Stephan; Fliess, Malte; Israelowitz, Meir; Kracht, Dietmar; Kuhbier, Jörn W.; Radtke, Christine; Reimers, Kerstin; Vogt, Peter M.

2012-01-01

The development of large tissue engineered bone remains a challenge in vitro, therefore the use of hybrid-implants might offer a bridge between tissue engineering and dense metal or ceramic implants. Especially the combination of the pseudoelastic implant material Nitinol (NiTi) with adipose derived stem cells (ASCs) opens new opportunities, as ASCs are able to differentiate osteogenically and therefore enhance osseointegration of implants. Due to limited knowledge about the effects of NiTi-structures manufactured by selective laser melting (SLM) on ASCs the study started with an evaluation of cytocompatibility followed by the investigation of the use of SLM-generated 3-dimensional NiTi-structures preseeded with ASCs as osteoimplant model. In this study we could demonstrate for the first time that osteogenic differentiation of ASCs can be induced by implant-mediated mechanical stimulation without support of osteogenic cell culture media. By use of an innovative implant design and synthesis via SLM-technique we achieved high rates of vital cells, proper osteogenic differentiation and mechanically loadable NiTi-scaffolds could be achieved. PMID:23236461

PCL-HA microscaffolds for in vitro modular bone tissue engineering.

PubMed

Totaro, Alessandra; Salerno, Aurelio; Imparato, Giorgia; Domingo, Concepción; Urciuolo, Francesco; Netti, Paolo Antonio

2017-06-01

The evolution of microscaffolds and bone-bioactive surfaces is a pivotal point in modular bone tissue engineering. In this study, the design and fabrication of porous polycaprolactone (PCL) microscaffolds functionalized with hydroxyapatite (HA) nanoparticles by means of a bio-safe and versatile thermally-induced phase separation process is reported. The ability of the as-prepared nanocomposite microscaffolds to support the adhesion, growth and osteogenic differentiation of human mesenchymal stem cells (hMSCs) in standard and osteogenic media and using dynamic seeding/culture conditions was investigated. The obtained results demonstrated that the PCL-HA nanocomposite microparticles had an enhanced interaction with hMSCs and induced their osteogenic differentiation, even without the exogenous addition of osteogenic factors. In particular, calcium deposition, alizarin red assay, histological analysis, osteogenic gene expression and collagen I secretion were assessed. The results of these tests demonstrated the formation of bone microtissue precursors after 28 days of dynamic culture. These findings suggest that PCL-HA nanocomposite microparticles represent an excellent platform for in vitro modular bone tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Self-Assembling Nanoclay Diffusion Gels for Bioactive Osteogenic Microenvironments.

PubMed

Shi, Pujiang; Kim, Yang-Hee; Mousa, Mohamed; Sanchez, Roxanna Ramnarine; Oreffo, Richard O C; Dawson, Jonathan I

2018-06-17

Laponite nanoparticles have attracted attention in the tissue engineering field for their protein interactions, gel-forming properties, and, more recently, osteogenic bioactivity. Despite growing interest in the osteogenic properties of Laponite, the application of Laponite colloidal gels to host the osteogenic differentiation of responsive stem cell populations remains unexplored. Here, the potential to harness the gel-forming properties of Laponite to generate injectable bioactive microenvironments for osteogenesis is demonstrated. A diffusion/dialysis gelation method allows the rapid formation of stable transparent gels from injectable, thixotropic Laponite suspensions in physiological fluids. Upon contact with buffered saline or blood serum, nanoporous gel networks exhibiting, respectively, fivefold and tenfold increases in gel stiffness are formed due to the reorganization of nanoparticle interactions. Laponite diffusion gels are explored as osteogenic microenvironments for skeletal stem cell containing populations. Laponite films support cell adhesion, proliferation, and differentiation of human bone marrow stromal cells in 2D. Laponite gel encapsulation significantly enhances osteogenic protein expression compared with 3D pellet culture controls. In both 2D and 3D conditions, cell associated mineralization is strongly enhanced. This study demonstrates that Laponite diffusion gels offer considerable potential as biologically active and clinically relevant bone tissue engineering scaffolds. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Canonical FGFs Prevent Osteogenic Lineage Commitment and Differentiation of Human Bone Marrow Stromal Cells Via ERK1/2 Signaling.

PubMed

Simann, Meike; Le Blanc, Solange; Schneider, Verena; Zehe, Viola; Lüdemann, Martin; Schütze, Norbert; Jakob, Franz; Schilling, Tatjana

2017-02-01

Controlling the adipo-osteogenic lineage decision of trabecular human bone marrow stromal cells (hBMSCs) in favor of osteogenesis represents a promising approach for osteoporosis therapy and prevention. Previously, Fibroblast Growth Factor 1 (FGF1) and its subfamily member FGF2 were scored as leading candidates to exercise control over skeletal precursor commitment and lineage decision albeit literature results are highly inconsistent. We show here that FGF1 and 2 strongly prevent the osteogenic commitment and differentiation of hBMSCs. Mineralization of extracellular matrix (ECM) and mRNA expression of osteogenic marker genes Alkaline Phosphatase (ALP), Collagen 1A1 (COL1A1), and Integrin-Binding Sialoprotein (IBSP) were significantly reduced. Furthermore, master regulators of osteogenic commitment like Runt-Related Transcription Factor 2 (RUNX2) and Bone Morphogenetic Protein 4 (BMP4) were downregulated. When administered under adipogenic culture conditions, canonical FGFs did not support osteogenic marker expression. Moreover despite the presence of osteogenic differentiation factors, FGFs even disabled the pro-osteogenic lineage decision of pre-differentiated adipocytic cells. In contrast to FGF Receptor 2 (FGFR2), FGFR1 was stably expressed throughout osteogenic and adipogenic differentiation and FGF addition. Moreover, FGFR1 and Extracellular Signal-Regulated Kinases 1 and 2 (ERK1/2) were found to be responsible for underlying signal transduction using respective inhibitors. Taken together, we present new findings indicating that canonical FGFR-ERK1/2 signaling entrapped hBMSCs in a pre-committed state and arrested further maturation of committed precursors. Our results might aid in unraveling and controlling check points relevant for ageing-associated aberrant adipogenesis with consequences for the treatment of degenerative diseases such as osteoporosis and for skeletal tissue engineering strategies. J. Cell. Biochem. 118: 263-275, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Assessment of cellular materials generated by co-cultured ‘inflamed’ and healthy periodontal ligament stem cells from patient-matched groups

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

Tang, Hao-Ning; Department of Stomatology, The First Affiliated Hospital of the Chinese PLA General Hospital, Beijing 100048; Xia, Yu

Recently, stem cells derived from the'inflamed’ periodontal ligament (PDL) tissue of periodontally diseased teeth (I-PDLSCs) have been increasingly suggested as a more readily accessible source of cells for regenerative therapies than those derived from healthy PDL tissue (H-PDLSCs). However, substantial evidence indicates that I-PDLSCs exhibit impaired functionalities compared with H-PDLSCs. In this study, patient-matched I-PDLSCs and H-PDLSCs were co-cultured at various ratios. Cellular materials derived from these cultures were investigated regarding their osteogenic potential in vitro and capacity to form new bone following in vivo transplantation. While patient-matched I-PDLSCs and H-PDLSCs could co-exist in co-culture systems, the proportion of I-PDLSCsmore » tended to increase during in vitro incubation. Compared with H-PDLSC monoculture, the presence of I-PDLSCs in the co-cultures appeared to enhance the overall cell proliferation. Although not completely rescued, the osteogenic and regenerative potentials of the cellular materials generated by co-cultured I-PDLSCs and H-PDLSCs were significantly improved compared with those derived from I-PDLSC monocultures. Notably, cells in co-cultures containing either 50% I-PDLSCs plus 50% H-PDLSCs or 25% I-PDLSCs plus 75% H-PDLSCs expressed osteogenesis-related proteins and genes at levels similar to those expressed in H-PDLSC monocultures (P>0.05). Irrespective of the percentage of I-PDLSCs, robust cellular materials were obtained from co-cultures with 50% or more H-PDLSCs, which exhibited equivalent potential to form new bone in vivo compared with sheets generated by H-PDLSC monocultures. These data suggest that the co-culture of I-PDLSCs with patient-matched H-PDLSCs is a practical and effective method for increasing the overall osteogenic and regenerative potentials of resultant cellular materials. - Highlights: • Co-culturing H-PDLSCs with I-PDLSCs led to rapid cell expansion. • H-PDLSCs and I-PDLSCs co-cultured at proper ratios retained cell differentiation. • Co-culturing proper ratios of H-PDLSCs and I-PDLSCs produced robust cell sheets. • I-PDLSCs can be used as an adjuvant to H-PDLSCs for yielding cellular materials.« less

Decellularized bone extracellular matrix and human dental pulp stem cells as a construct for bone regeneration.

PubMed

Paduano, Francesco; Marrelli, Massimo; Alom, Noura; Amer, Mahetab; White, Lisa J; Shakesheff, Kevin M; Tatullo, Marco

2017-06-01

Dental pulp tissue represents a source of mesenchymal stem cells that have a strong differentiation potential towards the osteogenic lineage. The objective of the current study was to examine in vitro osteogenic induction of dental pulp stem cells (DPSCs) cultured on hydrogel scaffolds derived from decellularized bone extracellular matrix (bECM) compared to collagen type I (Col-I), the major component of bone matrix. DPSCs in combination with bECM hydrogels were cultured under three different conditions: basal medium, osteogenic medium and medium supplemented with growth factors (GFs) and cell growth, mineral deposition, gene and protein expression were investigated. The DPSCs/bECM hydrogel constructs cultured in basal medium showed that cells were viable after three weeks and that the expression of runt-related transcription factor 2 (RUNX-2) and bone sialoprotein (BSP) were significantly upregulated in the absence of extra osteogenic inducers compared to Col-I hydrogel scaffolds. In addition, the protein expression levels of BSP and osteocalcin were higher on bECM with respect to Col-I hydrogel scaffolds. Furthermore, DPSCs/bECM hydrogels cultured with osteogenic or GFs supplemented medium displayed a higher upregulation of the osteo-specific markers compared to Col-I hydrogels in identical media. Collectively, our results demonstrate that bECM hydrogels might be considered as suitable scaffolds to support osteogenic differentiation of DPSCs.

Combined Use of Mesenchymal Stromal Cell Sheet Transplantation and Local Injection of SDF-1 for Bone Repair in a Rat Nonunion Model.

PubMed

Chen, Guangnan; Fang, Tingting; Qi, Yiying; Yin, Xiaofan; Di, Tuoyu; Feng, Gang; Lei, Zhong; Zhang, Yuxiang; Huang, Zhongming

2016-10-01

Bone nonunion treatments pose a challenge in orthopedics. This study investigated the joint effects of using mesenchymal stem cell (MSC) sheets with local injection of stromal cell-derived factor-1 (SDF-1) on bone formation. In vitro, we found that migration of MSCs was mediated by SDF-1 in a dose-dependent manner. Moreover, stimulation with SDF-1 had no direct effect on the proliferation or osteogenic differentiation of MSCs. Furthermore, the results indicated elevated expression levels of bone morphogenetic protein 2, alkaline phosphatase, osteocalcin, and vascular endothelial growth factor in MSC sheets compared with MSCs cultured in medium. New bone formation in fractures was evaluated by X-ray, micro-computed tomography (micro-CT), hematoxylin and eosin (H&E) staining, Safranin-O staining, and immunohistochemistry in vivo. In the rat bone fracture model, the MSC sheets transplanted into the injured site along with injection of SDF-1 showed significantly more new bone formation within the gap. Moreover, at 8 weeks, complete bone union was obtained in this group. In contrast, the control group showed nonunion of the bone. Our study suggests a new strategy involving the use of MSC sheets with a local injection of SDF-1 for hard tissue reconstruction, such as the healing of nonunions and bone defects.

Menaquinone-4 enhances osteogenic potential of human amniotic fluid mesenchymal stem cells cultured in 2D and 3D dynamic culture systems.

PubMed

Mandatori, Domitilla; Penolazzi, Letizia; Pipino, Caterina; Di Tomo, Pamela; Di Silvestre, Sara; Di Pietro, Natalia; Trevisani, Sara; Angelozzi, Marco; Ucci, Mariangela; Piva, Roberta; Pandolfi, Assunta

2018-02-01

Menaquinones, also known as Vitamin K2 family, regulate calcium homeostasis in a 'bone-vascular cross-talk' and recently received particular attention for their positive effect on bone formation. Given that the correlation between menaquinones and bone metabolism to date is still unclear, the objective of our study was to investigate the possible role of menaquinone-4 (MK-4), an isoform of the menaquinones family, in the modulation of osteogenesis. For this reason, we used a model of human amniotic fluid mesenchymal stem cells (hAFMSCs) cultured both in two-dimensional (2D) and three-dimensional (3D; RCCS™bioreactor) in vitro culture systems. Furthermore, to mimic the 'bone remodelling unit' in vitro, hAFMSCs were co-cultured in the 3D system with human monocyte cells (hMCs) as osteoclast precursors. The results showed that in a conventional 2D culture system, hAFMSCs were responsive to the MK-4, which significantly improved the osteogenic process through γ-glutamyl carboxylase-dependent pathway. The same results were obtained in the 3D dynamic system where MK-4 treatment supported the osteoblast-like formation promoting the extracellular bone matrix deposition and the expression of the osteogenic-related proteins (alkaline phosphatase, osteopontin, collagen type-1 and osteocalcin). Notably, when the hAFMSCs were co-cultured in a 3D dynamic system with the hMCs, the presence of MK-4 supported the cellular aggregate formation as well as the osteogenic function of hAFMSCs, but negatively affected the osteoclastogenic process. Taken together, our results demonstrate that MK-4 supported the aggregate formation of hAFMSCs and increased the osteogenic functions. Specifically, our data could help to optimize bone regenerative medicine combining cell-based approaches with MK-4 treatment. Copyright © 2017 John Wiley & Sons, Ltd.

Engineering tubular bone using mesenchymal stem cell sheets and coral particles

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

Geng, Wenxin; Ma, Dongyang; Yan, Xingrong

Highlights: • We developed a novel engineering strategy to solve the limitations of bone grafts. • We fabricated tubular constructs using cell sheets and coral particles. • The composite constructs showed high radiological density and compressive strength. • These characteristics were similar to those of native bone. -- Abstract: The development of bone tissue engineering has provided new solutions for bone defects. However, the cell-scaffold-based approaches currently in use have several limitations, including low cell seeding rates and poor bone formation capacity. In the present study, we developed a novel strategy to engineer bone grafts using mesenchymal stem cell sheetsmore » and coral particles. Rabbit bone marrow mesenchymal stem cells were continuously cultured to form a cell sheet with osteogenic potential and coral particles were integrated into the sheet. The composite sheet was then wrapped around a cylindrical mandrel to fabricate a tubular construct. The resultant tubular construct was cultured in a spinner-flask bioreactor and subsequently implanted into a subcutaneous pocket in a nude mouse for assessment of its histological characteristics, radiological density and mechanical property. A similar construct assembled from a cell sheet alone acted as a control. In vitro observations demonstrated that the composite construct maintained its tubular shape, and exhibited higher radiological density, compressive strength and greater extracellular matrix deposition than did the control construct. In vivo experiments further revealed that new bone formed ectopically on the composite constructs, so that the 8-week explants of the composite sheets displayed radiological density similar to that of native bone. These results indicate that the strategy of using a combination of a cell sheet and coral particles has great potential for bone tissue engineering and repairing bone defects.« less

TiO2 -enriched polymeric powder coatings support human mesenchymal cell spreading and osteogenic differentiation.

PubMed

Mozumder, Mohammad Sayem; Zhu, Jesse; Perinpanayagam, Hiran

2011-06-01

Novel polymeric powder coatings (PPC) were prepared by ultrafine powder coating technology and shown to support human mesenchymal cell attachment and growth. PPC surfaces enriched with nano-TiO(2) (nTiO(2)) showed enhanced cellular responses, and were compared to commercially pure titanium (cpTi). After cell attachment and growth, osteogenic differentiation and bone matrix formation ensures osseointegration for implantable biomaterials. Therefore, the objective of this study was to determine if mesenchymal cells grown on PPC could undergo osteogenic differentiation by inducing Runx2 and bone matrix proteins, and then initiate mineralization. Atomic force microscopy revealed intricate three-dimensional micro-topographies, and the measures of nano-roughness and porosity were similar for all PPC surfaces. Scanning electron microscopy showed that the cells attached and spread out over all of the surfaces. After 1 week in osteogenic media, RT-PCR analysis showed the induction of Runx2, the up-regulation of type I collagen, and the initial detection of alkaline phosphatase and bone sialoprotein. After 4 weeks, Alizarin Red staining showed mineral deposition. However, cell spreading and osteogenic differentiation were significantly (P < 0.05) higher on the cpTi controls than on the PPC surfaces. Furthermore, spreading and differentiation were consistently higher on the titanium-enriched PPC-2, -3 and -4 than on the titanium-free PPC-1. Therefore, despite the presence of complex micro-topographies and nano-features, titanium-enrichment enhanced the cellular response, and pure titanium still provided the best substrate. These findings confirm the cytocompatibility of these novel polymeric coatings and suggest that titanium-enrichment and nTiO(2) additives may enhance their performance.

Osteogenic differentiation of 3D cultured mesenchymal stem cells induced by bioactive peptides.

PubMed

Lukasova, Vera; Buzgo, Matej; Sovkova, Vera; Dankova, Jana; Rampichova, Michala; Amler, Evzen

2017-08-01

Bioactive peptides derived from receptor binding motifs of native proteins are a potent source of bioactive molecules that can induce signalling pathways. These peptides could substitute for osteogenesis promoting supplements. The work presented here compares three kinds of bioactive peptides derived from collagen III, bone morphogenetic protein 7 (BMP-7) and BMP-2 with their potential osteogenic activity on the model of porcine mesenchymal stem cells (pMSCs). pMSCs were cultured on electrospun polycaprolactone nanofibrous scaffolds with different concentrations of the bioactive peptides without addition of any osteogenic supplement. Analysis of pMSCs cultures included measurement of the metabolic activity and proliferation, immunofluorescence staining and also qPCR. Results showed no detrimental effect of the bioactive peptides to cultured pMSCs. Based on qPCR analysis, the bioactive peptides are specific for osteogenic differentiation with no detectable expression of collagen II. Our results further indicate that peptide derived from BMP-2 protein promoted the expression of mRNA for osteocalcin (OCN) and collagen I significantly compared to control groups and also supported deposition of OCN as observed by immunostaining method. The data suggest that bioactive peptide with an amino acid sequence of KIPKASSVPTELSAISTLYL derived from BMP-2 protein was the most potent for triggering osteogenic differentiation of pMSCs. © 2017 John Wiley & Sons Ltd.

Novel Osteogenic Ti-6Al-4V Device For Restoration Of Dental Function In Patients With Large Bone Deficiencies: Design, Development And Implementation

PubMed Central

Cohen, D. J.; Cheng, A.; Kahn, A.; Aviram, M.; Whitehead, A. J.; Hyzy, S. L.; Clohessy, R. M.; Boyan, B. D.; Schwartz, Z.

2016-01-01

Custom devices supporting bone regeneration and implant placement are needed for edentulous patients with large mandibular deficiencies where endosteal implantation is not possible. We developed a novel subperiosteal titanium-aluminum-vanadium bone onlay device produced by additive manufacturing (AM) and post-fabrication osteogenic micro-/nano-scale surface texture modification. Human osteoblasts produced osteogenic and angiogenic factors when grown on laser-sintered nano-/micro-textured surfaces compared to smooth surfaces. Surface-processed constructs caused higher bone-to-implant contact, vertical bone growth into disk pores (microCT and histomorphometry), and mechanical pull-out force at 5 and 10 w on rat calvaria compared to non surface-modified constructs, even when pre-treating the bone to stimulate osteogenesis. Surface-modified wrap-implants placed around rabbit tibias osseointegrated by 6 w. Finally, patient-specific constructs designed to support dental implants produced via AM and surface-processing were implanted on edentulous mandibular bone. 3 and 8 month post-operative images showed new bone formation and osseointegration of the device and indicated stability of the dental implants. PMID:26854193

Novel Osteogenic Ti-6Al-4V Device For Restoration Of Dental Function In Patients With Large Bone Deficiencies: Design, Development And Implementation.

PubMed

Cohen, D J; Cheng, A; Kahn, A; Aviram, M; Whitehead, A J; Hyzy, S L; Clohessy, R M; Boyan, B D; Schwartz, Z

2016-02-08

Custom devices supporting bone regeneration and implant placement are needed for edentulous patients with large mandibular deficiencies where endosteal implantation is not possible. We developed a novel subperiosteal titanium-aluminum-vanadium bone onlay device produced by additive manufacturing (AM) and post-fabrication osteogenic micro-/nano-scale surface texture modification. Human osteoblasts produced osteogenic and angiogenic factors when grown on laser-sintered nano-/micro-textured surfaces compared to smooth surfaces. Surface-processed constructs caused higher bone-to-implant contact, vertical bone growth into disk pores (microCT and histomorphometry), and mechanical pull-out force at 5 and 10 w on rat calvaria compared to non surface-modified constructs, even when pre-treating the bone to stimulate osteogenesis. Surface-modified wrap-implants placed around rabbit tibias osseointegrated by 6 w. Finally, patient-specific constructs designed to support dental implants produced via AM and surface-processing were implanted on edentulous mandibular bone. 3 and 8 month post-operative images showed new bone formation and osseointegration of the device and indicated stability of the dental implants.

Skeletogenesis in the swell shark Cephaloscyllium ventriosum.

PubMed

Eames, B Frank; Allen, Nancy; Young, Jonathan; Kaplan, Angelo; Helms, Jill A; Schneider, Richard A

2007-05-01

Extant chondrichthyans possess a predominantly cartilaginous skeleton, even though primitive chondrichthyans produced bone. To gain insights into this peculiar skeletal evolution, and in particular to evaluate the extent to which chondrichthyan skeletogenesis retains features of an osteogenic programme, we performed a histological, histochemical and immunohistochemical analysis of the entire embryonic skeleton during development of the swell shark Cephaloscyllium ventriosum. Specifically, we compared staining properties among various mineralizing tissues, including neural arches of the vertebrae, dermal tissues supporting oral denticles and Meckel's cartilage of the lower jaw. Patterns of mineralization were predicted by spatially restricted alkaline phosphatase activity earlier in development. Regarding evidence for an osteogenic programme in extant sharks, a mineralized tissue in the perichondrium of C. ventriosum neural arches, and to a lesser extent a tissue supporting the oral denticle, displayed numerous properties of bone. Although we uncovered many differences between tissues in Meckel's cartilage and neural arches of C. ventriosum, both elements impart distinct tissue characteristics to the perichondral region. Considering the evolution of osteogenic processes, shark skeletogenesis may illuminate the transition from perichondrium to periosteum, which is a major bone-forming tissue during the process of endochondral ossification.

Changes of vessel-cells complex in zones of adaptive remodeling of the bone tissue under microgravity conditions

NASA Astrophysics Data System (ADS)

Rodionova, N.; Oganov, V.; Nosova, L.

The development and differentiation of osteogenic cells in organism happen in closely topographical and functional connection with blood capillaries. We formerly proofed, that small-differentiated cells, which are in the population of perivascular cells are osteogenic cells -precursors . At the present time it is actually to clear up, how these biostructures react on conditions of less of biomechanical load on skeleton bones. We researched peculiarities of blood-bed structure and perivascular cells in metaphises of thighbones and tibial bones in rats, which were onboard the American space station SLS-2 and in experiments of modeling hypokinesia. There were used methods of cytochemistry, histology and electron microscopy. We established, that under the support and functional load decreasing in zones of bones adaptive remodeling, comparatively to control, on histosections the own volume of sinusoid capillaries reduces. The small vessels prevail here. The spaces of sinusoid capillaries are limited by 1 2 cells of the endothelia. Endotheliocytes in- general have the typical ultrastructure. Basal membranes are expressed not-distinctly. Perivascular cells don't create the unbroken layer. The population of these cells is not-homogeneous. It includes enclosed to endothelia small-differentiated forms and separating cells with sings of fibroblastic differentiation (the own volume of rough endoplasmic reticulum in cytoplasm induces). The part of these cells reacts on the alkaline phosphatase (the marker of the osteogenic differentiation). Under the conditions of support load decreasing (especially under the microgravity) there is a tendency to reducing of separating osteogenic cells number. We noted the priority of differentiating fibroblasts. It leads to further development in zones of bone remodeling of hearths of fibrous tissue, that doesn't mineralize. The obtained data are seen as one of mechanisms of osteoporosis and osteopenia development under the deficite of support load.

The impact of Wnt signalling and hypoxia on osteogenic and cementogenic differentiation in human periodontal ligament cells

PubMed Central

Li, Shuigen; Shao, Jin; Zhou, Yinghong; Friis, Thor; Yao, Jiangwu; Shi, Bin; Xiao, Yin

2016-01-01

Cementum is a periodontal support tissue that is directly connected to the periodontal ligament. It shares common traits with bone tissues, however, unlike bone, the cementum has a limited capacity for regeneration. As a result, following damage the cementum rarely, if ever, regenerates. Periodontal ligament cells (PDLCs) are able to differentiate into osteoblastic and cementogenic lineages according to specific local environmental conditions, including hypoxia, which is induced by inflammation or activation of the Wnt signalling pathway by local loading. The interactions between the Wnt signalling pathway and hypoxia during cementogenesis are of particular interest to improve the understanding of periodontal tissue regeneration. In the present study, osteogenic and cementogenic differentiation of PDLCs was investigated under hypoxic conditions in the presence and absence of Wnt pathway activation. Protein and gene expression of the osteogenic markers type 1 collagen (COL1) and runt-related transcription factor 2 (RUNX2), and cementum protein 1 (CEMP1) were used as markers for osteogenic and cementogenic differentiation, respectively. Wnt signalling activation inhibited cementogenesis, whereas hypoxia alone did not affect PDLC differentiation. However, hypoxia reversed the inhibition of cementogenesis that resulted from overexpression of Wnt signalling. Cross-talk between hypoxia and Wnt signalling pathways was, therefore, demonstrated to be involved in the differentiation of PDLCs to the osteogenic and cementogenic lineages. In summary, the present study suggests that the differentiation of PDLCs into osteogenic and cementogenic lineages is partially regulated by the Wnt signalling pathway and that hypoxia is also involved in this process. PMID:27840938

Poly(lactide-co-glycolide) nanofibrous scaffolds chemically coated with gold-nanoparticles as osteoinductive agents for osteogenesis

NASA Astrophysics Data System (ADS)

Lee, Donghyun; Heo, Dong Nyoung; Lee, Sang Jin; Heo, Min; Kim, Jeongho; Choi, Samjin; Park, Hun-Kuk; Park, Young Guk; Lim, Ho-Nam; Kwon, Il Keun

2018-02-01

Poly(lactide-co-glycolide) (PLGA) is a biocompatible and biodegradable polymer that has been widely used in devices for tissue engineering and drug delivery applications. Gold nanoparticles (GNPs) have also been used as biomaterials and have been found to have a positive effect on bone formation. In this study, we synthesized thiol end-capped PLGA (PLGA-SH) and used it for binding GNPs. This PLGA was processed into a sheet form via electrospinning. GNPs with an approximate size of 30 nm were attached onto the PLGA-SH sheet surfaces (PLGA-GNPs). This membrane was characterized by thermogravimetric analysis, ultraviolet/visible spectrophotometry, field emission scanning electron microscopy, energy dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, and confocal laser scanning microscopy. Characterization results show that the GNPs are well attached on the PLGA-SH sheet and it is possible to control the GNPs load. Additionally, in-vitro results showed that PLGA-GNPs have good biocompatibility. They were also found to enhance osteogenic differentiation of human adipose derived stem cells. From these results, we have determined that the PLGA-GNP fibers can be useful as materials for bone regeneration and can also potentially serve as drug carriers.

Enhancement of osteogenic differentiation of human adipose derived stem cells by the controlled release of platelet lysates from hybrid scaffolds produced by supercritical fluid foaming.

PubMed

Santo, Vítor E; Duarte, Ana Rita C; Popa, Elena G; Gomes, Manuela E; Mano, João F; Reis, Rui L

2012-08-20

A new generation of scaffolds capable of acting not only as support for cells but also as a source of biological cues to promote tissue regeneration is currently a hot topic of in bone Tissue Engineering (TE) research. The inclusion of growth factor (GF) controlled release functionalities in the scaffolds is a possible strategy to achieve such goal. Platelet Lysate (PL) is an autologous source of GFs, providing several bioactive agents known to act on bone regeneration. In this study, chitosan-chondroitin sulfate nanoparticles loaded with PL were included in a poly(D,L-lactic acid) foam produced by supercritical fluid foaming. The tridimensional (3D) structures were then seeded with human adipose-derived stem cells (hASCs) and cultured in vitro under osteogenic stimulus. The osteogenic differentiation of the seeded hASCs was observed earlier for the PL-loaded constructs, as shown by the earlier alkaline phosphatase peak and calcium detection and stronger Runx2 expression at day 7 of culture, in comparison with the control scaffolds. Osteocalcin gene expression was upregulated in presence of PL during all culture period, which indicates an enhanced osteogenic induction. These results suggest the synergistic effect of PL and hASCs in combinatory TE strategies and support the potential of PL to increase the multifunctionality of the 3D hybrid construct for bone TE applications. Copyright © 2012 Elsevier B.V. All rights reserved.

Adhesion of mesenchymal stem cells to polymer scaffolds occurs via distinct ECM ligands and controls their osteogenic differentiation.

PubMed

Chastain, Sara R; Kundu, Anup K; Dhar, Sanjay; Calvert, Jay W; Putnam, Andrew J

2006-07-01

The osteogenic potential of mesenchymal stem cells (MSCs) cultured on poly(lactide-co-glycolide) (PLGA) or poly(caprolactone) (PCL), two widely used polymeric biomaterials that have been reported to differentially support osteogenic differentiation, was compared in these studies. Here we report that MSCs cultured in 3-D PLGA scaffolds for up to 5 weeks significantly upregulate osteocalcin gene expression levels. By contrast, osteocalcin expression was markedly downregulated in 3-D PCL-based constructs over the same time course. We hypothesized that differential adsorption of extracellular matrix (ECM) proteins present in serum-containing culture medium and subsequent differences in integrin-mediated adhesion are responsible for these differences, and tested this hypothesis using thin (2-D) polymeric films. Supporting this hypothesis, significant amounts of fibronectin and vitronectin deposited onto both materials in serum-containing osteogenic media, with type-I collagen present in lower amounts. Adhesion-blocking studies revealed that MSCs adhere to PCL primarily via vitronectin, while type-I collagen mediates their attachment to PLGA. These adhesive mechanisms correlated with higher levels of alkaline phosphatase (ALP) activity after 2 weeks of monolayer culture on PLGA versus PCL. These data suggest that the initial adhesion of MSCs to PLGA via type-I collagen fosters osteogenesis while adhesion to PCL via vitronectin does not, and stress the need for an improved molecular understanding of cell-ECM interactions in stem cell-based therapies. Copyright (c) 2006 Wiley Periodicals, Inc.

Synergistic effect of defined artificial extracellular matrices and pulsed electric fields on osteogenic differentiation of human MSCs.

PubMed

Hess, Ricarda; Jaeschke, Anna; Neubert, Holger; Hintze, Vera; Moeller, Stephanie; Schnabelrauch, Matthias; Wiesmann, Hans-Peter; Hart, David A; Scharnweber, Dieter

2012-12-01

In vivo, bone formation is a complex, tightly regulated process, influenced by multiple biochemical and physical factors. To develop a vital bone tissue engineering construct, all of these individual components have to be considered and integrated to gain an in vivo-like stimulation of target cells. The purpose of the present studies was to investigate the synergistic role of defined biochemical and physical microenvironments with respect to osteogenic differentiation of human mesenchymal stem cells (MSCs). Biochemical microenvironments have been designed using artificial extracellular matrices (aECMs), containing collagen I (coll) and glycosaminoglycans (GAGs) like chondroitin sulfate (CS), or a high-sulfated hyaluronan derivative (sHya), formulated as coatings on three-dimensional poly(caprolactone-co-lactide) (PCL) scaffolds. As part of the physical microenvironment, cells were exposed to pulsed electric fields via transformer-like coupling (TC). Results showed that aECM containing sHya enhanced osteogenic differentiation represented by increases in ALP activity and gene-expression (RT-qPCR) of several bone-related proteins (RUNX-2, ALP, OPN). Electric field stimulation alone did not influence cell proliferation, but osteogenic differentiation was enhanced if osteogenic supplements were provided, showing synergistic effects by the combination of sHya and electric fields. These results will improve the understanding of bone regeneration processes and support the development of effective tissue engineered bone constructs. Copyright © 2012 Elsevier Ltd. All rights reserved.

Cell studies of hybridized carbon nanofibers containing bioactive glass nanoparticles using bone mesenchymal stromal cells

NASA Astrophysics Data System (ADS)

Zhang, Xiu-Rui; Hu, Xiao-Qing; Jia, Xiao-Long; Yang, Li-Ka; Meng, Qing-Yang; Shi, Yuan-Yuan; Zhang, Zheng-Zheng; Cai, Qing; Ao, Yin-Fang; Yang, Xiao-Ping

2016-12-01

Bone regeneration required suitable scaffolding materials to support the proliferation and osteogenic differentiation of bone-related cells. In this study, a kind of hybridized nanofibrous scaffold material (CNF/BG) was prepared by incorporating bioactive glass (BG) nanoparticles into carbon nanofibers (CNF) via the combination of BG sol-gel and polyacrylonitrile (PAN) electrospinning, followed by carbonization. Three types (49 s, 68 s and 86 s) of BG nanoparticles were incorporated. To understand the mechanism of CNF/BG hybrids exerting osteogenic effects, bone marrow mesenchymal stromal cells (BMSCs) were cultured directly on these hybrids (contact culture) or cultured in transwell chambers in the presence of these materials (non-contact culture). The contributions of ion release and contact effect on cell proliferation and osteogenic differentiation were able to be correlated. It was found that the ionic dissolution products had limited effect on cell proliferation, while they were able to enhance osteogenic differentiation of BMSCs in comparison with pure CNF. Differently, the proliferation and osteogenic differentiation were both significantly promoted in the contact culture. In both cases, CNF/BG(68 s) showed the strongest ability in influencing cell behaviors due to its fastest release rate of soluble silicium-relating ions. The synergistic effect of CNF and BG would make CNF/BG hybrids promising substrates for bone repairing.

Novel spinal instrumentation to enhance osteogenesis and fusion: a preliminary study.

PubMed

MacEwan, Matthew R; Talcott, Michael R; Moran, Daniel W; Leuthardt, Eric C

2016-09-01

OBJECTIVE Instrumented spinal fusion continues to exhibit high failure rates in patients undergoing multilevel lumbar fusion or pseudarthrosis revision; with Grade II or higher spondylolisthesis; or in those possessing risk factors such as obesity, tobacco use, or metabolic disorders. Direct current (DC) electrical stimulation of bone growth represents a unique surgical adjunct in vertebral fusion procedures, yet existing spinal fusion stimulators are not optimized to enhance interbody fusion. To develop an advanced method of applying DC electrical stimulation to promote interbody fusion, a novel osteogenic spinal system capable of routing DC through rigid instrumentation and into the vertebral bodies was fabricated. A pilot study was designed to assess the feasibility of osteogenic instrumentation and compare the ability of osteogenic instrumentation to promote successful interbody fusion in vivo to standard spinal instrumentation with autograft. METHODS Instrumented, single-level, posterior lumbar interbody fusion (PLIF) with autologous graft was performed at L4-5 in adult Toggenburg/Alpine goats, using both osteogenic spinal instrumentation (plus electrical stimulation) and standard spinal instrumentation (no electrical stimulation). At terminal time points (3 months, 6 months), animals were killed and lumbar spines were explanted for radiographic analysis using a SOMATOM Dual Source Definition CT Scanner and high-resolution Microcat II CT Scanner. Trabecular continuity, radiodensity within the fusion mass, and regional bone formation were examined to determine successful spinal fusion. RESULTS Quantitative analysis of average bone density in pedicle screw beds confirmed that electroactive pedicle screws used in the osteogenic spinal system focally enhanced bone density in instrumented vertebral bodies. Qualitative and quantitative analysis of high-resolution CT scans of explanted lumbar spines further demonstrated that the osteogenic spinal system induced solid bony fusion across the L4-5 disc space as early as 6 weeks postoperatively. In comparison, inactive spinal instrumentation with autograft was unable to promote successful interbody fusion by 6 months postoperatively. CONCLUSIONS Results of this study demonstrate that novel osteogenic spinal instrumentation supports interbody fusion through the focal delivery of DC electrical stimulation. With further technical development and scientific/clinical validation, osteogenic spinal instrumentation may offer a unique alternative to biological scaffolds and pharmaceutical adjuncts used in spinal fusion procedures.

Differentiation potentials of perivascular cells in the bone tissue remodeling zones under microgravity

NASA Astrophysics Data System (ADS)

Rodionova, Natalia; Katkova, Olena

Adaptive remodeling processes in the skeleton bones occur in the close topographical interconnection with blood capillaries followed by perivascular cells. Radioautographic studies with 3H- thymidine (Kimmel D.B., Fee W.S., 1980; Rodionova N.V., 1989, 2006) has shown that in osteogenesis zones there is sequential differentiation process of the perivascular cells into osteogenic ones. Using electron microscopy and cytochemistry we studied perivsacular cells in metaphysis of the rats femoral bones under conditions of modeling microgravity (28 days duration) and in femoral bones metaphyses of rats flown on board of the space laboratory (Spacelab - 2) It was revealed that population of the perivascular cells is not homogeneous in adaptive zones of the remodeling in both control and test groups (lowering support loading). This population comprises adjacent to endothelium little differentiated forms and isolated cells with differentiation features (specific volume of rough endoplasmic reticulum in cytoplasm is increased). Majority of the perivascular cells in the control group reveals reaction to alkaline phosphatase (marker of the osteogenic differentiation). In little differentiated cells this reaction is registered in nucleolus, nucleous and cytoplasm. In differentiating cells activity of the alkaline phosphatase is also detected on the outer surface of the cellular membrane. Unlike the control group in the bones of animals under microgravitaty reaction to the alkaline phosphatase is registered not for all cells of perivascular population. Part of the differentiating perivascular cells does not contain a product of the reaction. There is also visible trend of individual alkaline phosphatase containing perivascular cells amounts decrease (i.e. osteogenic cells-precursors). Under microgravity some little differentiated perivascular cells reveal destruction signs. Found decrease trend of the alkaline phosphatase containing cells (i.e. osteogenic cells) number in perivascular cells population. It is one of the mechanisms of the osteogenic process intensity decrease in bones due to lowering support loading on the bone skeleton. In particular this is confirmed by the fact that in the zones of adaptive remodeling we found fibroblasts and fibrosis zones - areas filled with non mineralized collagen fibrils on the bones surfaces. Hence it should be considered that lowering (removal) support loading slows down (or blocks) osteogenic differentiation of the perivascular cells part and stimulates differentiation of the fibroblast cells. Obtained data considered as one of the cellular mechanisms of the adaptive reactions development in spongy bone under microgravity which could lead to the bone mass loss.

Alpha-adrenergic blocker mediated osteoblastic stem cell differentiation

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

Choi, Yoon Jung; Lee, Jue Yeon; Research Center, Nano Intelligent Biomedical Engineering Corporation

Highlights: Black-Right-Pointing-Pointer Doxazocin directly up-regulated bone metabolism at a low dose. Black-Right-Pointing-Pointer Doxazocin induced osteoblastic stem cell differentiation without affecting cell proliferation. Black-Right-Pointing-Pointer This osteogenic stem cell differentiation is mediated by ERK-signal dependent pathway. -- Abstract: Recent researches have indicated a role for antihypertensive drugs including alpha- or beta-blockers in the prevention of bone loss. Some epidemiological studies reported the protective effects of those agents on fracture risk. However, there is limited information on the association with those agents especially at the mechanism of action. In the present study, we investigated the effects of doxazosin, an alpha-blocker that is clinicallymore » used for the treatment of benign prostatic hyperplasia (BPH) along with antihypertensive medication, on the osteogenic stem cell differentiation. We found that doxazosin increased osteogenic differentiation of human mesenchymal stem cells, detected by Alizarin red S staining and calcein. Doxazosin not only induced expression of alkaline phosphatase, type I collagen, osteopontin, and osteocalcin, it also resulted in increased phosphorylation of extracellular signal-regulated kinase (ERK1/2), a MAP kinase involved in osteoblastic differentiation. Treatment with U0126, a MAP kinase inhibitor, significantly blocked doxazosin-induced osteoblastic differentiation. Unrelated to activation of osteogenic differentiation by doxazosin, we found that there were no significant changes in adipogenic differentiation or in the expression of adipose-specific genes, including peroxisome proliferator-activated receptor {gamma}, aP2, or LPL. In this report, we suggest that doxazosin has the ability to increase osteogenic cell differentiation via ERK1/2 activation in osteogenic differentiation of adult stem cells, which supports the protective effects of antihypertensive drug on fracture risk and according to our data doxazosin might be useful for application in the field of bone metabolism.« less

Cytotoxicity and osteogenic potential of silicate calcium cements as potential protective materials for pulpal revascularization.

PubMed

Bortoluzzi, Eduardo A; Niu, Li-Na; Palani, Chithra D; El-Awady, Ahmed R; Hammond, Barry D; Pei, Dan-Dan; Tian, Fu-Cong; Cutler, Christopher W; Pashley, David H; Tay, Franklin R

2015-12-01

In pulpal revascularization, a protective material is placed coronal to the blood clot to prevent recontamination and to facilitate osteogenic differentiation of mesenchymal stem cells to produce new dental tissues. Although mineral trioxide aggregate (MTA) has been the material of choice for clot protection, it is easily displaced into the clot during condensation. The present study evaluated the effects of recently introduced calcium silicate cements (Biodentine and TheraCal LC) on the viability and osteogenic differentiation of human dental pulp stem cells (hDPSCs) by comparing with MTA Angelus. Cell viability was assessed using XTT assay and flow cytometry. The osteogenic potential of hDPSCs exposed to calcium silicate cements was examined using qRT-PCR for osteogenic gene expressions, alkaline phosphatase enzyme activity, Alizarin red S staining and transmission electron microscopy of extracellular calcium deposits. Parametric statistical methods were employed for analyses of significant difference among groups, with α=0.05. The cytotoxic effects of Biodentine and TheraCal LC on hDPSCs were time- and concentration-dependent. Osteogenic differentiation of hDPSCs was enhanced after exposure to Biodentine that was depleted of its cytotoxic components. This effect was less readily observed in hDPSCs exposed to TheraCal LC, although both cements supported extracellular mineralization better than the positive control (zinc oxide-eugenol-based cement). A favorable tissue response is anticipated to occur with the use of Biodentine as a blood clot-protecting material for pulpal revascularization. Further investigations with the use of in vivo animal models are required to validate the potential adverse biological effects of TheraCal LC on hDPSCs. Copyright © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Human Mesenchymal Stem Cell Behavior on Segmented Polyurethanes Prepared with Biologically Active Chain Extenders

PubMed Central

Kavanaugh, Taylor E.; Clark, Amy Y.; Chan-Chan, Lerma H.; Ramírez-Saldaña, Maricela; Vargas-Coronado, Rossana F.; Cervantes-Uc, José M.; Hernández-Sánchez, Fernando; García, Andrés J.; Cauich-Rodríguez, Juan V.

2016-01-01

The development of elastomeric, bioresorbable and biocompatible segmented polyurethanes (SPUs) for use in tissue-engineering applications has attracted considerable interest because of the existing need of mechanically tunable scaffolds for regeneration of different tissues, but the incorporation of osteoinductive molecules into SPUs has been limited. In this study, segmented polyurethanes were synthesized from poly (ε-caprolactone)diol, 4,4’-methylene bis(cyclohexyl isocyanate) (HMDI) using biologically active compounds such as ascorbic acid, L-glutamine, β-glycerol phosphate, and dexamethasone as chain extenders. Fourier Transform Infrared Spectroscopy (FTIR) revealed the formation of both urethanes and urea linkages while Differential Scanning Calorimetry, Dynamic Mechanical Analysis, X-ray Diffraction and mechanical testing showed that these polyurethanes were semi-crystalline polymers exhibiting high deformations. Cytocompatibility studies showed that only SPUs containing β-glycerol phosphate supported human mesenchymal stem cell (hMSC) adhesion, growth, and osteogenic differentiation, rendering them potentially suitable for bone tissue regeneration, whereas other SPUs failed to support either cell growth or osteogenic differentiation, or both. This study demonstrates that modification of SPUs with osteogenic compounds can lead to new cytocompatible polymers for regenerative medicine applications. PMID:26704555

Skeletogenesis in the swell shark Cephaloscyllium ventriosum

PubMed Central

Eames, B Frank; Allen, Nancy; Young, Jonathan; Kaplan, Angelo; Helms, Jill A; Schneider, Richard A

2007-01-01

Extant chondrichthyans possess a predominantly cartilaginous skeleton, even though primitive chondrichthyans produced bone. To gain insights into this peculiar skeletal evolution, and in particular to evaluate the extent to which chondrichthyan skeletogenesis retains features of an osteogenic programme, we performed a histological, histochemical and immunohistochemical analysis of the entire embryonic skeleton during development of the swell shark Cephaloscyllium ventriosum. Specifically, we compared staining properties among various mineralizing tissues, including neural arches of the vertebrae, dermal tissues supporting oral denticles and Meckel's cartilage of the lower jaw. Patterns of mineralization were predicted by spatially restricted alkaline phosphatase activity earlier in development. Regarding evidence for an osteogenic programme in extant sharks, a mineralized tissue in the perichondrium of C. ventriosum neural arches, and to a lesser extent a tissue supporting the oral denticle, displayed numerous properties of bone. Although we uncovered many differences between tissues in Meckel's cartilage and neural arches of C. ventriosum, both elements impart distinct tissue characteristics to the perichondral region. Considering the evolution of osteogenic processes, shark skeletogenesis may illuminate the transition from perichondrium to periosteum, which is a major bone-forming tissue during the process of endochondral ossification. PMID:17451531

Preparation and evaluation of polyurethane/cellulose nanowhisker bimodal foam nanocomposites for osteogenic differentiation of hMSCs.

PubMed

Shahrousvand, Ehsan; Shahrousvand, Mohsen; Ghollasi, Marzieh; Seyedjafari, Ehsan; Jouibari, Iman Sahebi; Babaei, Amir; Salimi, Ali

2017-09-01

Biocompatible and biodegradable polyurethanes (PUs) based on polycaprolactone diol (PCL) were prepared and filled with cellulose nanowhiskers (CNWs) obtained from wastepaper. The incorporated polyurethane nanocomposites were used to prepare foamed scaffolds with bimodal cell sizes through solvent casting/particulate leaching method. Sodium chloride and sugar porogens were also prepared to fabricate the scaffolds. The mechanical and thermal properties of PU/CNW nanocomposites were investigated. Incorporation of different CNWs resulted in various structures with tunable mechanical properties and biodegradability. All bimodal foam nanocomposites were biodegradable and also non-cytotoxic as revealed by MTT assay using SNL fibroblast cell line. PU/CNW foam scaffolds were used for osteogenic differentiation of human mesenchymal stem cells (hMSCs). Based on the results, such PU/CNW nanocomposites could support proliferation and osteogenic differentiation of hMSCs in three-dimensional synthetic extracellular matrix (ECM). Copyright © 2017 Elsevier Ltd. All rights reserved.

Three-dimensional culture and differentiation of human osteogenic cells in an injectable hydroxypropylmethylcellulose hydrogel.

PubMed

Trojani, Christophe; Weiss, Pierre; Michiels, Jean-François; Vinatier, Claire; Guicheux, Jérôme; Daculsi, Guy; Gaudray, Patrick; Carle, Georges F; Rochet, Nathalie

2005-09-01

The present work evaluates a newly developed silated hydroxypropylmethylcellulose (Si-HPMC)-based hydrogel as a scaffold for 3D culture of osteogenic cells. The pH variation at room temperature catalyzes the reticulation and self-hardening of the viscous polymer solution into a gelatine state. We designed reticulation time, final consistency and pH in order to obtain an easy handling matrice, suitable for in vitro culture and in vivo injection. Three human osteogenic cell lines and normal human osteogenic (HOST) cells were cultured in 3D inside this Si-HPMC hydrogel. We show here that osteosarcoma cells proliferate as clonogenic spheroids and that HOST colonies survive for at least 3 weeks. Mineralization assay and gene expression analysis of osteoblastic markers and cytokines, indicate that all the cells cultured in 3D into this hydrogel, exhibited a more mature differentiation status than cells cultured in monolayer on plastic. This study demonstrates that this Si-HPMC hydrogel is well suited to support osteoblastic survival, proliferation and differentiation when used as a new scaffold for 3D culture and represents also a potential basis for an innovative bone repair material.

Rapid Rapamycin-Only Induced Osteogenic Differentiation of Blood-Derived Stem Cells and Their Adhesion to Natural and Artificial Scaffolds

PubMed Central

Eliana, Cozzoli; Flavio, Acri; Marco, Ranalli; Giacomo, Diedenhofen

2017-01-01

Stem cells are a centerpiece of regenerative medicine research, and the recent development of adult stem cell-based therapy systems has vigorously expanded the scope and depth of this scientific field. The regeneration of damaged and/or degraded bone tissue in orthopedic, dental, or maxillofacial surgery is one of the main areas where stem cells and their regenerative potential could be used successfully, requiring tissue engineering solutions incorporating an ideal stem cell type paired with the correct mechanical support. Our contribution to this ongoing research provides a new model of in vitro osteogenic differentiation using blood-derived stem cells (BDSCs) and rapamycin, visibly expressing typical osteogenic markers within ten days of treatment. In depth imaging studies allowed us to observe the adhesion, proliferation, and differentiation of BDSCs to both titanium and bone scaffolds. We demonstrate that BDSCs can differentiate towards the osteogenic lineage rapidly, while readily adhering to the scaffolds we exposed them to. Our results show that our model can be a valid tool to study the molecular mechanisms of osteogenesis while tailoring tissue engineering solutions to these new insights. PMID:28814956

Polymeric vs hydroxyapatite-based scaffolds on dental pulp stem cell proliferation and differentiation

PubMed Central

Khojasteh, Arash; Motamedian, Saeed Reza; Rad, Maryam Rezai; Shahriari, Mehrnoosh Hasan; Nadjmi, Nasser

2015-01-01

AIM: To evaluate adhesion, proliferation and differentiation of human dental pulp stem cells (hDPSCs) on four commercially available scaffold biomaterials. METHODS: hDPSCs were isolated from human dental pulp tissues of extracted wisdom teeth and established in stem cell growth medium. hDPSCs at passage 3-5 were seeded on four commercially available scaffold biomaterials, SureOss (Allograft), Cerabone (Xenograft), PLLA (Synthetic), and OSTEON II Collagen (Composite), for 7 and 14 d in osteogenic medium. Cell adhesion and morphology to the scaffolds were evaluated by scanning electron microscopy (SEM). Cell proliferation and differentiation into osteogenic lineage were evaluated using DNA counting and alkaline phosphatase (ALP) activity assay, respectively. RESULTS: All scaffold biomaterials except SureOss (Allograft) supported hDPSC adhesion, proliferation and differentiation. hDPSCs seeded on PLLA (Synthetic) scaffold showed the highest cell proliferation and attachment as indicated with both SEM and DNA counting assay. Evaluating the osteogenic differentiation capability of hDPSCs on different scaffold biomaterials with ALP activity assay showed high level of ALP activity on cells cultured on PLLA (Synthetic) and OSTEON II Collagen (Composite) scaffolds. SEM micrographs also showed that in the presence of Cerabone (Xenograft) and OSTEON II Collagen (Composite) scaffolds, the hDPSCs demonstrated the fibroblastic phenotype with several cytoplasmic extension, while the cells on PLLA scaffold showed the osteoblastic-like morphology, round-like shape. CONCLUSION: PLLA scaffold supports adhesion, proliferation and osteogenic differentiation of hDPSCs. Hence, it may be useful in combination with hDPSCs for cell-based reconstructive therapy. PMID:26640621

Effects of flow configuration on bone tissue engineering using human mesenchymal stem cells in 3D chitosan composite scaffolds.

PubMed

Sellgren, Katelyn L; Ma, Teng

2015-08-01

Perfusion bioreactor plays important role in supporting 3D bone construct development. Scaffolds of chitosan composites have been studied to support bone tissue regeneration from osteogenic progenitor cells including human mesenchymal stem cells (hMSC). In this study, porous scaffolds of hydroxyapatite (H), chitosan (C), and gelatin (G) were fabricated by phase-separation and press-fitted in the perfusion bioreactor system where media flow is configured either parallel or transverse with respect to the scaffolds to investigate the impact of flow configuration on hMSC proliferation and osteogenic differentiation. The in vitro results showed that the interstitial flow in the transverse flow (TF) constructs reduced cell growth during the first week of culture but improved spatial cell distribution and early onset of osteogenic differentiation measured by alkaline phosphatase and expression of osteogenic genes. After 14 days of bioreactor culture, the TF constructs have comparable cell number but higher expression of bone markers genes and proteins compared to the parallel flow constructs. To evaluate ectopic bone formation, the HCG constructs seeded with hMSCs pre-cultured under two flow configurations for 7 days were implanted in CD-1 nude mice. While Masson's Trichrom staining revealed bone formation in both constructs, the TF constructs have improved spatial cell and osteoid distribution throughout the 2.0 mm constructs. The results highlight the divergent effects of media flow over the course of construct development and suggest that the flow configuration is an important parameter regulating the cellular events leading to bone construct formation in the HCG scaffolds. © 2014 Wiley Periodicals, Inc.

Engineering bioartificial tracheal tissue using hybrid fibroblast-mesenchymal stem cell cultures in collagen hydrogels.

PubMed

Naito, Hiroshi; Tojo, Takashi; Kimura, Michitaka; Dohi, Yoshiko; Zimmermann, Wolfram-Hubertus; Eschenhagen, Thomas; Taniguchi, Shigeki

2011-02-01

We aimed at providing the first in vitro and in vivo proof-of-concept for a novel tracheal tissue engineering technology. We hypothesized that bioartificial trachea (BT) could be generated from fibroblast and collagen hydrogels, mechanically supported by osteogenically-induced mesenchymal stem cells (MSC) in ring-shaped 3D-hydrogel cultures, and applied in an experimental model of rat trachea injury. Tube-shaped tissue was constructed from mixtures of rat fibroblasts and collagen in custom-made casting molds. The tissue was characterized histologically and mechanically. Ring-shaped tissue was constructed from mixtures of rat MSCs and collagen and fused to the tissue-engineered tubes to function as reinforcement. Stiffness of the biological reinforcement was enhanced by induction of osteogeneic differentiation in MSCs. Osteogenic differentiation was evaluated by assessment of osteocalcin (OC) secretion, quantification of calcium (Ca) deposit, and mechanical testing. Finally, BT was implanted to bridge a surgically-induced tracheal defect. A three-layer tubular tissue structure composed of an interconnected network of fibroblasts was constructed. Tissue collapse was prevented by the placement of MSC-containing ring-shaped tissue reinforcement around the tubular constructs. Osteogenic induction resulted in high OC secretion, high Ca deposit, and enhanced construct stiffness. Ultimately, when BT was implanted, recipient rats were able to breathe spontaneously.

Enhancement of stem cell differentiation to osteogenic lineage on hydroxyapatite-coated hybrid PLGA/gelatin nanofiber scaffolds.

PubMed

Sanaei-Rad, Parisa; Jafarzadeh Kashi, Tahereh-Sadat; Seyedjafari, Ehsan; Soleimani, Masoud

2016-11-01

A combination of polymeric materials and bioceramics has recently received a great deal of attention for bone tissue engineering applications. In the present study, hybrid nanofibrous scaffolds were fabricated from PLGA and gelatin via electrospinning and then were coated with hydroxyapatite (HA). They were then characterized and used in stem cell culture studies for the evaluation of their biological behavior and osteogenic differentiation in vitro. This study showed that all PLGA, hybrid PLGA/gelatin and HA-PLGA/gelatin scaffolds were composed of ultrafine fibers with smooth morphology and interconnected pores. The MTT assay confirmed that the scaffolds can support the attachment and proliferation of stem cells. During osteogenic differentiation, bone-related gene expression, ALP activity and biomineralization on HA-PLGA/gelatin scaffolds were higher than those observed on other scaffolds and TCPS. PLGA/gelatin electrospun scaffolds also showed higher values of these markers than TCPS. Taking together, it was shown that nanofibrous structure enhanced osteogenic differentiation of adipose-tissue derived stem cells. Furthermore, surface-coated HA stimulated the effect of nanofibers on the commitment of stem cells toward osteolineage. In conclusion, HA-PLGA/gelatin electrospun scaffolds were demonstrated to have significant potential for bone tissue engineering applications. Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

3D chitosan-gelatin-chondroitin porous scaffold improves osteogenic differentiation of mesenchymal stem cells.

PubMed

Machado, C B; Ventura, J M G; Lemos, A F; Ferreira, J M F; Leite, M F; Goes, A M

2007-06-01

A porous 3D scaffold was developed to support and enhance the differentiation process of mesenchymal stem cells (MSC) into osteoblasts in vitro. The 3D scaffold was made with chitosan, gelatin and chondroitin and it was crosslinked by EDAC. The scaffold physicochemical properties were evaluated. SEM revealed the high porosity and interconnection of pores in the scaffold; rheological measurements show that the scaffold exhibits a characteristic behavior of strong gels. The elastic modulus found in compressive tests of the crosslinked scaffold was about 50 times higher than the non-crosslinked one. After 21 days, the 3D matrix submitted to hydrolytic degradation loses above 40% of its weight. MSC were collected from rat bone marrow and seeded in chitosan-gelatin-chondroitin 3D scaffolds and in 2D culture plates as well. MSC were differentiated into osteoblasts for 21 days. Cell proliferation and alkaline phosphatase activity were followed weekly during the osteogenic process. The osteogenic differentiation of MSC was improved in 3D culture as shown by MTT assay and alkaline phosphatase activity. On the 21st day, bone markers, osteopontin and osteocalcin, were detected by the PCR analysis. This study shows that the chitosan-gelatin-chondroitin 3D structure provides a good environment for the osteogenic process and enhances cellular proliferation.

Targeted Reduction of Vascular Msx1 and Msx2 Mitigates Arteriosclerotic Calcification and Aortic Stiffness in LDLR-Deficient Mice Fed Diabetogenic Diets

PubMed Central

Cheng, Su-Li; Behrmann, Abraham; Shao, Jian-Su; Ramachandran, Bindu; Krchma, Karen; Bello Arredondo, Yoanna; Kovacs, Attila; Mead, Megan; Maxson, Robert

2014-01-01

When fed high-fat diets, male LDLR−/− mice develop obesity, hyperlipidemia, hyperglycemia, and arteriosclerotic calcification. An osteogenic Msx-Wnt regulatory program is concomitantly upregulated in the vasculature. To better understand the mechanisms of diabetic arteriosclerosis, we generated SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR−/− mice, assessing the impact of Msx1+Msx2 gene deletion in vascular myofibroblast and smooth muscle cells. Aortic Msx2 and Msx1 were decreased by 95% and 34% in SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR−/− animals versus Msx1(fl/fl);Msx2(fl/fl);LDLR−/− controls, respectively. Aortic calcium was reduced by 31%, and pulse wave velocity, an index of stiffness, was decreased in SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR−/− mice vs. controls. Fasting blood glucose and lipids did not differ, yet SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR−/− siblings became more obese. Aortic adventitial myofibroblasts from SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR−/− mice exhibited reduced osteogenic gene expression and mineralizing potential with concomitant reduction in multiple Wnt genes. Sonic hedgehog (Shh) and Sca1, markers of aortic osteogenic progenitors, were also reduced, paralleling a 78% reduction in alkaline phosphatase (TNAP)–positive adventitial myofibroblasts. RNA interference revealed that although Msx1+Msx2 supports TNAP and Wnt7b expression, Msx1 selectively maintains Shh and Msx2 sustains Wnt2, Wnt5a, and Sca1 expression in aortic adventitial myofibroblast cultures. Thus, Msx1 and Msx2 support vascular mineralization by directing the osteogenic programming of aortic progenitors in diabetic arteriosclerosis. PMID:25056439

Effects of ß-TCP scaffolds on neurogenic and osteogenic differentiation of human embryonic stem cells.

PubMed

Arpornmaeklong, Premjit; Pressler, Michael J

2018-01-01

Extracellular matrix (ECM) and adhesion molecules play crucial roles in regulating growth and differentiation of stem cells. The current study aimed to investigate the effects of beta-tricalcium phosphate (ß-TCP) scaffolds on differentiation and expression of ECM and adhesion molecules of human embryonic stem cells (hESCs). Undifferentiated hESCs were seeded on ß-TCP scaffolds and cell culture plates and cultured in growth and osteogenic medium for 21 days. Scanning electron microscopy (SEM) displayed adhesion and growth of hESCs on the porous ß-TCP scaffolds. Histological analysis, immunohistochemical staining and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) demonstrated that the scaffolds supported growth and differentiation of hESCs. Expression levels of neural crest related genes (AP2a, FoxD3, HNK1, P75, Sox1, Sox10) and osteoblast-related genes (Runx2, SPP1 and BGLA) on the scaffolds in osteogenic medium were significantly higher than on the scaffolds in growth and cell culture plates in osteogenic medium, respectively (p<0.05). Polymerase chain reaction array experiments demonstrated increased expression of ECM and adhesion molecule-related genes on the scaffolds. In conclusion, osteoconductive scaffolds such as ß-TCP scaffolds promoted differentiation of hESCs, particularly expression of genes related to neural crest stem cell and osteoblastic differentiations. Beta-TCP scaffolds could be an alternative cell culture substrate for neural crest and osteogenic differentiation of hESCs. Optimization of culture medium may be necessary to enhance lineage restriction of hESCs on the ß-TCP scaffolds. Copyright © 2017 Elsevier GmbH. All rights reserved.

Prolonged exposure to bacterial toxins downregulated expression of toll-like receptors in mesenchymal stromal cell-derived osteoprogenitors

PubMed Central

Mo, Irene Fung Ying; Yip, Kevin Hak Kong; Chan, Wing Keung; Law, Helen Ka Wai; Lau, Yu Lung; Chan, Godfrey Chi Fung

2008-01-01

Background Human mesenchymal stromal cells (MSCs, also known as mesenchymal stem cells) are multipotent cells with potential therapeutic value. Owing to their osteogenic capability, MSCs may be clinically applied for facilitating osseointegration in dental implants or orthopedic repair of bony defect. However, whether wound infection or oral microflora may interfere with the growth and osteogenic differentiation of human MSCs remains unknown. This study investigated whether proliferation and osteogenic differentiation of MSCs would be affected by potent gram-positive and gram-negative derived bacterial toxins commonly found in human settings. Results We selected lipopolysaccharide (LPS) from Escherichia coli and lipoteichoic acid (LTA) from Streptococcus pyogenes as our toxins of choice. Our findings showed both LPS and LTA did not affect MSC proliferation, but prolonged LPS challenge upregulated the osteogenic differentiation of MSCs, as assessed by alkaline phosphatase activity and calcium deposition. Because toll-like receptors (TLRs), in particularly TLR4 and TLR2, are important for the cellular responsiveness to LPS and LTA respectively, we evaluated their expression profiles serially from MSCs to osteoblasts by quantitative PCR. We found that during osteogenic differentiation, MSC-derived osteoprogenitors gradually expressed TLR2 and TLR4 by Day 12. But under prolonged incubation with LPS, MSC-derived osteoprogenitors had reduced TLR2 and TLR4 gene expression. This peculiar response to LPS suggests a possible adaptive mechanism when MSCs are subjected to continuous exposure with bacteria. Conclusion In conclusion, our findings support the potential of using human MSCs as a biological graft, even under a bacterial toxin-rich environment. PMID:18799018

In vitro comparison of the efficacy of TGF-β1 and PDGF-BB in combination with freeze-dried bone allografts for induction of osteogenic differentiation in MG-63 osteoblast-like cells.

PubMed

Vahabi, Surena; Torshabi, Maryam; Esmaeil Nejad, Azadeh

2016-12-01

Predictable regeneration of alveolar bone defects has always been a challenge in implant dentistry. Bone allografts are widely used bone substitutes with controversial osteoinductive activity. This in vitro study aimed to assess the osteogenic potential of some commercially available freeze-dried bone allografts supplemented with human recombinant platelet-derived growth factor-BB and transforming growth factor beta-1. Cell viability, mineralization, and osteogenic gene expression of MG-63 osteoblast-like cells were compared among the allograft alone, allograft/platelet-derived growth factor-BB, allograft/transforming growth factor beta-1, and allograft/platelet-derived growth factor-BB/transforming growth factor beta-1 groups. The methyl thiazol tetrazolium assay, real-time quantitative reverse transcription polymerase chain reaction and alizarin red staining were performed, respectively, for assessment of cell viability, differentiation, and mineralization at 24-72 h post treatment. The allograft with greater cytotoxic effect on MG-63 cells caused the lowest differentiation among the groups. In comparison with allograft alone, allograft/transforming growth factor beta-1, and allograft/transforming growth factor beta-1/platelet-derived growth factor-BB caused significant upregulation of bone sialoprotein and osteocalcin osteogenic mid-late marker genes, and resulted in significantly higher amounts of calcified nodules especially in mineralized non-cytotoxic allograft group. Supplementation of platelet-derived growth factor-BB alone in 5 ng/mL concentration had no significant effect on differentiation or mineralization markers. According to the results, transforming growth factor beta-1 acts synergistically with bone allografts to enhance the osteogenic differentiation potential. Therefore, this combination may be useful for rapid transformation of undifferentiated cells into bone-forming cells for bone regeneration. However, platelet-derived growth factor-BB supplementation did not support this synergistic ability to enhance osteogenic differentiation and thus, further investigations are required.

Design of electrospun nanofibrous mats for osteogenic differentiation of mesenchymal stem cells.

PubMed

Wang, Shige; Hu, Fei; Li, Jingchao; Zhang, Shuping; Shen, Mingwu; Huang, Mingxian; Shi, Xiangyang

2017-05-26

The clinical translation potential of mesenchymal stem cells (MSCs) in regenerative medicine has been greatly exploited. With the merits of high surface area to volume ratio, facile control of components, well retained topography, and the capacity to mimic the native extracellular matrix (ECM), nanofibers have received a great deal of attention as bone tissue engineering scaffolds. Electrospinning has been considered as an efficient approach for scale-up fabrication of nanofibrous materials. Electrospun nanofibers are capable of stimulating cell-matrix interaction to form a cell niche, directing cellular behavior, and promoting the MSCs adhesion and proliferation. In this review, we give a comprehensive literature survey on the mechanisms of electrospun nanofibers in supporting the MSCs differentiation. Specifically, the influences of biological and physical osteogenic inductive cues on the MSCs osteogenic differentiation are reviewed. Along with the significant advances in the field, current research challenges and future perspectives are also discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

MSC/ECM Cellular Complexes Induce Periodontal Tissue Regeneration.

PubMed

Takewaki, M; Kajiya, M; Takeda, K; Sasaki, S; Motoike, S; Komatsu, N; Matsuda, S; Ouhara, K; Mizuno, N; Fujita, T; Kurihara, H

2017-08-01

Transplantation of mesenchymal stem cells (MSCs), which possess self-renewing properties and multipotency, into a periodontal defect is thought to be a useful option for periodontal tissue regeneration. However, developing more reliable and predictable implantation techniques is still needed. Recently, we generated clumps of an MSC/extracellular matrix (ECM) complex (C-MSC), which consisted of cells and self-produced ECM. C-MSCs can regulate their cellular functions in vitro and can be grafted into a defect site, without any artificial scaffold, to induce bone regeneration. Accordingly, this study aimed to evaluate the effect of C-MSC transplantation on periodontal tissue regeneration in beagle dogs. Seven beagle dogs were employed to generate a premolar class III furcation defect model. MSCs isolated from dog ilium were seeded at a density of 7.0 × 10 4 cells/well into 24-well plates and cultured in growth medium supplemented with 50 µg/mL ascorbic acid for 4 d. To obtain C-MSCs, confluent cells were scratched using a micropipette tip and were then torn off as a cellular sheet. The sheet was rolled up to make round clumps of cells. C-MSCs were maintained in growth medium or osteoinductive medium (OIM) for 5 or 10 d. The biological properties of C-MSCs were evaluated in vitro, and their periodontal tissue regenerative activity was tested by using a dog class III furcation defect model. Immunofluorescence analysis revealed that type I collagen fabricated the form of C-MSCs. OIM markedly elevated calcium deposition in C-MSCs at day 10, suggesting its osteogenic differentiation capacity. Both C-MSCs and C-MSCs cultured with OIM transplantation without an artificial scaffold into the dog furcation defect induced periodontal tissue regeneration successfully compared with no graft, whereas osteogenic-differentiated C-MSCs led to rapid alveolar bone regeneration. These findings suggested that the use of C-MSCs refined by self-produced ECM may represent a novel predictable periodontal tissue regenerative therapy.

Gene expression of runx2, Osterix, c-fos, DLX-3, DLX-5, and MSX-2 in dental follicle cells during osteogenic differentiation in vitro.

PubMed

Morsczeck, C

2006-02-01

Recently, osteogenic precursor cells were isolated from human dental follicles, which differentiate into cementoblast- or osteoblast- like cells under in vitro conditions. However, mechanisms for osteogenic differentiation are not known in detail. Dental follicle cell long-term cultures supplemented with dexamethasone or with insulin resulted in mineralized nodules, whereas no mineralization or alkaline phosphatase activity was detected in the control culture without an osteogenic stimulus. A real-time reverse-transcriptase polymerase chain reaction (PCR) analysis was developed to investigate gene expression during osteogenic differentiation in vitro. Expression of the alkaline phosphatase (ALP) gene was detected during differentiation in the control culture and was similar to that in cultures with dexamethasone and insulin. DLX-3, DLX-5, runx2, and MSX-2 are differentially expressed during osteogenic differentiation in bone marrow mesenchymal stem cells. In dental follicle cells, gene expression of runx2, DLX-5, and MSX-2 was unaffected during osteogenic differentiation in vitro. Osteogenic differentiation appeared to be independent of MSX-2 expression; the same was true of runx2 and DLX-5, which were protagonists of osteogenic differentiation and osteocalcin promoter activity in bone marrow mesenchymal stem cells. Like in bone marrow-derived stem cells, DLX-3 gene expression was increased in dental follicle cells during osteogenic differentiation but similar to control cultures. However, gene expression of osterix was not detected in dental follicle cells during osteogenic differentiation; this gene is expressed during osteogenic differentiation in bone marrow stem cells. These real-time PCR results display molecular mechanisms in dental follicle precursor cells during osteogenic differentiation that are different from those in bone marrow-derived mesenchymal stem cells.

Use of RUNX2 Expression to Identify Osteogenic Progenitor Cells Derived from Human Embryonic Stem Cells

PubMed Central

Zou, Li; Kidwai, Fahad K.; Kopher, Ross A.; Motl, Jason; Kellum, Cory A.; Westendorf, Jennifer J.; Kaufman, Dan S.

2015-01-01

Summary We generated a RUNX2-yellow fluorescent protein (YFP) reporter system to study osteogenic development from human embryonic stem cells (hESCs). Our studies demonstrate the fidelity of YFP expression with expression of RUNX2 and other osteogenic genes in hESC-derived osteoprogenitor cells, as well as the osteogenic specificity of YFP signal. In vitro studies confirm that the hESC-derived YFP+ cells have similar osteogenic phenotypes to osteoprogenitor cells generated from bone-marrow mesenchymal stem cells. In vivo studies demonstrate the hESC-derived YFP+ cells can repair a calvarial defect in immunodeficient mice. Using the engineered hESCs, we monitored the osteogenic development and explored the roles of osteogenic supplements BMP2 and FGF9 in osteogenic differentiation of these hESCs in vitro. Taken together, this reporter system provides a novel system to monitor the osteogenic differentiation of hESCs and becomes useful to identify soluble agents and cell signaling pathways that mediate early stages of human bone development. PMID:25680477

Overexpression of Insulin-Like Growth Factor 1 Enhanced the Osteogenic Capability of Aging Bone Marrow Mesenchymal Stem Cells.

PubMed

Chen, Ching-Yun; Tseng, Kuo-Yun; Lai, Yen-Liang; Chen, Yo-Shen; Lin, Feng-Huei; Lin, Shankung

2017-01-01

Many studies have indicated that loss of the osteoblastogenic potential in bone marrow mesenchymal stem cells (bmMSCs) is the major component in the etiology of the aging-related bone deficit. But how the bmMSCs lose osteogenic capability in aging is unclear. Using 2-dimentional cultures, we examined the dose response of human bmMSCs, isolated from adult and aged donors, to exogenous insulin-like growth factor 1 (IGF-1), a growth factor regulating bone formation. The data showed that the mitogenic activity and the osteoblastogenic potential of bmMSCs in response to IGF-1 were impaired with aging, whereas higher doses of IGF-1 increased the proliferation rate and osteogenic potential of aging bmMSCs. Subsequently, we seeded IGF-1-overexpressing aging bmMSCs into calcium-alginate scaffolds and incubated in a bioreactor with constant perfusion for varying time periods to examine the effect of IGF-1 overexpression to the bone-forming capability of aging bmMSCs. We found that IGF-1 overexpression in aging bmMSCs facilitated the formation of cell clusters in scaffolds, increased the cell survival inside the cell clusters, induced the expression of osteoblast markers, and enhanced the biomineralization of cell clusters. These results indicated that IGF-1 overexpression enhanced cells' osteogenic capability. Thus, our data suggest that the aging-related loss of osteogenic potential in bmMSCs can be attributed in part to the impairment in bmMSCs' IGF-1 signaling, and support possible application of IGF-1-overexpressing autologous bmMSCs in repairing bone defect of the elderly and in producing bone graft materials for repairing large scale bone injury in the elderly.

Cytotoxicity and Osteogenic Potential of Silicate Calcium Cements as Potential Protective Materials for Pulpal Revascularization

PubMed Central

Bortoluzzi, Eduardo A.; Niu, Li-na; Palani, Chithra D.; El-Awady, Ahmed R.; Hammond, Barry D.; Pei, Dan-dan; Tian, Fu-cong; Cutler, Christopher W.; Pashley, David H.; Tay, Franklin R.

2016-01-01

Objectives In pulpal revascularization, a protective material is placed coronal to the blood clot to prevent recontamination and to facilitate osteogenic differentiation of mesenchynal stem cells to produce new dental tissues. Although mineral trioxide aggregate (MTA) has been the material of choice for clot protection, it is easily displaced into the clot during condensation. The present study evaluated the effects of recently-introduced calcium silicate cements (Biodentine and TheraCal LC) on the viability and osteogenic differentiation of human dental pulp stem cells (hDPSCs) by comparing with MTA Angelus. Methods Cell viability was assessed using XTT assay and flow cytometry. The osteogenic potential of hDPSCs exposed to calcium silicate cements was examined using qRT-PCR for osteogeic gene expressions, alkaline phosphatase enzyme activity, Alizarin red S staining and transmission electron microscopy of extracellular calcium deposits. Parametric statistical methods were employed for analyses of significant difference among groups, with α=0.05. Results The cytotoxic effects of Biodentine and TheraCal LC on hDPSCs were time- and concentration-dependent. Osteogenic differentiation of hDPSCs was enhanced after exposure to Biodentine that was depleted of its cytotoxic components. This effect was less readily observed in hDPSCs exposed to TheraCal LC, although both cements supported extracelluar mineralization better than the positive control (zinc oxide-eugenol–based cement). Significance A favorable tissue response is anticipated to occur with the use of Biodentine as a blood clot-protecting material for pulpal revascularizaiton. Further investigations with the use of in vivo animal models are required to validate the potential adverse biological effects of TheraCal LC on hDPSCs. PMID:26494267

Pretreating mesenchymal stem cells with electrical stimulation causes sustained long-lasting pro-osteogenic effects.

PubMed

Eischen-Loges, Maria; Oliveira, Karla M C; Bhavsar, Mit B; Barker, John H; Leppik, Liudmila

2018-01-01

Electrical stimulation (ES) has a long history of successful use in the clinical treatment of refractory, non-healing bone fractures and has recently been proposed as an adjunct to bone tissue-engineering treatments to optimize their therapeutic potential. This idea emerged from ES's demonstrated positive effects on stem cell migration, proliferation, differentiation and adherence to scaffolds, all cell behaviors recognized to be advantageous in Bone Tissue Engineering (BTE). In previous in vitro experiments we demonstrated that direct current ES, administered daily, accelerates Mesenchymal Stem Cell (MSC) osteogenic differentiation. In the present study, we sought to define the optimal ES regimen for maximizing this pro-osteogenic effect. Rat bone marrow-derived MSC were exposed to 100 mV/mm, 1 hr/day for three, seven, and 14 days, then osteogenic differentiation was assessed at Day 14 of culture by measuring collagen production, calcium deposition, alkaline phosphatase activity and osteogenic marker gene expression. We found that exposing MSC to ES for three days had minimal effect, while seven and 14 days resulted in increased osteogenic differentiation, as indicated by significant increases in collagen and calcium deposits, and expression of osteogenic marker genes Col1a1 , Osteopontin , Osterix and Calmodulin . We also found that cells treated with ES for seven days, maintained this pro-osteogenic activity long (for at least seven days) after discontinuing ES exposure. This study showed that while three days of ES is insufficient to solicit pro-osteogenic effects, seven and 14 days significantly increases osteogenic differentiation. Importantly, we found that cells treated with ES for only seven days, maintained this pro-osteogenic activity long after discontinuing ES exposure. This sustained positive osteogenic effect is likely due to the enhanced expression of RunX2 and Calmodulin we observed. This prolonged positive osteogenic effect, long after discontinuing ES treatment, if incorporated into BTE treatment protocols, could potentially improve outcomes and in doing so help BTE achieve its full therapeutic potential.

Cell behavior of human mesenchymal stromal cells in response to silica/collagen based xerogels and calcium deficient culture conditions.

PubMed

Wagner, Alena-Svenja; Glenske, Kristina; Henß, Anja; Kruppke, Benjamin; Rößler, Sina; Hanke, Thomas; Moritz, Andreas; Rohnke, Marcus; Kressin, Monika; Arnhold, Stefan; Schnettler, Reinhard; Wenisch, Sabine

2017-07-04

Herein, we aim to elucidate osteogenic effects of two silica-based xerogels with different degrees of bioactivity on human bone-derived mesenchymal stromal cells by means of scanning electron microscopy, quantitative PCR enhanced osteogenic effects and the formation of an extracellular matrix which could be ascribed to the sample with lower bioactivity. Given the high levels of bioactivity, the cells revealed remarkable sensitivity to extremely low calcium levels of the media. Therefore, additional experiments were performed to elucidate cell behavior under calcium deficient conditions. The results refer to capacity of the bone-derived stromal cells to overcome calcium deficiency even though proliferation, migration and osteogenic differentiation capabilities were diminished. One reason for the differences of the cellular response (on tissue culture plates versus xerogels) to calcium deficiency seems to be the positive effect of silica. The silica could be detected intracellularly as shown by time of flight-secondary ion mass spectrometry after cultivation of primary cells for 21 days on the surfaces of the xerogels. Thus, the present findings refer to different osteogenic differentiation potentials of the xerogels according to the different degrees of bioactivity, and to the role of silica as a stimulator of osteogenesis. Finally, the observed pattern of connexin-based hemichannel gating supports the assumption that connexin 43 is a key factor for calcium-mediated osteogenesis in bone-derived mesenchymal stromal cells.

Application of whey protein isolate in bone regeneration: Effects on growth and osteogenic differentiation of bone-forming cells.

PubMed

Douglas, Timothy E L; Vandrovcová, Marta; Kročilová, Nikola; Keppler, Julia K; Zárubová, Jana; Skirtach, Andre G; Bačáková, Lucie

2018-01-01

Recently, milk-derived proteins have attracted attention for applications in the biomedical field such as tissue regeneration. Whey protein isolate (WPI), especially its main component β-lactoglobulin, can modulate immunity and acts as an antioxidant, antitumor, antiviral, and antibacterial agent. There are very few reports of the application of WPI in tissue engineering, especially in bone tissue engineering. In this study, we tested the influence of different concentrations of WPI on behavior of human osteoblast-like Saos-2 cells, human adipose tissue-derived stem cells (ASC), and human neonatal dermal fibroblasts (FIB). The positive effect on growth was apparent for Saos-2 cells and FIB but not for ASC. However, the expression of markers characteristic for early osteogenic cell differentiation [type-I collagen (COL1) and alkaline phosphatase (ALP)] as well as ALP activity, increased dose-dependently in ASC. Importantly, Saos-2 cells were able to deposit calcium in the presence of WPI, even in a proliferation medium without other supplements that support osteogenic cell differentiation. The results indicate that, depending on the cell type, WPI can act as an enhancer of cell proliferation and osteogenic differentiation. Therefore, enrichment of biomaterials for bone regeneration with WPI seems a promising approach, especially due to the low cost of WPI. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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.

Stimulation of osteogenic differentiation in human osteoprogenitor cells by pulsed electromagnetic fields: an in vitro study

PubMed Central

2010-01-01

Background Although pulsed electromagnetic field (PEMF) stimulation may be clinically beneficial during fracture healing and for a wide range of bone disorders, there is still debate on its working mechanism. Mesenchymal stem cells are likely mediators facilitating the observed clinical effects of PEMF. Here, we performed in vitro experiments to investigate the effect of PEMF stimulation on human bone marrow-derived stromal cell (BMSC) metabolism and, specifically, whether PEMF can stimulate their osteogenic differentiation. Methods BMSCs derived from four different donors were cultured in osteogenic medium, with the PEMF treated group being continuously exposed to a 15 Hz, 1 Gauss EM field, consisting of 5-millisecond bursts with 5-microsecond pulses. On culture day 1, 5, 9, and 14, cells were collected for biochemical analysis (DNA amount, alkaline phosphatase activity, calcium deposition), expression of various osteoblast-relevant genes and activation of extracellular signal-regulated kinase (ERK) signaling. Differences between treated and control groups were analyzed using the Wilcoxon signed rank test, and considered significant when p < 0.05. Results Biochemical analysis revealed significant, differentiation stage-dependent, PEMF-induced differences: PEMF increased mineralization at day 9 and 14, without altering alkaline phosphatase activity. Cell proliferation, as measured by DNA amounts, was not affected by PEMF until day 14. Here, DNA content stagnated in PEMF treated group, resulting in less DNA compared to control. Quantitative RT-PCR revealed that during early culture, up to day 9, PEMF treatment increased mRNA levels of bone morphogenetic protein 2, transforming growth factor-beta 1, osteoprotegerin, matrix metalloproteinase-1 and -3, osteocalcin, and bone sialoprotein. In contrast, receptor activator of NF-κB ligand expression was primarily stimulated on day 14. ERK1/2 phosphorylation was not affected by PEMF stimulation. Conclusions PEMF exposure of differentiating human BMSCs enhanced mineralization and seemed to induce differentiation at the expense of proliferation. The osteogenic stimulus of PEMF was confirmed by the up-regulation of several osteogenic marker genes in the PEMF treated group, which preceded the deposition of mineral itself. These findings indicate that PEMF can directly stimulate osteoprogenitor cells towards osteogenic differentiation. This supports the theory that PEMF treatment may recruit these cells to facilitate an osteogenic response in vivo. PMID:20731873

RIA fractions contain mesenchymal stroma cells with high osteogenic potency.

PubMed

Kuehlfluck, Pamela; Moghaddam, Arash; Helbig, Lars; Child, Christopher; Wildemann, Britt; Schmidmaier, Gerhard

2015-12-01

The gold standard for treatment of non-union is the transplantation of autologous bone from iliac crest. As an alternative, material can be harvested by femoral reaming with the Reamer-Irrigator-Aspirator(®) (RIA)-System. This material might be a source for human mesenchymal stroma cells (MSCs) with osteogenic potency. The aim of this study was the characterisation of cells harvested with the RIA system and the comparison of their properties with cells isolated from bone marrow ("BM") and fat tissue ("adipose"). The RIA material was separated into the liquid aspiration fraction ("liquid") and the solid RIA fraction. From the solid RIA fraction the cells were cultured either directly ("native") or after collagenase digestion and filtration ("filtrate"). Stem cell characteristics were analysed and the osteogenic potential was investigated in vitro and in vivo. Fat tissue and bone marrow were harvested from nine patients (three women, six males, with a mean of 48.1 years) with atrophic non-union RIA material. The cells were isolated and characterised by flow cytometry, three lineage differentiation capacities and colony-forming unit fibroblast assay. Gene expression profiles were performed and osteogenic differentiation in vivo was analysed. All three RIA fractions contained mesenchymal stromal cells (MSCs) as demonstrated by CFU-F assay, three linage differentiation and surface marker analysis. The RIA-MSCs exhibited a significantly higher osteogenic potential in vitro compared to adipose-MSCs, whereas no difference was seen compared to BM-MSCs. Quantitative RT-PCR analysis revealed an expression of osteogenic markers in all isolated cells. The implantation of MSCs with β-TCP scaffolds into the mice muscle showed significantly higher bone formation for the filtrate RIA-MSC, native RIA-MSC and BM-MSC groups compared to the adipose-MSC group. The filtrate RIA-MSCs formed twice as much new bone in vivo compared to BM-MSCs. The present study showed high potency of cells isolated by reaming. Even in the irrigation fluid, which is normally discarded, cells with the characteristics of stromal stem cells were isolated. In comparison to adipose-MSCs and BM-MSCs, the RIA-MSCs showed a similar or even better osteogenic potential in vitro and in vivo and this supports their usability in orthopaedic surgery. Copyright © 2015 Elsevier Ltd. All rights reserved.

Screening of Osteogenic-Enhancing Short Peptides from BMPs for Biomimetic Material Applications

PubMed Central

Kanie, Kei; Kurimoto, Rio; Tian, Jing; Ebisawa, Katsumi; Narita, Yuji; Honda, Hiroyuki; Kato, Ryuji

2016-01-01

Bone regeneration is an important issue in many situations, such as bone fracture and surgery. Umbilical cord mesenchymal stem cells (UC-MSCs) are promising cell sources for bone regeneration. Bone morphogenetic proteins and their bioactive peptides are biomolecules known to enhance the osteogenic differentiation of MSCs. However, fibrosis can arise during the development of implantable biomaterials. Therefore, it is important to control cell organization by enhancing osteogenic proliferation and differentiation and inhibiting fibroblast proliferation. Thus, we focused on the screening of such osteogenic-enhancing peptides. In the present study, we developed new peptide array screening platforms to evaluate cell proliferation and alkaline phosphatase activity in osteoblasts, UC-MSCs and fibroblasts. The conditions for the screening platform were first defined using UC-MSCs and an osteogenic differentiation peptide known as W9. Next, in silico screening to define the candidate peptides was carried out to evaluate the homology of 19 bone morphogenetic proteins. Twenty-five candidate 9-mer peptides were selected for screening. Finally, the screening of osteogenic-enhancing (osteogenic cell-selective proliferation and osteogenic differentiation) short peptide was carried out using the peptide array method, and three osteogenic-enhancing peptides were identified, confirming the validity of this screening. PMID:28773850

Osteogenic differentiation of immature osteoblasts: Interplay of cell culture media and supplements.

PubMed

Brauer, A; Pohlemann, T; Metzger, W

2016-01-01

Differentiation of immature osteoblasts to mature osteoblasts in vitro initially was induced by supplementing the medium with β-gylcerophosphate and dexamethasone. Later, ascorbic acid, vitamin D3, vitamin K3 and TGFβ1 were used in varying concentrations as supplements to generate a mature osteoblast phenotype. We tested the effects of several combinations of cell culture media, seeding protocols and osteogenic supplements on osteogenic differentiation of human primary osteoblasts. Osteogenic differentiation was analyzed by staining alkaline phosphatase (ALP) with 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) and by von Kossa staining of deposited calcium phosphate. The combinations of culture media and supplements significantly influenced osteogenic differentiation, but the seeding protocol did not. Staining of ALP and calcium phosphate could be achieved only if our own mix of osteogenic supplements was used in combination with Dulbecco's modified Eagle medium or if a commercial mix of osteogenic supplements was used in combination with osteoblast growth medium. Especially for von Kossa, we observed great variations in the staining intensity. Because osteogenic differentiation is a complex process, the origin of the osteoblasts, cell culture media and osteogenic supplements should be established by preliminary experiments to achieve optimal differentiation. Staining of ALP or deposited calcium phosphate should be supplemented with qRT-PCR studies to learn more about the influence of specific supplements on osteogenic markers.

Multipotency of skeletal muscle stem cells on their native substrate and the expression of Connexin 43 during adoption of adipogenic and osteogenic fate.

PubMed

Elashry, Mohamed I; Heimann, Manuela; Wenisch, Sabine; Patel, Ketan; Arnhold, Stefan

2017-10-01

Muscle regeneration is performed by resident muscle stem cells called satellite cells (SC). However they are multipotent, being able to adopt adipogenic and osteogenic fate under the correct stimuli. Since SC behavior can be regulated by the extra-cellular matrix, we examined the robustness of the myogenic programme of SC on their native substrate-the surface of a myofiber. We show that the native substrate supports myogenic differentiation judged by the expression of members of the Myogenic Determination Factor (MRF) family. However SC even on their native substrate can be induced into adopting adipogenic or osteogenic fate. Furthermore conditions that support adipose or bone formation inhibit the proliferation of SC progeny as well as their migration. We show that Connexin43 (Cx43), a gap junction complex protein, is only expressed by activated and not quiescent SC. Furthermore, it is not expressed by SC that are in the process of changing their fate. Lastly we show that intact adult mouse muscle contains numerous cells expressing Cx43 and that the density of these cells seems to be related to capillary density. We suggest the Cx43 expression is localized to angioblasts and is more prominent in oxidative slow muscle compared to glycolytic fast muscle. Crown Copyright © 2017. Published by Elsevier GmbH. All rights reserved.

Modeling and analysis of molecularinteraction between Smurf1-WW2 domain and various isoforms of LIM mineralization protein.

PubMed

Sangadala, Sreedhara; Boden, Scott D; Metpally, Raghu Prasad Rao; Reddy, Boojala Vijay B

2007-08-15

LIM Mineralization Protein-1 (LMP-1) has been cloned and shown to be osteoinductive. Our efforts to understand the mode of action of LMP-1 led to the determination that LMP-1 interacts with Smad Ubiquitin Regulatory Factor-1 (Smurf1). Smurf1 targets osteogenic Smads, Smad1/5, for ubiquitin-mediated proteasomal degradation. Smurf1 interaction with LMP-1 or Smads is based on the presence of unique WW-domain interacting motif in these target molecules. By performing site-directed mutagenesis and binding studies in vitro on purified recombinant proteins, we identified a specific motif within the osteogenic region of several LMP isoforms that is necessary for Smurf1 interaction. Similarly, we have identified that the WW2 domain of Smurf1 is necessary for target protein interaction. Here, we present a homology-based modeling of the Smurf1 WW2 domain and its interacting motif of LMP-1. We performed computational docking of the interacting domains in Smurf1 and LMPs to identify the key amino acid residues involved in their binding regions. In support of the computational predictions, we also present biochemical evidence supporting the hypothesis that the physical interaction of Smurf1 and osteoinductive forms of LMP may prevent Smurf1 from targeting osteogenic Smads by ubiquitin-mediated proteasomal degradation.

Epigallocatechin-3-gallate (EGCG) as a pro-osteogenic agent to enhance osteogenic differentiation of mesenchymal stem cells from human bone marrow: an in vitro study.

PubMed

Jin, Pan; Wu, Huayu; Xu, Guojie; Zheng, Li; Zhao, Jinmin

2014-05-01

The proliferation and osteogenic capacity of mesenchymal stem cells (MSCs) needs to be improved for their use in cell-based therapy for osteoporosis. (-)-Epigallocatechin-3-gallate (EGCG), one of the green tea catechins, has been widely investigated in studies of osteoblasts and osteoclasts. However, no consensus on its role as an osteogenic inducer has been reached, possibly because of the various types of cell lines examined and the range of concentrations of EGCG used. In this study, the osteogenic effects of EGCG are studied in primary human bone-marrow-derived MSCs (hBMSCs) by detecting cell proliferation, alkaline phosphatase (ALP) activity and the expression of relevant osteogenic markers. Our results show that EGCG has a strong stimulatory effect on hBMSCs developing towards the osteogenic lineage, especially at a concentration of 5 μM, as evidenced by an increased ALP activity, the up-regulated expression of osteogenic genes and the formation of bone-like nodules. Further exploration has indicated that EGCG directes osteogenic differentiation via the continuous up-regulation of Runx2. The underlying mechanism might involve EGCG affects on osteogenic differentiation through the modulation of bone morphogenetic protein-2 expression. EGCG has also been found to promote the proliferation of hBMSCs in a dose-dependent manner. This might be associated with its antioxidative effect leading to favorable amounts of reactive oxygen species in the cellular environment. Our study thus indicates that EGCG can be used as a pro-osteogenic agent for the stem-cell-based therapy of osteoporosis.

Assessment of cellular materials generated by co-cultured 'inflamed' and healthy periodontal ligament stem cells from patient-matched groups.

PubMed

Tang, Hao-Ning; Xia, Yu; Xu, Jie; Tian, Bei-Min; Zhang, Xi-Yu; Chen, Fa-Ming

2016-08-01

Recently, stem cells derived from the'inflamed' periodontal ligament (PDL) tissue of periodontally diseased teeth (I-PDLSCs) have been increasingly suggested as a more readily accessible source of cells for regenerative therapies than those derived from healthy PDL tissue (H-PDLSCs). However, substantial evidence indicates that I-PDLSCs exhibit impaired functionalities compared with H-PDLSCs. In this study, patient-matched I-PDLSCs and H-PDLSCs were co-cultured at various ratios. Cellular materials derived from these cultures were investigated regarding their osteogenic potential in vitro and capacity to form new bone following in vivo transplantation. While patient-matched I-PDLSCs and H-PDLSCs could co-exist in co-culture systems, the proportion of I-PDLSCs tended to increase during in vitro incubation. Compared with H-PDLSC monoculture, the presence of I-PDLSCs in the co-cultures appeared to enhance the overall cell proliferation. Although not completely rescued, the osteogenic and regenerative potentials of the cellular materials generated by co-cultured I-PDLSCs and H-PDLSCs were significantly improved compared with those derived from I-PDLSC monocultures. Notably, cells in co-cultures containing either 50% I-PDLSCs plus 50% H-PDLSCs or 25% I-PDLSCs plus 75% H-PDLSCs expressed osteogenesis-related proteins and genes at levels similar to those expressed in H-PDLSC monocultures (P>0.05). Irrespective of the percentage of I-PDLSCs, robust cellular materials were obtained from co-cultures with 50% or more H-PDLSCs, which exhibited equivalent potential to form new bone in vivo compared with sheets generated by H-PDLSC monocultures. These data suggest that the co-culture of I-PDLSCs with patient-matched H-PDLSCs is a practical and effective method for increasing the overall osteogenic and regenerative potentials of resultant cellular materials. Copyright © 2016 Elsevier Inc. All rights reserved.

Impact of electromagnetic fields on stem cells: common mechanisms at the crossroad between adult neurogenesis and osteogenesis

PubMed Central

Leone, Lucia; Podda, Maria Vittoria; Grassi, Claudio

2015-01-01

In the recent years adult neural and mesenchymal stem cells have been intensively investigated as effective resources for repair therapies. In vivo and in vitro studies have provided insights on the molecular mechanisms underlying the neurogenic and osteogenic processes in adulthood. This knowledge appears fundamental for the development of targeted strategies to manipulate stem cells. Here we review recent literature dealing with the effects of electromagnetic fields on stem cell biology that lends support to their use as a promising tool to positively influence the different steps of neurogenic and osteogenic processes. We will focus on recent studies revealing that extremely-low frequency electromagnetic fields enhance adult hippocampal neurogenesis by inducing epigenetic modifications on the regulatory sequences of genes responsible for neural stem cell proliferation and neuronal differentiation. In light of the emerging critical role played by chromatin modifications in maintaining the stemness as well as in regulating stem cell differentiation, we will also attempt to exploit epigenetic changes that can represent common targets for electromagnetic field effects on neurogenic and osteogenic processes. PMID:26124705

Naringin Stimulates Osteogenic Differentiation of Rat Bone Marrow Stromal Cells via Activation of the Notch Signaling Pathway

PubMed Central

Yu, Guo-yong; Zheng, Gui-zhou; Chang, Bo; Hu, Qin-xiao; Lin, Fei-xiang; Liu, De-zhong; Wu, Chu-cheng; Du, Shi-xin

2016-01-01

Naringin is a major flavonoid found in grapefruit and is an active compound extracted from the Chinese herbal medicine Rhizoma Drynariae. Naringin is a potent stimulator of osteogenic differentiation and has potential application in preventing bone loss. However, the signaling pathway underlying its osteogenic effect remains unclear. We hypothesized that the osteogenic activity of naringin involves the Notch signaling pathway. Rat bone marrow stromal cells (BMSCs) were cultured in osteogenic medium containing-naringin, with or without DAPT (an inhibitor of Notch signaling), the effects on ALP activity, calcium deposits, osteogenic genes (ALP, BSP, and cbfa1), adipogenic maker gene PPARγ2 levels, and Notch expression were examined. We found that naringin dose-dependently increased ALP activity and Alizarin red S staining, and treatment at the optimal concentration (50 μg/mL) increased mRNA levels of osteogenic genes and Notch1 expression, while decreasing PPARγ2 mRNA levels. Furthermore, treatment with DAPT partly reversed effects of naringin on BMSCs, as judged by decreases in naringin-induced ALP activity, calcium deposits, and osteogenic genes expression, as well as upregulation of PPARγ2 mRNA levels. These results suggest that the osteogenic effect of naringin partly involves the Notch signaling pathway. PMID:27069482

Basic fibroblastic growth factor affects the osteogenic differentiation of dental pulp stem cells in a treatment-dependent manner.

PubMed

Qian, J; Jiayuan, W; Wenkai, J; Peina, W; Ansheng, Z; Shukai, S; Shafei, Z; Jun, L; Longxing, N

2015-07-01

To determine how basic fibroblastic growth factor (bFGF) affected the osteogenic differentiation of human dental pulp stem cells (DPSCs) in vitro and in vivo. Basic fibroblastic growth factor stimulation of DPSCs was divided into a pre-treatment period and an osteogenic differentiation period. Alizarin red quantification experiments and alkaline phosphatase activity quantification assay were performed to examine the osteogenic differentiation of DPSCs after different bFGF stimulation. Quantification reverse transcription polymerase chain reaction was used to analyze the osteogenic gene expression of DPSCs after different bFGF stimulation. In addition, DPSCs that received the 1 and 2 weeks bFGF pre-treatments as in the in vitro experiments were mineralized for 1 week and seeded into hydroxyapatite/tricalcium phosphate (HA/TCP) pills and subcutaneously transplanted into naked mice for 2 or 3 months. The transplants were removed, sliced and stained using Modified Ponceau Trichrome Stain to observe the formation of mineralized tissue. Basic fibroblastic growth factor stimulation in the osteogenic differentiation period decreased the in vitro osteogenic differentiation ability of DPSCs. One week pre-treatment with bFGF increased the in vitro osteogenic differentiation ability of DPSCs, whereas 2 weeks pre-treatment with bFGF decreased the in vitro osteogenic differentiation ability of DPSCs. The pre-treatment period was vital for the osteogenic differentiation of DPSCs in vitro. The in vivo results were similar to the in vitro results. Basic fibroblastic growth factor affected the osteogenic differentiation of DPSCs in a treatment-dependent manner both in vitro and in vivo. © 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Bone marrow stromal cells on a three-dimensional bioactive fiber mesh undergo osteogenic differentiation in the absence of osteogenic media supplements: the effect of silanol groups.

PubMed

Rodrigues, Márcia T; Leonor, Isabel B; Gröen, Nathalie; Viegas, Carlos A; Dias, Isabel R; Caridade, Sofia G; Mano, João F; Gomes, Manuela E; Reis, Rui L

2014-10-01

Osteogenic differentiation is a tightly regulated process dependent on the stimuli provided by the micro-environment. Silicon-substituted materials are known to have an influence on the osteogenic phenotype of undifferentiated and bone-derived cells. This study aims to investigate the bioactivity profile as well as the mechanical properties of a blend of starch and poly-caprolactone (SPCL) polymeric fiber mesh scaffolds functionalized with silanol (Si-OH) groups as key features for bone tissue engineering strategies. The scaffolds were made from SPCL by a wet spinning technique. A calcium silicate solution was used as a non-solvent to develop an in situ functionalization with Si-OH groups in a single-step approach. We also explored the relevance of silicon incorporated in SPCL-Si scaffolds to the in vitro osteogenic process of goat bone marrow stromal cells (gBMSCs) with and without osteogenic supplements in the culture medium. We hypothesized that SPCL-Si scaffolds could act as physical and chemical millieus to induce per se the osteogenic differentiation of gBMSCs. Results show that osteogenic differentiation of gBMSCs and the production of a mineralized extracellular matrix on bioactive SPCL-Si scaffolds occur for up to 2weeks, even in the absence of osteogenic supplements in the culture medium. The omission of media supplements to induce osteogenic differentiation is a promising feature towards simplified and cost-effective cell culturing procedures of a potential bioengineered product, and concomitant translation into the clinical field. Thus, the present work demonstrates that SPCL-Si scaffolds and their intrinsic properties sustain gBMSC osteogenic features in vitro, even in the absence of osteogenic supplements to the culture medium, and show great potential for bone regeneration strategies. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Targeted reduction of vascular Msx1 and Msx2 mitigates arteriosclerotic calcification and aortic stiffness in LDLR-deficient mice fed diabetogenic diets.

PubMed

Cheng, Su-Li; Behrmann, Abraham; Shao, Jian-Su; Ramachandran, Bindu; Krchma, Karen; Bello Arredondo, Yoanna; Kovacs, Attila; Mead, Megan; Maxson, Robert; Towler, Dwight A

2014-12-01

When fed high-fat diets, male LDLR(-/-) mice develop obesity, hyperlipidemia, hyperglycemia, and arteriosclerotic calcification. An osteogenic Msx-Wnt regulatory program is concomitantly upregulated in the vasculature. To better understand the mechanisms of diabetic arteriosclerosis, we generated SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) mice, assessing the impact of Msx1+Msx2 gene deletion in vascular myofibroblast and smooth muscle cells. Aortic Msx2 and Msx1 were decreased by 95% and 34% in SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) animals versus Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) controls, respectively. Aortic calcium was reduced by 31%, and pulse wave velocity, an index of stiffness, was decreased in SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) mice vs. controls. Fasting blood glucose and lipids did not differ, yet SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) siblings became more obese. Aortic adventitial myofibroblasts from SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) mice exhibited reduced osteogenic gene expression and mineralizing potential with concomitant reduction in multiple Wnt genes. Sonic hedgehog (Shh) and Sca1, markers of aortic osteogenic progenitors, were also reduced, paralleling a 78% reduction in alkaline phosphatase (TNAP)-positive adventitial myofibroblasts. RNA interference revealed that although Msx1+Msx2 supports TNAP and Wnt7b expression, Msx1 selectively maintains Shh and Msx2 sustains Wnt2, Wnt5a, and Sca1 expression in aortic adventitial myofibroblast cultures. Thus, Msx1 and Msx2 support vascular mineralization by directing the osteogenic programming of aortic progenitors in diabetic arteriosclerosis. © 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

The effect of low static magnetic field on osteogenic and adipogenic differentiation potential of human adipose stromal/stem cells

NASA Astrophysics Data System (ADS)

Marędziak, Monika; Śmieszek, Agnieszka; Tomaszewski, Krzysztof A.; Lewandowski, Daniel; Marycz, Krzysztof

2016-01-01

The aim of this work was to investigate the effects of static magnetic field (SMF) on the osteogenic properties of human adipose derived mesenchymal stem cells (hASCs). In this study in seven days viability assay we examined the impact of SMF on cells proliferation rate, population doubling time, and ability to form single-cell derived colonies. We have also examined cells' morphology, ultrastructure and osteogenic properties on the protein as well as mRNA level. We established a complex approach, which enabled us to obtain information about SMF and hASCs potential in the context of differentiation into osteogenic and adipogenic lineages. We demonstrated that SMF enhances both viability and osteogenic properties of hASCs through higher proliferation factor and shorter population doubling time. We have also observed asymmetrically positioned nuclei and organelles after SMF exposition. With regards to osteogenic properties we observed increased levels of osteogenic markers i.e. osteopontin, osteocalcin and increased ability to form osteonodules with positive reaction to Alizarin Red dye. We have also shown that SMF besides enhancing osteogenic properties of hASCs, simultaneously decreases their ability to differentiate into adipogenic lineage. Our results clearly show a direct influence of SMF on the osteogenic potential of hASCs. These results provide key insights into the role of SMF on their cellular fate and properties.

The effect of noncoherent red light irradiation on proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells.

PubMed

Peng, Fei; Wu, Hua; Zheng, Yadong; Xu, Xiqiang; Yu, Jizhe

2012-05-01

Mesenchymal stem cells (MSCs) are promising for use in regenerative medicine. Low-level light irradiation (LLLI) has been shown to modulate various processes in different biological systems. The aim of our study was to investigate the effect of red light emitted from a light-emitting diode (LED) on bone marrow MSCs with or without osteogenic supplements. MSCs both with and without osteogenic supplements were divided into four groups, and each group was irradiated at doses of 0, 1, 2 and 4 J/cm(2). Cellular proliferation was evaluated using WST-8 and 5-ethynyl-2'-deoxyuridine (EdU) fluorescence staining. The alkaline phosphatase activity, mineralization, and expression of osteoblast master genes (Col1α1, Alpl, Bglap and Runx2) were monitored as indicators of MSC differentiation towards osteoblasts. In groups without osteogenic supplements, red light at all doses significantly stimulated cellular proliferation, whereas the osteogenic phenotype of the MSCs was not enhanced. In groups with osteogenic supplements, red light increased alkaline phosphatase activity and mineralized nodule formation, and stimulated the expression of Bglap and Runx2, but decreased cellular proliferation. In conclusion, nonconherent red light can promote proliferation but cannot induce osteogenic differentiation of MSCs in normal media, while it enhances osteogenic differentiation and decreases proliferation of MSCs in media with osteogenic supplements.

Mesenchymal stem cell-derived exosomes have altered microRNA profiles and induce osteogenic differentiation depending on the stage of differentiation

PubMed Central

Wang, Xiaoqin; Omar, Omar; Vazirisani, Forugh; Thomsen, Peter

2018-01-01

Human mesenchymal stem cell (hMSC)-derived exosomes have shown regenerative effects, but their role in osteogenesis and the underlying mechanism are yet to be determined. In this study, we examined the time-course secretion of exosomes by hMSCs during the entire process of osteogenic differentiation. Exosomes derived from hMSCs in various stages of osteogenic differentiation committed homotypic cells to differentiate towards osteogenic lineage, but only exosomes from late stages of osteogenic differentiation induced extracellular matrix mineralisation. Exosomes from expansion and early and late stages of osteogenic differentiation were internalised by a subpopulation of hMSCs. MicroRNA profiling revealed a set of differentially expressed exosomal microRNAs from the late stage of osteogenic differentiation, which were osteogenesis related. Target prediction demonstrated that these microRNAs enriched pathways involved in regulation of osteogenic differentiation and general mechanisms how exosomes exert their functions, such as “Wnt signalling pathway” and “endocytosis”. Taken together, the results show that MSCs secrete exosomes with different biological properties depending on differentiation stage of their parent cells. The exosomal cargo transferred from MSCs in the late stage of differentiation induces osteogenic differentiation and mineralisation. Moreover, it is suggested that the regulatory effect on osteogenesis by exosomes is at least partly exerted by exosomal microRNA. PMID:29447276

The influence of stereolithographic scaffold architecture and composition on osteogenic signal expression with rat bone marrow stromal cells

PubMed Central

Kim, Kyobum; Dean, David; Wallace, Jonathan; Breithaupt, Rob; Mikos, Antonios G.; Fisher, John P.

2011-01-01

Scaffold design parameters, especially physical construction factors such as mechanical stiffness of substrate materials, pore size of 3D porous scaffolds, and channel geometry, are known to influence the osteogenic signal expression and subsequent differentiation of a transplanted cell population. In this study of photocrosslinked poly(propylene fumarate) (PPF) and diethyl fumarate (DEF) scaffolds, the effect of DEF incorporation ratio and pore size on the osteogenic signal expression of rat bone marrow stromal cells (BMSCs) was investigated. Results demonstrated that DEF concentrations and pore sizes that led to increased scaffold mechanical stiffness also upregulated osteogenic signal expression, including bone morphogenic protein-2 (BMP-2), fibroblast growth factors-2 (FGF-2), transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF), and Runx2 transcriptional factor. Similar scaffold fabrication parameters supported rapid BMSC osteoblastic differentiation, as demonstrated by increased alkaline phosphatase (ALP) and osteocalcin expression. When scaffolds with random architecture, fabricated by porogen leaching, were compared to those with controlled architecture, fabricated by stereolithography (SLA), results showed that SLA scaffolds with the highly permeable and porous channels also have significantly higher expression of FGF-2, TGF-β1, and VEGF. Subsequent ALP expression and osteopontin secretion were also significantly increased in SLA scaffolds. Based upon these results, we conclude that scaffold properties provided by additive manufacturing techniques such as SLA fabrication, particularly increased mechanical stiffness and high permeability, may stimulate dramatic BMSC responses that promote rapid bone tissue regeneration. PMID:21396709

Osteogenic Differentiation of Human and Ovine Bone Marrow Stromal Cells in response to β-Glycerophosphate and Monosodium Phosphate.

PubMed

Bottagisio, Marta; Lovati, Arianna B; Lopa, Silvia; Moretti, Matteo

2015-08-01

Bone defects are severe burdens in clinics, and thus cell therapy offers an alternative strategy exploiting the features of bone marrow stromal cells (BMSCs). Sheep are a suitable orthopedic preclinical model for similarities with humans. This study compares the influence of two phosphate sources combined with bone morphogenetic protein-2 (BMP-2) on the osteogenic potential of human and ovine BMSCs. β-Glycerophosphate (β-GlyP) and monosodium phosphate (NaH2PO4) were used as organic and inorganic phosphate sources. Osteogenic differentiation of the BMSCs was assessed by calcified matrix, alkaline phosphatase (ALP) activity, and gene expression analysis. A higher calcified matrix deposition was detected in BMSCs cultured with NaH2PO4. Although no significant differences were detected among media for human BMSCs, β-GlyP with or without BMP-2 determined a positive trend in ALP levels compared to NaH2PO4. In contrast, NaH2PO4 had a positive effect on ALP levels in ovine BMSCs. β-GlyP better supported the expression of COL1A1 in human BMSCs, whereas all media enhanced RUNX2 and SPARC expression. Ovine BMSCs responded poorly to any media for RUNX2, COL1A1, and SPARC expression. NaH2PO4 improved calcified matrix deposition without upregulating the transcriptional expression of osteogenic markers. A further optimization of differentiation protocols needs to be performed to translate the procedures from preclinical to clinical models.

Osteogenic potency of a 3-dimensional scaffold-free bonelike sphere of periodontal ligament stem cells in vitro.

PubMed

Singhatanadgit, Weerachai; Varodomrujiranon, Manatsanan

2013-12-01

The present study aimed to investigate the osteogenic potency of scaffold-free 3-dimensional (3D) spheres of periodontal ligament stem cells (PDLSCs). The osteogenic potency of PDLSC spheres was determined by the ability to form mineralization and to express key osteogenesis-associated genes. The alkaline phosphatase (ALP) activity and the protein content of PDLSC spheres were also measured. The 3D sphere developed its osteogenic potency in a time-dependent manner, containing approximately 10-fold higher mineralization, 5-fold higher protein content, and 4-fold greater ALP activity than those in the controls. The expression of key osteogenic genes was also upregulated in the 3D PDLSC spheres. Cellular outgrowth was observed when reintroduced into 2D culture. PDLSCs were able to undergo osteogenic differentiation in a scaffold-free 3D culture, producing bonelike mineralization in vitro. This suggests, at least in vitro, the osteogenic potency of the 3D PDLSC spheres. Copyright © 2013 Elsevier Inc. All rights reserved.

Synthesis, characterization, and in-vitro cytocompatibility of amorphous β-tri-calcium magnesium phosphate ceramics.

PubMed

Singh, Satish S; Roy, Abhijit; Lee, Boeun; Banerjee, Ipsita; Kumta, Prashant N

2016-10-01

Biphasic mixtures of crystalline β-tricalcium magnesium phosphate (β-TCMP) and an amorphous calcium magnesium phosphate have been synthesized and reported to support enhanced hMSC differentiation in comparison to β-tricalcium phosphate (β-TCP) due to the release of increased amounts of bioactive ions. In the current study, completely amorphous β-TCMP has been synthesized which is capable of releasing increased amounts of Mg(2+) and PO4(3-) ions, rather than a biphasic mixture as earlier reported. The amorphous phase formed was observed to crystallize between temperatures of 400-600°C. The scaffolds prepared with amorphous β-TCMP were capable of supporting enhanced hMSC proliferation and differentiation in comparison to commercially available β-TCP. However, a similar gene expression of mature osteoblast markers, OCN and COL-1, in comparison to biphasic β-TCMP was observed. To further study the role of Mg(2+) and PO4(3-) ions in regulating hMSC osteogenic differentiation, the capability of hMSCs to mineralize in growth media supplemented with Mg(2+) and PO4(3-) ions was studied. Interestingly, 5mM PO4(3-) supported mineralization while the addition of 5mM Mg(2+) to 5mM PO4(3-) inhibited mineralization. It was therefore concluded that the release of Ca(2+) ions from β-TCMP scaffolds also plays a role in regulating osteogenic differentiation on these scaffolds and it is noted that further work is required to more accurately determine the exact role of Mg(2+) in regulating hMSC osteogenic differentiation. Copyright © 2016 Elsevier B.V. All rights reserved.

Role of the unfolded protein response in topography-induced osteogenic differentiation in rat bone marrow mesenchymal stem cells.

PubMed

Shi, Mengqi; Song, Wen; Han, Tianxiao; Chang, Bei; Li, Guangwen; Jin, Jianfeng; Zhang, Yumei

2017-05-01

The topography of biomaterials can significantly influence the osteogenic differentiation of cells. Understanding topographical signal transduction is critical for developing biofunctional surfaces, but the current knowledge is insufficient. Recently, numerous reports have suggested that the unfolded protein response (UPR) and osteogenic differentiation are inter-linked. Therefore, we hypothesize that the UPR pathway may be involved in the topography-induced osteogenesis. In the present study, different surface topographies were fabricated on pure titanium foils and the endoplasmic reticulum (ER) stress and UPR pathway were systematically investigated. We found that ER stress and the PERK-eIF2α-ATF4 pathway were activated in a time- and topography-dependent manner. Additionally, the activation of the PERK-eIF2α-ATF4 pathway by different topographies was in line with their osteogenic induction capability. More specifically, the osteogenic differentiation could be enhanced or weakened when the PERK-eIF2α-ATF4 pathway was promoted or inhibited, respectively. Furthermore, tuning of the degree of ER stress with different concentrations of thapsigargin revealed that mild ER stress promotes osteogenic differentiation, whereas excessive ER stress inhibits osteogenic differentiation and causes apoptosis. Taken together, our findings suggest that the UPR may play a critical role in topography-induced osteogenic differentiation, which may help to provide new insights into topographical signal transduction. Suitable implant surface topography can effectively improve bioactivity and eventual bone affinity. However, the mechanism of topographical signaling transduction is unclear and criteria for designation of an appropriate implant surface topography is lacking. This study shows that the ER stress and PERK-eIF2α-ATF4 pathway were activated by micro- and micro/nano-topographies, which is corresponding to the osteogenic induction abilities of these topographies. Furthermore, we have found that mild ER stress improves osteogenic differentiation, whereas excessive ER stress inhibits osteogenic differentiation and causes apoptosis. Our findings demonstrate that the UPR plays a critical role in the topography induced osteogenic differentiation, which may help to provide new insights into the topographical signaling transduction. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Mesenchymal Stem Cell Spheroids Retain Osteogenic Phenotype Through α2β1 Signaling

PubMed Central

Murphy, Kaitlin C.; Hoch, Allison I.; Harvestine, Jenna N.; Zhou, Dejie

2016-01-01

The induction of mesenchymal stem cells (MSCs) toward the osteoblastic lineage using osteogenic supplements prior to implantation is one approach under examination to enhance their bone-forming potential. MSCs rapidly lose their induced phenotype upon removal of the soluble stimuli; however, their bone-forming potential can be sustained when provided with continued instruction via extracellular matrix (ECM) cues. In comparison with dissociated cells, MSC spheroids exhibit improved survival and secretion of trophic factors while maintaining their osteogenic potential. We hypothesized that entrapment of MSC spheroids formed from osteogenically induced cells would exhibit better preservation of their bone-forming potential than would dissociated cells from monolayer culture. Spheroids exhibited comparable osteogenic potential and increased proangiogenic potential with or without osteogenic preconditioning versus monolayer-cultured MSCs. Spheroids were then entrapped in collagen hydrogels, and the osteogenic stimulus was removed. In comparison with entrapped dissociated MSCs, spheroids exhibited significantly increased markers of osteogenic differentiation. The capacity of MSC spheroids to retain their osteogenic phenotype upon withdrawal of inductive cues was mediated by α2β1 integrin binding to cell-secreted ECM. These results demonstrate the capacity of spheroidal culture to sustain the mineral-producing phenotype of MSCs, thus enhancing their contribution toward bone formation and repair. Significance Despite the promise of mesenchymal stem cells (MSCs) for cell-based therapies for tissue repair and regeneration, there is little evidence that transplanted MSCs directly contribute to new bone formation, suggesting that induced cells rapidly lose their osteogenic phenotype or undergo apoptosis. In comparison with dissociated cells, MSC spheroids exhibit increased trophic factor secretion and improved cell survival. The loss of phenotype represents a significant clinical challenge for cell therapies, yet there is no evidence for whether MSC spheroids retain their osteogenic phenotype upon entrapment in a clinically relevant biomaterial. These findings demonstrate that MSC spheroids retain their osteogenic phenotype better than do dissociated MSCs, and this is due to integrin engagement with the cell-secreted extracellular matrix. These data provide evidence for a novel approach for potentiating the use of MSCs in bone repair. PMID:27365484

The Osteogenic Niche Promotes Early-Stage Bone Colonization of Disseminated Breast Cancer Cells

PubMed Central

Wang, Hai; Yu, Cuijuan; Gao, Xia; Welte, Thomas; Muscarella, Aaron M.; Tian, Lin; Zhao, Hong; Zhao, Zhen; Du, Shiyu; Tao, Jianning; Lee, Brendan; Westbrook, Thomas F.; Wong, Stephen T. C.; Jin, Xin; Rosen, Jeffrey M.; Osborne, C. Kent; Zhang, Xiang H.-F.

2014-01-01

Summary Breast cancer bone micrometastases can remain asymptomatic for years before progressing into overt lesions. The biology of this process, including the microenvironment niche and supporting pathways, is unclear. We find that bone micrometastases predominantly reside in a niche that exhibits features of osteogenesis. Niche interactions are mediated by heterotypic adherens junctions (hAJs) involving cancer-derived E-cadherin and osteogenic N-cadherin, the disruption of which abolishes niche-conferred advantages. We further elucidate that hAJ activates the mTOR pathway in cancer cells, which drives the progression from single cells to micrometastases. Human datasets analyses support the roles of AJ and the mTOR pathway in bone colonization. Our study illuminates the initiation of bone colonization, and provides potential therapeutic targets to block progression toward osteolytic metastases. Significance In advanced stages, breast cancer bone metastases are driven by paracrine crosstalk among cancer cells, osteoblasts, and osteoclasts, which constitute a vicious osteolytic cycle. Current therapies targeting this process limit tumor progression, but do not improve patient survival. On the other hand, bone micrometastases may remain indolent for years before activating the vicious cycle, providing a therapeutic opportunity to prevent macrometastases. Here, we show that bone colonization is initiated in a microenvironment niche exhibiting active osteogenesis. Cancer and osteogenic cells form heterotypic adherens junctions, which enhance mTOR activity and drive early-stage bone colonization prior to osteolysis. These results reveal a strong connection between osteogenesis and micrometastasis and suggest potential therapeutic targets to prevent bone macrometastases. PMID:25600338

Bone marrow-derived mesenchymal stem cells assembled with low-dose BMP-2 in a three-dimensional hybrid construct enhances posterolateral spinal fusion in syngeneic rats.

PubMed

Hu, Tao; Abbah, Sunny Akogwu; Toh, Soo Yein; Wang, Ming; Lam, Raymond Wing Moon; Naidu, Mathanapriya; Bhakta, Gajadhar; Cool, Simon M; Bhakoo, Kishore; Li, Jun; Goh, James Cho-Hong; Wong, Hee-Kit

2015-12-01

The combination of potent osteoinductive growth factor, functional osteoblastic cells, and osteoconductive materials to induce bone formation is a well-established concept in bone tissue engineering. However, supraphysiological dose of growth factor, such as recombinant human bone morphogenetic protein 2 (rhBMP-2), which is necessary in contemporary clinical application, have been reported to result in severe side effects. We hypothesize that the synergistic osteoinductive capacity of low-dose bone morphogenetic protein 2 (BMP-2) combined with undifferentiated bone marrow-derived stromal cells (BMSCs) is comparable to that of osteogenically differentiated BMSCs when used in a rodent model of posterolateral spinal fusion. A prospective study using a rodent model of posterolateral spinal fusion was carried out. Thirty-six syngeneic Fischer rats comprised the patient sample. Six groups of implants were evaluated as follows (n=6): (1) 10 µg BMP-2 with undifferentiated BMSCs; (2) 10 µg BMP-2 with osteogenic-differentiated BMSCs; (3) 2.5 µg BMP-2 with undifferentiated BMSCs; (4) 2.5 µg BMP-2 with osteogenic-differentiated BMSCs; (5) 0.5 µg BMP-2 with undifferentiated BMSCs; and (6) 0.5 µg BMP-2 with osteogenic-differentiated BMSCs. Optimal in vitro osteogenic differentiation of BMSCs was determined by quantitative real-time polymerase chain reaction (qRT-PCR) gene analysis whereas in vivo bone formation capacity was evaluated by manual palpation, micro-computed tomography, and histology. Rat BMSCs cultured in fibrin matrix that was loaded into the pores of medical-grade poly epsilon caprolactone tricalcium phosphate scaffolds differentiated toward osteogenic lineage by expressing osterix, runt-related transcription factor 2, and osteocalcium mRNA when supplemented with dexamethasone, ascorbic acid, and β-glycerophosphate. Whereas qRT-PCR revealed optimal increase in osteogenic genes expression after 7 days of in vitro culture, in vivo transplantation study showed that pre-differentiation of BMSCs before transplantation failed to promote posterolateral spinal fusion when co-delivered with low-dose BMP-2 (1/6 or 17% fusion rate). In contrast, combined delivery of undifferentiated BMSCs with low-dose BMP-2 (2.5 µg) demonstrated significantly higher fusion rate (4/6 or 67%) as well as significantly increased volume of new bone formation (p<.05). In summary, this study supports the combination of undifferentiated BMSCs and low-dose rhBMP-2 for bone tissue engineering construct. Copyright © 2015 Elsevier Inc. All rights reserved.

Implications of adipose-derived stromal cells in a 3D culture system for osteogenic differentiation: an in vitro and in vivo investigation.

PubMed

Shen, Francis H; Werner, Brian C; Liang, Haixiang; Shang, Hulan; Yang, Ning; Li, Xudong; Shimer, Adam L; Balian, Gary; Katz, Adam J

2013-01-01

Healthy mammalian cells in normal tissues are organized in complex three-dimensional (3D) networks that display nutrient and signaling gradients. Conventional techniques that grow cells in a 2D monolayer fail to reproduce the environment that is observed in vivo. In recent years, 3D culture systems have been used to mimic tumor microenvironments in cancer research and to emulate embryogenesis in stem cell cultures. However, there have been no studies exploring the ability for adipose-derived stromal (ADS) cells in a 3D culture system to undergo osteogenic differentiation. To characterize and investigate the in vitro and in vivo potential for human ADS cells in a novel 3D culture system to undergo osteogenic differentiation. Basic science and laboratory study. Human ADS cells were isolated and prepared as either a 2D monolayer or 3D multicellular aggregates (MAs). Multicellular aggregates were formed using the hanging droplet technique. Cells were treated in osteogenic medium in vitro, and cellular differentiation was investigated using gene expression, histology, and microCT at 1-, 2-, and 4-week time points. In vivo investigation involved creating a muscle pouch by developing the avascular muscular interval in the vastus lateralis of male athymic rats. Specimens were then pretreated with osteogenic medium and surgically implanted as (1) carrier (Matrigel) alone (control), (2) carrier with human ADS cells in monolayer, or (3) human ADS cells as MAs. In vivo evidence of osteogenic differentiation was evaluated with micro computed tomography and histologic sectioning at a 2-week time point. Human ADS cells cultured by the hanging droplet technique successfully formed MAs at the air-fluid interface. Adipose-derived stromal cells cultured in monolayer or as 3D MAs retain their ability to self-replicate and undergo multilineage differentiation as confirmed by increased runx2/Cbfa2, ALP, and OCN and increased matrix mineralization on histologic sectioning. Multicellular aggregate cells expressed increased differentiation potential and extracellular matrix production over the same human ADS cells cultured in monolayer. Furthermore, MAs reseeded onto monolayer retained their stem cell capabilities. When implanted in vivo, significantly greater bone volume and extracellular matrix were present in the implanted specimens of MAs confirmed on both microCT and histological sectioning. This is the first study to investigate the capability of human ADS cells in a 3D culture system to undergo osteogenic differentiation. The results confirm that MAs maintain their stem cell characteristics. Compared with analogous cells in monolayer culture, the human ADS cells as MAs exhibit elevated levels of osteogenic differentiation and increased matrix mineralization. Furthermore, the creation of uniform spheroids allows for improved handling and manipulation during transplantation. These findings strongly support the concept that 3D culture systems remain not only a viable option for stem cell culture but also possibly a more attractive alternative to traditional culture techniques to improve the osteogenic potential of human adipose stem cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Osteogenic differentiation of equine adipose tissue derived mesenchymal stem cells using CaCl2.

PubMed

Elashry, Mohamed I; Baulig, Nadine; Heimann, Manuela; Bernhardt, Caroline; Wenisch, Sabine; Arnhold, Stefan

2018-04-01

Adipose tissue derived mesenchymal stem cells (ASCs) may be used to cure bone defects after osteogenic differentiation. In this study we tried to optimize osteogenic differentiation for equine ASCs using various concentrations of CaCl 2 in comparison to the standard osteogenic protocol. ASCs were isolated from subcutaneous adipose tissue from mixed breed horses. The osteogenic induction protocols were (1) the standard osteogenic medium (OM) composed of dexamethasone, ascorbic acid and β-glycerol phosphate; (2) CaCl 2 based protocol composed of 3, 5 and 7.5mM CaCl 2 . Differentiation and proliferation were evaluated at 7, 10, 14 and 21days post-differentiation induction using the alizarin red staining (ARS) detecting matrix calcification. Semi-quantification of cell protein content, ARS and alkaline phosphatase activity (ALP) were performed using an ELISA reader. Quantification of the transcription level for the common osteogenic markers alkaline phosphatase (ALP) and Osteopontin (OP) was performed using RT-qPCR. In the presence of CaCl 2 , a concentration dependent effect on the osteogenic differentiation capacity was evident by the ARS evaluation and OP gene expression. We provide evidence that 5 and 7mM CaCl 2 enhance the osteogenic differentiation compared to the OM protocol. Although, there was a clear commitment of ASCs to the osteogenic fate in the presence of 5 and 7mM CaCl 2 , cell proliferation was increased compared to OM. We report that an optimized CaCl 2 protocol reliably influences ASCs osteogenesis while conserving the proliferation capacity. Thus, using these protocols provide a platform for using ASCs as a cell source in bone tissue engineering. Copyright © 2017 Elsevier Ltd. All rights reserved.

Silibinin alleviates high glucose-suppressed osteogenic differentiation of human bone marrow stromal cells via antioxidant effect and PI3K/Akt signaling.

PubMed

Ying, Xiaozhou; Chen, Xiaowei; Liu, Haixiao; Nie, Pengfei; Shui, Xiaolong; Shen, Yue; Yu, Kehe; Cheng, Shaowen

2015-10-15

High glucose is one of the possible causes for osteoporosis and fracture in diabetes mellitus. Our previous study showed that silibinin can increase osteogenic effect by stimulating osteogenic genes expression in human bone marrow stem cells (hBMSCs). However, no study has yet investigated the effect of silibinin on osteogenic differentiation of hBMSCs cultured with high glucose. The aim of this study was to evaluate the influence of high glucose on osteogenic differentiation of hBMSCs and to determine if silibinin can alleviate those effects. In this study, the hBMSCs were cultured in an osteogenic medium with physiological (normal glucose, NG, 5.5mM) or diabetic (high glucose, HG, 30mM). The effects of silibinin on HG-induced osteogenic differentiation were evaluated by alkaline phosphatas (ALP) activity assay, Von Kossa staining and real time-polymerase chain reaction. HG-induced oxidative damage was also assessed. Western blot were performed to examine the role of PI3K/Akt pathway. We demonstrated that HG suppressed osteogenic differentiation of hBMSCs, manifested by a decrease in expression of osteogenic markers and an increase of oxidative damage markers including reactive oxygen species and lipid peroxide (MDA). Remarkably, all of the observed oxidative damage and osteogenic dysfunction induced by HG were inhibited by silibinin. Furthermore, the PI3K/Akt pathway was activated by silibinin. These results demonstrate that silibinin may attenuate HG-mediated hBMSCs dysfunction through antioxidant effect and modulation of PI3K/Akt pathway, suggesting that silibinin may be a superior drug candidate for the treatment of diabetes related bone diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

Down-regulated non-coding RNA (lncRNA-ANCR) promotes osteogenic differentiation of periodontal ligament stem cells.

PubMed

Jia, Qian; Jiang, Wenkai; Ni, Longxing

2015-02-01

Our studies aimed to figure out how anti-differentiation noncoding RNA (ANCR) regulates the proliferation and osteogenic differentiation of periodontal ligament stem cells (PDLSCs). In this study, we used lentivirus infection to down-regulate the expression of ANCR in PDLSCs. Then we compared the proliferation of control cells and PDLSC/ANCR-RNAi cells by Cell Counting Kit-8. And the osteogenic differentiation of control cells and PDLSC/ANCR-RNAi cells were evaluated by Alkaline phosphatase (ALP) activity quantification and Alizarin red staining. WNT inhibitor was used to analyze the relationship between ANCR and canonical WNT signalling pathway. The expression of osteogenic differentiation marker mRNAs, DKK1, GSK3-β and β-catenin were evaluated by qRT-PCR. The results showed that down-regulated ANCR promoted proliferation of PDLSCs. Down-regulated ANCR also promoted osteogenic differentiation of PDLSCs by up-regulating osteogenic differentiation marker genes. After the inhibition of canonical WNT signalling pathway, the osteogenic differentiation of PDLSC/ANCR-RNAi cells was inhibited too. qRT-PCR results also demonstrated that canonical WNT signalling pathway was activated for ANCR-RNAi on PDLSCs during the procedure of proliferation and osteogenic induction. These results indicated that ANCR was a key regulator of the proliferation and osteogenic differentiation of PDLSCs, and its regulating effects was associated with the canonical WNT signalling pathway, thus offering a new target for oral stem cell differentiation studies that could also facilitate oral tissue engineering. Copyright © 2014. Published by Elsevier Ltd.

Aging Reduces an ERRalpha-Directed Mitochondrial Glutaminase Expression Suppressing Glutamine Anaplerosis and Osteogenic Differentiation of Mesenchymal Stem Cells.

PubMed

Huang, Tongling; Liu, Renzhong; Fu, Xuekun; Yao, Dongsheng; Yang, Meng; Liu, Qingli; Lu, William W; Wu, Chuanyue; Guan, Min

2017-02-01

Aging deteriorates osteogenic capacity of mesenchymal stem/stromal cells (MSCs), contributing to imbalanced bone remodeling and osteoporosis. Glutaminase (Gls) catabolizes glutamine into glutamate at the first step of mitochondrial glutamine (Gln)-dependent anaplerosis which is essential for MSCs upon osteogenic differentiation. Estrogen-related receptor α (ERRα) regulates genes required for mitochondrial function. Here, we found that ERRα and Gls are upregulated by osteogenic induction in human MSCs (hMSCs). In contrast, osteogenic differentiation capacity and glutamine consumption of MSCs, as well as ERRα, Gls and osteogenic marker genes are significantly reduced with age. We demonstrated that ERRα binds to response elements on Gls promoter and affects glutamine anaplerosis through transcriptional induction of Gls. Conversely, mTOR inhibitor rapamycin, ERRα inverse agonist compound 29 or Gls inhibitor BPTES leads to reduced Gln anaplerosis and deteriorated osteogenic differentiation of hMSCs. Importantly, overexpression of ERRα or Gls restored impairment by these inhibitors. Finally, we proved that compensated ERRα or Gls expression indeed potentiated Gln anaplerosis and osteogenic capability of elderly mice MSCs in vitro. Together, we establish that Gls is a novel ERRα target gene and ERRα/Gls signaling pathway plays an important role in osteogenic differentiation of MSCs, providing new sights into novel regenerative therapeutics development. Our findings suggest that restoring age-related mitochondrial Gln-dependent anaplerosis may be beneficial for degenerative bone disorders such as osteoporosis. Stem Cells 2017;35:411-424. © 2016 AlphaMed Press.

Transfer of fibroblast sheets cultured on thermoresponsive dishes with membranes.

PubMed

Kawecki, Marek; Kraut, Małgorzata; Klama-Baryła, Agnieszka; Łabuś, Wojciech; Kitala, Diana; Nowak, Mariusz; Glik, Justyna; Sieroń, Aleksander L; Utrata-Wesołek, Alicja; Trzebicka, Barbara; Dworak, Andrzej; Szweda, Dawid

2016-06-01

In cell or tissue engineering, it is essential to develop a support for cell-to-cell adhesion, which leads to the generation of cell sheets connected by extracellular matrix. Such supports must be hydrophobic and should result in a detachable cell sheet. A thermoresponsive support that enables the cultured cell sheet to detach using only a change in temperature could be an interesting alternative in regenerative medicine. The aim of this study was to evaluate plates covered with thermoresponsive polymers as supports for the formation of fibroblast sheets and to develop a damage-free procedure for cell sheet transfer with the use of membranes as transfer tools. Human skin fibroblasts were seeded on supports coated with a thermoresponsive polymer: commercial UpCell™ dishes (NUNC™) coated with thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and dishes coated with thermoresponsive poly(tri(ethylene glycol) monoethyl ether methacrylate) (P(TEGMA-EE)). Confluent fibroblast sheets were effectively cultured and harvested from both commercial PNIPAM-coated dishes and laboratory P(TEGMA-EE)-coated dishes. To transfer a detached cell sheet, two membranes, Immobilon-P(®) and SUPRATHEL(®), were examined. The use of SUPRATHEL for relocating the cell sheets opens a new possibility for the clinical treatment of wounds. This study established the background for implementing thermoresponsive supports for transplanting in vitro cultured fibroblasts.

Tenascin-C mimetic Peptide nanofibers direct stem cell differentiation to osteogenic lineage.

PubMed

Sever, Melike; Mammadov, Busra; Guler, Mustafa O; Tekinay, Ayse B

2014-12-08

Extracellular matrix contains various signals for cell surface receptors that regulate cell fate through modulation of cellular activities such as proliferation and differentiation. Cues from extracellular matrix components can be used for development of new materials to control the stem cell fate. In this study, we achieved control of stem cell fate toward osteogenic commitment by using a single extracellular matrix element despite the contradictory effect of mechanical stiffness. For this purpose, we mimicked bone extracellular matrix by incorporating functional sequence of fibronectin type III domain from native tenascin-C on self-assembled peptide nanofibers. When rat mesenchymal stem cells (rMSCs) were cultured on these peptide nanofibers, alkaline phosphatase (ALP) activity and alizarin red staining indicated osteogenic differentiation even in the absence of osteogenic supplements. Moreover, expression levels of osteogenic marker genes were significantly enhanced revealed by quantitative real-time polymerase chain reaction (qRT-PCR), which showed the remarkable bioactive role of this nanofiber system on osteogenic differentiation. Overall, these results showed that tenascin-C mimetic peptides significantly enhanced the attachment, proliferation, and osteogenic differentiation of rMSCs even in the absence of any external bioactive factors and regardless of the suitable stiff mechanical properties normally required for osteogenic differentiation. Thus, these peptide nanofibers provide a promising new platform for bone regeneration.

Effects of microgravity modeled by large gradient high magnetic field on the osteogenic initiation of human mesenchymal stem cells.

PubMed

Shi, Dongyan; Meng, Rui; Deng, Wanglong; Ding, Wenchao; Zheng, Qiang; Yuan, Wenji; Liu, Liyue; Zong, Chen; Shang, Peng; Wang, Jinfu

2010-12-01

Microgravity (MG) leads to a decrease in osteogenic potential of human bone marrow-derived mesenchymal stem cells (hMSCs). In the present study, we used large gradient high magnetic field (LGHMF) produced by a superconducting magnet to model MG (LGHMF-MG) and analyzed the effects of LGHMF-MG on survival, cytoskeleton and osteogenic potential of hMSCs. Results showed that the LGHMF-MG treatment for 6 h disrupted the cytoskeleton of hMSCs, and the LGHMF-MG treatment for 24 h led to cell death. LGHMF-MG treatments for 6 h in early stages of osteogenic induction (the pre-treatment before osteogenic induction, the beginning-treatment in the beginning-stage of osteogenic induction and the middle-treatment in the middle-stage of osteogenic induction) resulted in suppression on osteogenesis of hMSCs. The suppression intensity was reduced gradually as the treatment stage of LGHMF-MG was postponed. The LGHMF-MG treatment for 6 h in the ending-stage of osteogenic induction (the ending-treatment) had no obvious effect on osteogenesis of hMSCs. These results indicated that LGHMF-MG should affect the initiation of osteogenesis. Finally, the possible mechanism for the inhibition effect of LGHMF-MG on osteogenesis of hMSCs is discussed.

Evaluation of the osteoinductive potential of a bio-inspired scaffold mimicking the osteogenic niche for bone augmentation.

PubMed

Minardi, Silvia; Corradetti, Bruna; Taraballi, Francesca; Sandri, Monica; Van Eps, Jeffrey; Cabrera, Fernando J; Weiner, Bradley K; Tampieri, Anna; Tasciotti, Ennio

2015-09-01

Augmentation of regenerative osteogenesis represents a premier clinical need, as hundreds of thousands of patients are left with insufficient healing of bony defects related to a host of insults ranging from congenital abnormalities to traumatic injury to surgically-induced deficits. A synthetic material that closely mimics the composition and structure of the human osteogenic niche represents great potential to successfully address this high demand. In this study, a magnesium-doped hydroxyapatite/type I collagen scaffold was fabricated through a biologically-inspired mineralization process and designed to mimic human trabecular bone. The composition of the scaffold was fully characterized by XRD, FTIR, ICP and TGA, and compared to human bone. Also, the scaffold microstructure was evaluated by SEM, while its nano-structure and nano-mechanical properties were evaluated by AFM. Human bone marrow-derived mesenchymal stem cells were used to test the in vitro capability of the scaffold to promote osteogenic differentiation. The cell/scaffold constructs were cultured up to 7 days and the adhesion, organization and proliferation of the cells were evaluated. The ability of the scaffold to induce osteogenic differentiation of the cells was assessed over 3 weeks and the correlate gene expression for classic genes of osteogenesis was assessed. Finally, when tested in an ectopic model in rabbit, the scaffold produced a large volume of trabecular bone in only two weeks, that subsequently underwent maturation over time as expected, with increased mature cortical bone formation, supporting its ability to promote bone regeneration in clinically-relevant scenarios. Altogether, these results confirm a high level of structural mimicry by the scaffold to the composition and structure of human osteogenic niche that translated to faster and more efficient osteoinduction in vivo--features that suggest such a biomaterial may have great utility in future clinical applications where bone regeneration is required. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Smurf1 plays a role in EGF inhibition of BMP2-induced osteogenic differentiation

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

Lee, Hye-Lim; Park, Hyun-Jung; Kwon, Arang

2014-05-01

It has been demonstrated that epidermal growth factor (EGF) plays a role in supporting the proliferation of bone marrow stromal cells in bone but inhibits their osteogenic differentiation. However, the mechanism underlying EGF inhibition of osteoblast differentiation remains unclear. Smurf1 is an E3 ubiquitin ligase that targets Smad1/5 and Runx2, which are critical transcription factors for bone morphogenetic protein 2 (BMP2)-induced osteoblast differentiation. In this study, we investigated the effect of EGF on the expression of Smurf1, and the role of Smurf1 in EGF inhibition of osteogenic differentiation using C2C12 cells, a murine myoblast cell line. EGF increased Smurf1 expression,more » which was blocked by inhibiting the activity of either JNK or ERK. Chromatin immunoprecipitation and Smurf1 promoter assays demonstrated that c-Jun and Runx2 play roles in the EGF induction of Smurf1 transcription. EGF suppressed BMP2-induced expression of osteogenic marker genes, which were rescued by Smurf1 knockdown. EGF downregulated the protein levels of Runx2 and Smad1 in a proteasome-dependent manner. EGF decreased the transcriptional activity of Runx2 and Smurf1, which was partially rescued by Smurf1 silencing. Taken together, these results suggest that EGF increases Smurf1 expression via the activation of JNK and ERK and the subsequent binding of c-Jun and Runx2 to the Smurf1 promoter and that Smurf1 mediates the inhibitory effect of EGF on BMP2-induced osteoblast differentiation. - Highlights: • EGF increases the expression level of Smurf1 in mesenchymal precursor cells. • EGF reduces the protein levels and transcriptional activity of Runx2 and Smad1. • EGF suppresses BMP2-induced osteogenic differentiation, which is rescued by Smurf1 knockdown.« less

Non-dermatological complications and genetic aspects of the Rothmund-Thomson syndrome.

PubMed

Starr, D G; McClure, J P; Connor, J M

1985-01-01

We report two new cases of Rothmund-Thomson syndrome which emphasize the less well-known non-dermatological complications, namely: hypodontia, soft tissue contractures, proportionate short stature, hypogonadism, anaemia and osteogenic sarcoma. Genetic analysis of these and previously reported pedigrees supports autosomal recessive inheritance.

MiR-132 regulates osteogenic differentiation via downregulating Sirtuin1 in a peroxisome proliferator-activated receptor β/δ–dependent manner

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

Gong, Kai; Qu, Bo; Liao, Dongfa

MicroRNAs (miRNAs) play significant roles in multiple diseases by regulating the expression of their target genes. Type 2 diabetes mellitus (T2DM) is a chronic endocrine and metabolic disease with complex mechanisms. T2DM can result in diabetic osteoporosis (DO), which is characterized by bone loss, decreased bone mineral density and increased bone fractures. The promotion of osteogenic differentiation of osteoblasts is an effective way to treat osteoporosis. In the present study, high glucose (HG) and free fatty acids (FFA) were employed to mimic T2DM in MC3T3-E1 cells. To induce osteogenic differentiation, MC3T3-E1 cells were cultured in osteogenic medium. The results showedmore » that osteogenic differentiation was significantly suppressed by HG and FFA. We found that miR-132 expression was significantly upregulated and much higher in HG-FFA–induced cells than other selected miRNAs, indicating that miR-132 might play an important role in DO. Furthermore, overexpression of miR-132 markedly inhibited the expression of key markers of osteogenic differentiation and alkaline phosphatase (ALP) activity. Reciprocally, inhibition of miR-132 restored osteogenic differentiation, even under treatment with HG-FFA. We also showed that Sirtuin 1 (Sirt1) was one of the target genes of miR-132, whose expression was controlled by miR-132. Ectopic expression of Sirt1 reversed the decrease in osteogenic differentiation caused by miR-132 and HG-FFA. These results demonstrated the direct role of miR-132 in suppressing osteogenic differentiation through downregulating Sirt1. Moreover, we demonstrated that peroxisome proliferator-activated receptor β/δ (PPARβ/δ) was a downstream molecule of Sirt1, and its knockout by PPARβ/δ siRNA significantly abolished the promotive effects of Sirt1 on osteogenic differentiation, indicating that Sirt1 functioned in a PPARβ/δ–dependent manner. Taken together, we provide crucial evidence that miR-132 plays a key role in regulating osteogenic differentiation through Sirt1 in a PPARβ/δ–dependent manner, indicating that miR-132 and Sirt1-PPARβ/δ may act as potential therapeutic targets for T2DM–induced osteoporosis. - Highlights: • MiR-132 participates in regulating osteogenic differentiation of MC3T3-E1 cells. • Sirt1 is a target gene of miR-132. • Sirt1 is the effector of miR-132 in regulating osteogenic differentiation. • MiR-132-Sirt1 regulates osteogenic differentiation in a PPARβ/δ–dependent manner.« less

Osteogenic differentiation of human mesenchymal bone marrow cells in silk scaffolds is regulated by nitric oxide.

PubMed

Damoulis, Petros D; Drakos, Dimitrios E; Gagari, Eleni; Kaplan, David L

2007-11-01

Bone marrow-derived mesenchymal stem cells (BMSC) are a powerful tool for tissue engineering and can be used in the regeneration of bone and other tissues. Nitric oxide (NO) produced by the endothelial NO synthase (eNOS) plays an important role in bone development and healing. We hypothesized that NO plays a role in osteogenic differentiation of BMSC cultured in three-dimensional silk scaffolds. eNOS protein was measured by Western Analysis and its activity was assessed by measuring nitrite in culture supernatants. Mineralization was evaluated through calcium deposition and the expression of genes associated with osteogenic differentiation (collagen I, RUNX2, and osteocalcin) was quantified using real-time RT-PCR. eNOS was consistently expressed with minor fluctuations, but NO production significantly increased at later time points (weeks 4 and 5). Addition of a competitive NOS inhibitor (L-NAME) resulted in a modest decrease in calcium deposition, which became statistically significant in week 5. This was preceded by a dramatic decrease in RUNX2 and osteocalcin expression in week 4. These results support our hypothesis and implicate NO as an important player in bone tissue engineering.

Osteogenic differentiation of periosteum-derived stromal cells in blast-associated traumatic loading

NASA Astrophysics Data System (ADS)

Sory, David R.; Amin, Harsh D.; Rankin, Sara M.; Proud, William G.

2017-06-01

One of the most recurrent medical complications resulting from blast trauma includes blast-induced heterotopic ossification. Heterotopic ossification refers to the pathologic formation of extraskeletal bone in non-osseous tissue. Although a number of studies have established the interaction between mechanics and biology in bone formation following shock trauma, the exact nature of the mechanical stimuli associated to blast-loading and their influence on the activation of osteogenic differentiation of cells remain unanswered. Here we present the design and calibration of a loading platform compatible with living cells to examine the effects of mechanical stress pulses of blast-associated varying strain rates on the activation of osteogenic differentiation of periosteum (PO) cells. Multiaxial compression loadings of PO cells are performed at different magnitudes of stress and ranges of strain rate. A proof of concept is presented so as to establish a new window to address fundamental questions regarding blast injuries at the cellular level. This work was conducted under the auspices of the Royal British Legion Centre for Blast Injury Studies at Imperial College London. The authors would like to acknowledge the financial support of the Royal British Legion.

Osteogenic differentiation of stem cells derived from human periodontal ligaments and pulp of human exfoliated deciduous teeth.

PubMed

Chadipiralla, Kiranmai; Yochim, Ji Min; Bahuleyan, Bindu; Huang, Chun-Yuh Charles; Garcia-Godoy, Franklin; Murray, Peter E; Stelnicki, Eric J

2010-05-01

Multipotent stem cells derived from periodontal ligaments (PDLSC) and pulp of human exfoliated deciduous teeth (SHED) represent promising cell sources for bone regeneration. Recent studies have demonstrated that retinoic acid (RA) and dexamethasone (Dex) induce osteogenesis of postnatal stem cells. The objective of this study was to examine the effects of RA and Dex on the proliferation and osteogenic differentiation of SHED and PDLSC and to compare the osteogenic characteristics of SHED and PDLSC under RA treatment. SHED and PDLSC were treated with serum-free medium either alone or supplemented with RA or Dex for 21 days. The proliferation of SHED and PDLSC was significantly inhibited by both RA and Dex. RA significantly upregulated gene expression and the activity of alkaline phosphatase in SHED and PDLSC. Positive Alizarin red and von Kossa staining of calcium deposition was seen on the RA-treated SHED and PDLSC after 21 days of culture. The influences of RA on the osteogenic differentiation of SHED and PDLSC were significantly stronger than with Dex. Supplementation with insulin enhanced RA-induced osteogenic differentiation of SHED. Thus, RA is an effective inducer of osteogenic differentiation of SHED and PDLSC, whereas RA treatment in combination with insulin supplementation might be a better option for inducing osteogenic differentiation. Significantly higher cell proliferation of PDLSC results in greater calcium deposition after 3-week culture, suggesting that PDLSC is a better osteogenic stem cell source. This study provides valuable information for efficiently producing osteogenically differentiated SHED or PDLSC for in vivo bone regeneration.

Naringin enhances osteogenic differentiation through the activation of ERK signaling in human bone marrow mesenchymal stem cells.

PubMed

Wang, Huichao; Li, Chunbo; Li, Jianming; Zhu, Yingjie; Jia, Yudong; Zhang, Ying; Zhang, Xiaodong; Li, Wenlong; Cui, Lei; Li, Wuyin; Liu, Youwen

2017-04-01

Naringin has been reported to regulate bone metabolism. However, its effect on osteogenesis remains unclear. The aim was to investigate the effect of naringin on osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) through the activation of the ERK signaling pathway in osteogenic differentiation. Annexin V-FITC assay and MTT assay were used to measure the effect of naringin on cytotoxicity and proliferation of hBMSCs, respectively. Alkaline phosphatase activity analysis, Alizarin Red S staining, Western blotting, and real-time PCR assay were used to evaluate both the potential effect of naringin on osteogenic differentiation and the role of ERK signaling pathway in osteogenic differentiation. Our results showed that naringin had no obvious toxicity on hBMSCs, and could significantly promote the proliferation of hBMSCs. Naringin also enhanced the osteogenic differentiation of hBMSCs and increased the protein and mRNA expression levels of osteogenic markers such as Runx-2, OXS, OCN, and Col1 in a dose-dependent manner. In addition, we found that the enhancing effect of naringin on osteogenic differentiation was related to the activation of phosphor-ERK, with an increase in duration of activity from 30 min to 120 min. More importantly, both the enhancing effect of naringin on osteogenic differentiation and the activity effect of naringin on ERK signaling pathway were reversed by U0126 addition. Our findings demonstrated that naringin promoted proliferation and osteogenesis of hBMSCs by activating the ERK signaling pathway and it might be a potential therapeutic agent for treating or preventing osteoporosis.

Bioactive glass ions as strong enhancers of osteogenic differentiation in human adipose stem cells.

PubMed

Ojansivu, Miina; Vanhatupa, Sari; Björkvik, Leena; Häkkänen, Heikki; Kellomäki, Minna; Autio, Reija; Ihalainen, Janne A; Hupa, Leena; Miettinen, Susanna

2015-07-01

Bioactive glasses are known for their ability to induce osteogenic differentiation of stem cells. To elucidate the mechanism of the osteoinductivity in more detail, we studied whether ionic extracts prepared from a commercial glass S53P4 and from three experimental glasses (2-06, 1-06 and 3-06) are alone sufficient to induce osteogenic differentiation of human adipose stem cells. Cells were cultured using basic medium or osteogenic medium as extract basis. Our results indicate that cells stay viable in all the glass extracts for the whole culturing period, 14 days. At 14 days the mineralization in osteogenic medium extracts was excessive compared to the control. Parallel to the increased mineralization we observed a decrease in the cell amount. Raman and Laser Induced Breakdown Spectroscopy analyses confirmed that the mineral consisted of calcium phosphates. Consistently, the osteogenic medium extracts also increased osteocalcin production and collagen Type-I accumulation in the extracellular matrix at 13 days. Of the four osteogenic medium extracts, 2-06 and 3-06 induced the best responses of osteogenesis. However, regardless of the enhanced mineral formation, alkaline phosphatase activity was not promoted by the extracts. The osteogenic medium extracts could potentially provide a fast and effective way to differentiate human adipose stem cells in vitro. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Mineralogenic characteristics of osteogenic lineage-committed human dental pulp stem cells following their exposure to a discoloration-free calcium aluminosilicate cement.

PubMed

Niu, Li-Na; Pei, Dan-Dan; Morris, Matthew; Jiao, Kai; Huang, Xue-Qing; Primus, Carolyn M; Susin, Lisiane F; Bergeron, Brian E; Pashley, David H; Tay, Franklin R

2016-10-01

An experimental discoloration-free calcium aluminosilicate cement has been developed with the intention of maximizing the beneficial attributes of tricalcium silicate cements and calcium aluminate cements. The present study examined the effects of this experimental cement (Quick-Set2) on the mineralogenic characteristics of osteogenic lineage-committed human dental pulp stem cells (hDPSCs), by comparing the cellular responses with a commercially available tricalcium silicate cement (white mineral trioxide aggregate (ProRoot(®) MTA); WMTA). The osteogenic potential of hDPSCs exposed to the cements was examined using qRT-PCR for osteogenic gene expressions, Western blot for osteogenic-related protein expressions, alkaline phosphatase enzyme activity, Alizarin red S staining, Fourier transform infrared spectroscopy and transmission electron microscopy of extracellular calcium deposits. Results of the six assays indicated that osteogenic differentiation of hDPSCs was significantly enhanced after exposure to the tricalcium silicate cement or the experimental calcium aluminosilicate cement, with the former demonstrating better mineralogenic stimulation capacity. The better osteogenic stimulating effect of the tricalcium silicate cement on hDPSCs may be due to its relatively higher silicate content, or higher OH(-) and Ca(2+) release. Further investigations with the use of in vivo animal models are required to validate the potential augmenting osteogenic effects of the experimental discoloration-free calcium aluminosilicate cement. Published by Elsevier Ltd.

Effects of hypoxia on osteogenic differentiation of mesenchymal stromal cells used as a cell therapy for avascular necrosis of the femoral head.

PubMed

Ciapetti, Gabriela; Granchi, Donatella; Fotia, Caterina; Savarino, Lucia; Dallari, Dante; Del Piccolo, Nicola; Donati, Davide Maria; Baldini, Nicola

2016-09-01

Avascular necrosis of the femoral head (AVN) occurs as common result of various conditions or develops as a primary entity, with a high freqency in young adults. Because of its tendency toward osteoarthritis requiring total hip arthroplasty, alternative treatments are being advocated, including cell therapy with mesenchymal stromal cells (MSCs). Because osteonecrotic bone is a severely hypoxic tissue, with a 1-3% oxygen tension, the survival and function of multipotent cells is questionable. In this study, the proliferative, immunophenotypic and osteogenic properties of bone marrow (BM)-derived MSCs from a clinical series of patients with AVN were evaluated under in vitro conditions mimicking the hypoxic milieu of AVN to verify the rationale for cell therapy. MSCs retrieved from the iliac crest (BM-MSC) were isolated, expanded and induced to osteogenic differentiation under a 2% pO2 atmosphere (hypoxia) in comparison with the standard 21% pO2 (normoxia) that is routinely used in cell culture assays. Both proliferation and colony-forming ability were significantly enhanced in hypoxia-exposed BM-MSCs compared with BM-MSCs under normoxia. The expression of bone-related genes, including alkaline phosphatase, Type I collagen, and osteocalcin was significantly increased under hypoxia. Moreover, mineral deposition after osteogenic induction was not hampered, but in some cases even enhanced under low oxygen tension. These findings support autologous cell therapy as an effective treatment to stimulate bone healing in the hypoxic microenvironment of AVN. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Sirtuin1 promotes osteogenic differentiation through downregulation of peroxisome proliferator-activated receptor γ in MC3T3-E1 cells

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

Qu, Bo; Ma, Yuan; Yan, Ming

Osteoporosis is a skeletal disorder characterized by bone loss, resulting in architectural deterioration of the skeleton, decreased bone strength and an increased risk of fragility fractures. Strengthening osteogenesis is an effective way to relieve osteoporosis. Sirtuin1 (Sirt1) is a nicotinamide adenine dinucleotide (NAD{sup +})-dependent deacetylase, which is reported to be involved in improving osteogenesis. Sirt1 targets peroxisome proliferator-activated receptor γ (PPARγ) in the regulation of adipose tissues; however, the molecular mechanism of Sirt1 in osteogenic differentiation is still unknown. PPARγ tends to induce more adipogenic differentiation rather than osteogenic differentiation. Hence, we hypothesized that Sirt1 facilitates osteogenic differentiation through downregulationmore » of PPARγ signaling. Mouse pre-osteoblastic MC3T3-E1 cells were cultured under osteogenic medium. Sirt1 was overexpressed through plasmid transfection. The results showed that high expression of Sirt1 was associated with increased osteogenic differentiation, as indicated by quantitative PCR and Western blot analysis of osteogenic markers, and Von Kossa staining. Sirt1 overexpression also directly and negatively regulated the expression of PPARγ and its downstream molecules. Use of the PPARγ agonist Rosiglitazone, reversed the effects of Sirt1 on osteogenic differentiation. Using constructed luciferase plasmids, we demonstrated a role of Sirt1 in inhibiting PPARγ–induced activity and expression of adipocyte–specific genes, including acetyl-coenzyme A carboxylase (Acc) and fatty acid binding protein 4 (Fabp4). The interaction between Sirt1 and PPARγ was further confirmed using co-immunoprecipitation analysis. Together, these results reveal a novel mechanism for Sirt1 in osteogenic differentiation through downregulation of PPARγ activity. These findings suggest that the Sirt1–PPARγ pathway may represent a potential target for enhancement of osteogenesis and treatment of osteoporosis. - Highlights: • Sirt1 inhibits PPARγ signaling in MC3T3-E1 cells. • PPARγ negatively regulates osteogenic differentiation of MC3T3-E1 cells. • Sirt1 promotes osteogenic differentiation through downregulation of PPARγ.« less

Utilizing boron nitride sheets as thin supports for high resolution imaging of nanocrystals.

PubMed

Wu, Yimin A; Kirkland, Angus I; Schäffel, Franziska; Porfyrakis, Kyriakos; Young, Neil P; Briggs, G Andrew D; Warner, Jamie H

2011-05-13

We demonstrate the use of thin BN sheets as supports for imaging nanocrystals using low voltage (80 kV) aberration-corrected high resolution transmission electron microscopy. This provides an alternative to the previously utilized 2D crystal supports of graphene and graphene oxide. A simple chemical exfoliation method is applied to get few layer boron nitride (BN) sheets with micrometer-sized dimensions. This generic approach of using BN sheets as supports is shown by depositing Mn doped ZnSe nanocrystals directly onto the BN sheets and resolving the atomic structure from both the ZnSe nanocrystals and the BN support. Phase contrast images reveal moiré patterns of interference between the beams diffracted by the nanocrystals and the BN substrate that are used to determine the relative orientation of the nanocrystals with respect to the BN sheets and interference lattice planes. Double diffraction is observed and has been analyzed.

Graphene supports in vitro proliferation and osteogenic differentiation of goat adult mesenchymal stem cells: potential for bone tissue engineering.

PubMed

Elkhenany, Hoda; Amelse, Lisa; Lafont, Andersen; Bourdo, Shawn; Caldwell, Marc; Neilsen, Nancy; Dervishi, Enkeleda; Derek, Oshin; Biris, Alexandru S; Anderson, David; Dhar, Madhu

2015-04-01

Current treatments for bone loss injuries involve autologous and allogenic bone grafts, metal alloys and ceramics. Although these therapies have proved useful, they suffer from inherent challenges, and hence, an adequate bone replacement therapy has not yet been found. We hypothesize that graphene may be a useful nanoscaffold for mesenchymal stem cells and will promote proliferation and differentiation into bone progenitor cells. In this study, we evaluate graphene, a biocompatible inert nanomaterial, for its effect on in vitro growth and differentiation of goat adult mesenchymal stem cells. Cell proliferation and differentiation are compared between polystyrene-coated tissue culture plates and graphene-coated plates. Graphitic materials are cytocompatible and support cell adhesion and proliferation. Importantly, cells seeded on to oxidized graphene films undergo osteogenic differentiation in fetal bovine serum-containing medium without the addition of any glucocorticoid or specific growth factors. These findings support graphene's potential to act as an osteoinducer and a vehicle to deliver mesenchymal stem cells, and suggest that the combination of graphene and goat mesenchymal stem cells provides a promising construct for bone tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.

Osteogenic differentiation capacity of human skeletal muscle-derived progenitor cells.

PubMed

Oishi, Teruyo; Uezumi, Akiyoshi; Kanaji, Arihiko; Yamamoto, Naoki; Yamaguchi, Asami; Yamada, Harumoto; Tsuchida, Kunihiro

2013-01-01

Heterotopic ossification (HO) is defined as the formation of ectopic bone in soft tissue outside the skeletal tissue. HO is thought to result from aberrant differentiation of osteogenic progenitors within skeletal muscle. However, the precise origin of HO is still unclear. Skeletal muscle contains two kinds of progenitor cells, myogenic progenitors and mesenchymal progenitors. Myogenic and mesenchymal progenitors in human skeletal muscle can be identified as CD56(+) and PDGFRα(+) cells, respectively. The purpose of this study was to investigate the osteogenic differentiation potential of human skeletal muscle-derived progenitors. Both CD56(+) cells and PDGFRα(+) cells showed comparable osteogenic differentiation potential in vitro. However, in an in vivo ectopic bone formation model, PDGFRα(+) cells formed bone-like tissue and showed successful engraftment, while CD56(+) cells did not form bone-like tissue and did not adapt to an osteogenic environment. Immunohistological analysis of human HO sample revealed that many PDGFRα(+) cells were localized in proximity to ectopic bone formed in skeletal muscle. MicroRNAs (miRNAs) are known to regulate many biological processes including osteogenic differentiation. We investigated the participation of miRNAs in the osteogenic differentiation of PDGFRα(+) cells by using microarray. We identified miRNAs that had not been known to be involved in osteogenesis but showed dramatic changes during osteogenic differentiation of PDGFRα(+) cells. Upregulation of miR-146b-5p and -424 and downregulation of miR-7 during osteogenic differentiation of PDGFRα(+) cells were confirmed by quantitative real-time RT-PCR. Inhibition of upregulated miRNAs, miR-146b-5p and -424, resulted in the suppression of osteocyte maturation, suggesting that these two miRNAs have the positive role in the osteogenesis of PDGFRα(+) cells. Our results suggest that PDGFRα(+) cells may be the major source of HO and that the newly identified miRNAs may regulate osteogenic differentiation process of PDGFRα(+) cells.

Primary osteogenic sarcoma of the breast

PubMed Central

Ogundiran, Temidayo O; Ademola, Samuel A; Oluwatosin, Odunayo M; Akang, Effiong E; Adebamowo, Clement A

2006-01-01

Background Primary extra-osseous osteogenic sarcomas have been reported in many tissues of the body but their occurrence in the breast is extremely rare. It can arise as a result of osseous metaplasia in a pre-existing benign or malignant neoplasm of the breast or as non-phylloides sarcoma from the soft tissue of a previously normal breast. Case presentation A 40 year-old Nigerian woman was clinically diagnosed to have carcinoma of the left breast. The histology report of core-needle biopsy of the mass showed a malignant neoplasm comprising islands of chondroblastic and osteoblastic stromal cells. This report changed the diagnosis from carcinoma to osteogenic sarcoma of the breast. She had a left modified radical mastectomy, however there was significant post surgery skin deficit. A latissimus dorsi musculocutaneous flap was used to cover the anterior chest wall defect. Sections from the mastectomy specimen confirmed the diagnosis of osteogenic sarcoma. She died six months after mastectomy. Conclusion A diagnosis of osteogenic sarcoma of the breast was made based on histology report and after excluding an osteogenic sarcoma arising from underlying ribs and sternum. This is the second documented case of primary osteogenic sarcoma of the breast coming from Nigeria PMID:17156481

Insulin-like growth factor 1 can promote proliferation and osteogenic differentiation of human dental pulp stem cells via mTOR pathway.

PubMed

Feng, Xingmei; Huang, Dan; Lu, Xiaohui; Feng, Guijuan; Xing, Jing; Lu, Jun; Xu, Ke; Xia, Weiwei; Meng, Yan; Tao, Tao; Li, Liren; Gu, Zhifeng

2014-12-01

Insulin-like growth factor 1 (IGF-1) is a multifunctional peptide that can enhance osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs). However, it remains unclear whether IGF-1 can promote osteogenic differentiation of human dental pulp stem cells (DPSCs). In our study, DPSCs were isolated from the impacted third molars, and treated with IGF-1. Osteogenic differentiation abilities were investigated. We found that IGF-1 activated the mTOR signaling pathway during osteogenic differentiation of DPSCs. IGF-1 also increased the expression of runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), osterix (OSX) and collagen type I (COL I) during this process. Rapamycin, an mTOR inhibitor, blocked osteogenic differentiation induced by IGF-1. Meanwhile, CCK-8 assay and flow cytometry results demonstrated that 10-200 ng/mL IGF-1 could enhance proliferation ability of DPSCs and 100 ng/mL was the optimal concentration. In summary, IGF-1 could promote proliferation and osteogenic differentiation of DPSCs via mTOR pathways, which might have clinical implications for osteoporosis. © 2014 The Authors Development, Growth & Differentiation © 2014 Japanese Society of Developmental Biologists.

Changes in the population of perivascular cells in the bone tissue remodeling zones under microgravity

NASA Astrophysics Data System (ADS)

Katkova, Olena; Rodionova, Natalia; Shevel, Ivan

2016-07-01

Microgravity and long-term hypokinesia induce reduction both in bone mass and mineral saturation, which can lead to the development of osteoporosis and osteopenia. (Oganov, 2003). Reorganizations and adaptive remodeling processes in the skeleton bones occur in the topographical interconnection with blood capillaries and perivascular cells. Radioautographic studies with 3H- thymidine (Kimmel, Fee, 1980; Rodionova, 1989, 2006) have shown that in osteogenesis zones there is sequential differentiation process of the perivascular cells into osteogenic. Hence the study of populations of perivascular stromal cells in areas of destructive changes is actual. Perivascular cells from metaphysis of the rat femoral bones under conditions of modeling microgravity were studied using electron microscopy and cytochemistry (hindlimb unloading, 28 days duration) and biosatellite «Bion-M1» (duration of flight from April 19 till May 19, 2013 on C57, black mice). It was revealed that both control and test groups populations of the perivascular cells are not homogeneous in remodeling adaptive zones. These populations comprise of adjacent to endothelium poorly differentiated forms and isolated cells with signs of differentiation (specific increased volume of rough endoplasmic reticulum in cytoplasm). Majority of the perivascular cells in the control group (modeling microgravity) reveals reaction to alkaline phosphatase (marker of the osteogenic differentiation). In poorly differentiated cells this reaction is registered in nucleolus, nucleous and cytoplasm. In differentiating cells activity of the alkaline phosphatase is also detected on the outer surface of the cellular membrane. Unlike the control group in the bones of experimental animals reaction to the alkaline phosphatase is registered not in all cells of perivascular population. Part of the differentiating perivascular cells does not contain a product of the reaction. Under microgravity some poorly differentiated perivascular cells reveal signs of destruction. Thus it was found that number of the alkaline phosphatase containing cells (i.e. osteogenic cells) declines in perivascular cells population. It is one of the mechanisms of the osteogenic process decrease of intensity in bones because of lessening support loading on the bone skeleton. In the adaptive remodeling zones of bone tissue (near the vascular canals) in experiments fibroblasts and fibrosis zones were found - areas filled with non-mineralized collagen fibrils on the bones surfaces. Hence it should be considered that decrease (removal) of support loading slows down osteogenic differentiation of the part of perivascular cells and stimulates differentiation of the fibroblast cells. Obtained data is considered as one of the cellular mechanisms of the adaptive reactions development in spongy bone under microgravity which could lead to the bone mass loss.

Effect of activated autologous platelet-rich plasma on proliferation and osteogenic differentiation of human adipose-derived stem cells in vitro

PubMed Central

Xu, Fang-Tian; Li, Hong-Mian; Yin, Qing-Shui; Liang, Zhi-Jie; Huang, Min-Hong; Chi, Guang-Yi; Huang, Lu; Liu, Da-Lie; Nan, Hua

2015-01-01

To investigate whether activated autologous platelet-rich plasma (PRP) can promote proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs) in vitro. hASCs were isolated from lipo-aspirates, and characterized by specific cell markers and multilineage differentiation capacity after culturing to the 3rd passage. PRP was collected and activated from human peripheral blood of the same patient. Cultured hASCs were treated with normal osteogenic inductive media alone (group A, control) or osteogenic inductive media plus 5%, 10%, 20%, 40%PRP (group B, C, D, E, respectively). Cell proliferation was assessed by CCK-8 assay. mRNA expression of osteogenic marker genes including alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OCN) and core binding factor alpha 1 (Cbfa1) were determined by Real-Time Quantitative PCR Analysis (qPCR). Data revealed that different concentrations of activated autologous PRP significantly promoted hASCs growth in the proliferation phase compared to the without PRP group and resulted in a dose-response relationship. At 7-d and 14-d time point of the osteogenic induced stage, ALP activity in PRP groups gradually increased with the increasing of concentrations of PRP and showed that dose-response relationship. At 21-d time point of the osteogenic induced stage, PRP groups make much more mineralization and mRNA relative expression of ALP, OPN, OCN and Cbfa1 than that without PRP groups and show that dose-response relationship. This study indicated that different concentrations of activated autologous PRP can promote cell proliferation at earlier stage and promote osteogenic differentiation at later stage of hASCs in vitro. Moreover, it displayed a dose-dependent effect of activated autologous PRP on cell proliferation and osteogenic differentiation of hASCs in vitro. PMID:25901195

Dexamethasone Enhances Osteogenic Differentiation of Bone Marrow- and Muscle-Derived Stromal Cells and Augments Ectopic Bone Formation Induced by Bone Morphogenetic Protein-2

PubMed Central

Yuasa, Masato; Yamada, Tsuyoshi; Taniyama, Takashi; Masaoka, Tomokazu; Xuetao, Wei; Yoshii, Toshitaka; Horie, Masaki; Yasuda, Hiroaki; Uemura, Toshimasa; Okawa, Atsushi; Sotome, Shinichi

2015-01-01

We evaluated whether dexamethasone augments the osteogenic capability of bone marrow-derived stromal cells (BMSCs) and muscle tissue-derived stromal cells (MuSCs), both of which are thought to contribute to ectopic bone formation induced by bone morphogenetic protein-2 (BMP-2), and determined the underlying mechanisms. Rat BMSCs and MuSCs were cultured in growth media with or without 10-7 M dexamethasone and then differentiated under osteogenic conditions with dexamethasone and BMP-2. The effects of dexamethasone on cell proliferation and osteogenic differentiation, and also on ectopic bone formation induced by BMP-2, were analyzed. Dexamethasone affected not only the proliferation rate but also the subpopulation composition of BMSCs and MuSCs, and subsequently augmented their osteogenic capacity during osteogenic differentiation. During osteogenic induction by BMP-2, dexamethasone also markedly affected cell proliferation in both BMSCs and MuSCs. In an in vivo ectopic bone formation model, bone formation in muscle-implanted scaffolds containing dexamethasone and BMP-2 was more than two fold higher than that in scaffolds containing BMP-2 alone. Our results suggest that dexamethasone potently enhances the osteogenic capability of BMP-2 and may thus decrease the quantity of BMP-2 required for clinical application, thereby reducing the complications caused by excessive doses of BMP-2. Highlights: 1. Dexamethasone induced selective proliferation of bone marrow- and muscle-derived cells with higher differentiation potential. 2. Dexamethasone enhanced the osteogenic capability of bone marrow- and muscle-derived cells by altering the subpopulation composition. 3. Dexamethasone augmented ectopic bone formation induced by bone morphogenetic protein-2. PMID:25659106

Notch signaling pathways in human thoracic ossification of the ligamentum flavum.

PubMed

Qu, Xiaochen; Chen, Zhongqiang; Fan, Dongwei; Sun, Chuiguo; Zeng, Yan; Hou, Xiaofei; Ning, Shanglong

2016-08-01

This study investigated the pathological process of Notch signaling in the osteogenesis of ligamentum flavum tissues and cells, and the associated regulatory mechanisms. Notch receptors, ligands, and target genes were identified by quantitative polymerase chain reaction (qPCR) in ligamentum flavum cells and immunohistochemistry in ligamentum flavum sections from ossification of the ligamentum flavum (OLF) patients and controls. The temporospatial expression patterns of JAG1/Notch2/HES1 in human ligamentum flavum cells during osteogenic differentiation were determined by qPCR. Lentiviral vectors for Notch2 overexpression and knockdown were constructed and transfected into ligamentum flavum cells before osteogenic differentiation to examine the function of Notch signaling pathways in the osteogenic differentiation of ligamentum flavum cells. Alkaline phosphatase, Runx2, Osterix, osteocalcin, and osteopontin mRNA levels, alkaline phosphatase activity, and Alizarin Red staining were used as indicators of osteogenic differentiation. JAG1/Notch2/HES1 mRNA levels were up-regulated in ligamentum flavum cells from OLF patients, which increased during osteogenic differentiation. Immunohistochemical analysis suggested positive Notch2 expression at the ossification front. Down-regulation of Notch2 expression decelerated osteogenic differentiation of ligamentum flavum cells, and Notch2 overexpression promoted osteogenic differentiation of ligamentum flavum cells. Expression of Runx2 and Osterix increased in a manner similar to that of Notch2 during osteogenic differentiation of ligamentum flavum cells, and Notch2 knockdown and overexpression influenced their expression levels. Notch signaling plays an important role in OLF, and Notch may affect the osteogenic differentiation of ligamentum flavum cells via interactions with Runx2 and Osterix.© 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1481-1491, 2016. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Modification of Silk Fibroin Using Diazonium Coupling Chemistry and the Effects on hMSC Proliferation and Differentiation

PubMed Central

Murphy, Amanda R.; John, Peter St.; Kaplan, David L.

2009-01-01

A simple chemical modification method using diazonium coupling chemistry was developed to tailor the structure and hydrophilicity of silk fibroin protein. The extent of modification using several aniline derivatives was characterized using UV/vis and 1H NMR spectroscopy, and the resulting protein structure was analyzed with ATR-FTIR spectroscopy. Introduction of hydrophobic functional groups facilitated rapid conversion of the protein from a random coil to a β-sheet structure, while addition of hydrophilic groups inhibited this process. hMSCs were grown on these modified silks to assess the biocompatibility of these materials. The hydrophilicity of the silk derivatives was found to affect the growth rate and morphology, but hMSCs were able to attach, proliferate and differentiate into an osteogenic lineage on all of the silk derivatives. PMID:18417206

Coculture with endothelial cells enhances osteogenic differentiation of periodontal ligament stem cells via cyclooxygenase-2/prostaglandin E2/vascular endothelial growth factor signaling under hypoxia.

PubMed

Zhao, Lixing; Wu, Yeke; Tan, Lijun; Xu, Zhenrui; Wang, Jun; Zhao, Zhihe; Li, Xiaoyu; Li, Yu; Yang, Pu; Tang, Tian

2013-12-01

During periodontitis and orthodontic tooth movement, periodontal vasculature is severely impaired, leading to a hypoxic microenvironment of periodontal cells. However, the impact of hypoxia on periodontal cells is poorly defined. The present study investigates responses of cocultured endothelial cells (ECs) and periodontal ligament stem cells (PDLSCs) to hypoxia. Osteogenic differentiation, molecular characterization, and various behaviors of PDLSCs and human umbilical venous ECs under hypoxia were assessed by quantitative real-time reverse-transcription polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. Moreover, the effect of ECs on PDLSC osteogenic differentiation was tested using NS398 (cyclooxygenase 2 blocker), SU5416 (vascular endothelial growth factor [VEGF] receptor inhibitor), AH6809, L-798106, and L-161982 (EP1/2/3/4 antagonists). First, hypoxia promoted osteogenic differentiation in PDLSCs and enhanced EC migration, whereas PD98059 (extracellular signal-regulated protein kinase [ERK] inhibitor) blocked, and cocultured ECs further enhanced, hypoxia-induced osteogenic differentiation. Second, NS398 impaired EC migration and prostaglandin E2 (PGE2)/VEGF release, whereas cocultured PDLSCs and exogenous PGE2 partially reversed it. Third, NS398 (pretreated ECs) decreased PGE2/VEGF concentrations. NS398-treated ECs and AH6809/SU5416-treated PDLSCs impaired cocultured EC-induced enhancement of PDLSC osteogenic differentiation. Hypoxia enhances ERK-mediated osteogenic differentiation in PDLSCs. Coculture with EC further augments PDLSC osteogenic differentiation via cyclooxygenase-2/PGE2/VEGF signaling.

Fibrinogen Induces RUNX2 Activity and Osteogenic Development from Human Pluripotent Stem Cells

PubMed Central

Kidwai, Fahad; Edwards, Jessica; Zou, Li; Kaufman, Dan S.

2016-01-01

Pluripotent stem cells, both human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC), provide an important resource to produce specialized cells such as osteogenic cells for therapeutic applications such as repair or replacement of injured, diseased or damaged bone. hESCs and iPSCs can also be used to better define basic cellular and genetic mechanisms that regulate the earliest stages of human bone development. However, current strategies to mediate osteogenic differentiation of hESC and iPSC are typically limited by the use of xenogeneic components such as fetal bovine serum (FBS) that make defining specific agents that mediate human osteogenesis difficult. Runt-related transcription factor 2 (RUNX2) is a key regulator required for osteogenic differentiation. Here, we used a RUNX2-YFP reporter system to characterize the novel ability of fibrinogen to mediate human osteogenic development from hESC and iPSC in defined (serum-free) conditions. These studies demonstrate that fibrinogen mediates significant osteo-induction potential. Specifically, fibrinogen binds to the surface integrin (α9β1) to mediate RUNX2 gene expression through the SMAD1/5/8 signaling pathway. Additional studies characterize the fibrinogen-induced hESC/iPSC-derived osteogenic cells to demonstrate these osteogenic cells retain the capacity to express typical mature osteoblastic markers. Together, these studies define a novel fibrinogen-α9β1-SMAD1/5/8-RUNX2 signaling axis can efficiently induce osteogenic differentiation from hESCs and iPSCs. PMID:27331788

DNA Demethylation Rescues the Impaired Osteogenic Differentiation Ability of Human Periodontal Ligament Stem Cells in High Glucose

PubMed Central

Liu, Zhi; Chen, Tian; Sun, Wenhua; Yuan, Zongyi; Yu, Mei; Chen, Guoqing; Guo, Weihua; Xiao, Jingang; Tian, Weidong

2016-01-01

Diabetes mellitus, characterized by abnormally high blood glucose levels, gives rise to impaired bone remodeling. In response to high glucose (HG), the attenuated osteogenic differentiation capacity of human periodontal ligament stem cells (hPDLSCs) is associated with the loss of alveolar bone. Recently, DNA methylation was reported to affect osteogenic differentiation of stem cells in pathological states. However, the intrinsic mechanism linking DNA methylation to osteogenic differentiation ability in the presence of HG is still unclear. In this study, we found that diabetic rats with increased DNA methylation levels in periodontal ligaments exhibited reduced bone mass and density. In vitro application of 5-aza-2′-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, to decrease DNA methylation levels in hPDLSCs, rescued the osteogenic differentiation capacity of hPDLSCs under HG conditions. Moreover, we demonstrated that the canonical Wnt signaling pathway was activated during this process and, under HG circumstances, the 5-aza-dC-rescued osteogenic differentiation capacity was blocked by Dickkopf-1, an effective antagonist of the canonical Wnt signaling pathway. Taken together, these results demonstrate for the first time that suppression of DNA methylation is able to facilitate the osteogenic differentiation capacity of hPDLSCs exposed to HG, through activation of the canonical Wnt signaling pathway. PMID:27273319

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.

Osteogenic Treatment Initiating a Tissue-Engineered Cartilage Template Hypertrophic Transition.

PubMed

Fu, J Y; Lim, S Y; He, P F; Fan, C J; Wang, D A

2016-10-01

Hypertrophic chondrocytes play a critical role in endochondral bone formation as well as the progress of osteoarthritis (OA). An in vitro cartilage hypertrophy model can be used as a platform to study complex molecular mechanisms involved in these processes and screen new drugs for OA. To develop an in vitro cartilage hypertrophy model, we treated a tissue-engineered cartilage template, living hyaline cartilaginous graft (LhCG), with osteogenic medium for hypertrophic induction. In addition, endothelial progenitor cells (EPCs) were seeded onto LhCG constructs to mimic vascular invasion. The results showed that osteogenic treatment significantly inhibited the synthesis of endostatin in LhCG constructs and enhanced expression of hypertrophic marker-collagen type X (Col X) and osteogenic markers, as well as calcium deposition in vitro. Upon subcutaneous implantation, osteogenic medium-treated LhCG constructs all stained positive for Col X and showed significant calcium deposition and blood vessel invasion. Col X staining and calcium deposition were most obvious in osteogenic medium-treated only group, while there was no difference between EPC-seeded and non-seeded group. These results demonstrated that osteogenic treatment was of the primary factor to induce hypertrophic transition of LhCG constructs and this model may contribute to the establishment of an in vitro cartilage hypertrophy model.

Effect of lactoferrin on osteogenic differentiation of human adipose stem cells.

PubMed

Ying, Xiaozhou; Cheng, Shaowen; Wang, Wei; Lin, Zhongqin; Chen, Qingyu; Zhang, Wei; Kou, Dongquan; Shen, Yue; Cheng, Xiaojie; Peng, Lei; Zi Xu, Hua; Zhu Lu, Chuan

2012-03-01

Many in vitro studies of the analysis of the lactoferrin (LF) effect on cells have been reported. However, no study has yet investigated the effect of LF on osteogenic differentiation of human adipose-derived stem cells (hADSCs). The aim of this study was to evaluate the effect of LF on osteogenic differentiation of human adipose stem cells. The hADSCs were cultured in an osteogenic medium with 0, 10, 50 and 100 μg/ml LF, respectively. hADSC proliferation was analysed by Cell Counting Kit-8 (CCK-8) assay, and cell osteogenic differentiation was evaluated by alkaline phosphatase (ALP) activity assay, von Kossa staining and real-time polymerase chain reaction (RT-PCR). Cell proliferation was significantly increased by LF in a dose-dependent manner from days 4 to 14. Cells cultured with 100 μg/ml LF presented a higher activity compared with the control. The deposition of calcium was increased after the addition of LF. The mRNA expression of type I collagen (COL-I), ALP, osteocalcin (OCN) and RUNX2 increased markedly as a result of LF treatment. We have shown for the first time that LF could promote the proliferation and osteogenic differentiation of hADSCs, which could be a promising approach for enhancing osteogenic capacity of cell-based construction in bone tissue engineering.

Design, synthesis, and osteogenic activity of daidzein analogs on human mesenchymal stem cells

USDA-ARS?s Scientific Manuscript database

Osteoporosis, defined by the loss of bone mass and strength, results in the loss of structural and mechanical support in bone, and leads to an increased risk of fractures. In the adult skeleton, the bone undergoes continuous resorption carried out by osteoclast cells, and formation by osteoblast cel...

A comparative study of the proliferation and osteogenic differentiation of human periodontal ligament cells cultured on β-TCP ceramics and demineralized bone matrix with or without osteogenic inducers in vitro.

PubMed

An, Shaofeng; Gao, Yan; Huang, Xiangya; Ling, Junqi; Liu, Zhaohui; Xiao, Yin

2015-05-01

The repair of bone defects that result from periodontal diseases remains a clinical challenge for periodontal therapy. β-tricalcium phosphate (β-TCP) ceramics are biodegradable inorganic bone substitutes with inorganic components that are similar to those of bone. Demineralized bone matrix (DBM) is an acid-extracted organic matrix derived from bone sources that consists of the collagen and matrix proteins of bone. A few studies have documented the effects of DBM on the proliferation and osteogenic differentiation of human periodontal ligament cells (hPDLCs). The aim of the present study was to investigate the effects of inorganic and organic elements of bone on the proliferation and osteogenic differentiation of hPDLCs using three-dimensional porous β-TCP ceramics and DBM with or without osteogenic inducers. Primary hPDLCs were isolated from human periodontal ligaments. The proliferation of the hPDLCs on the scaffolds in the growth culture medium was examined using a Cell-Counting kit-8 (CCK-8) and scanning electron microscopy (SEM). Alkaline phosphatase (ALP) activity and the osteogenic differentiation of the hPDLCs cultured on the β-TCP ceramics and DBM were examined in both the growth culture medium and osteogenic culture medium. Specific osteogenic differentiation markers were examined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). SEM images revealed that the cells on the β-TCP were spindle-shaped and much more spread out compared with the cells on the DBM surfaces. There were no significant differences observed in cell proliferation between the β-TCP ceramics and the DBM scaffolds. Compared with the cells that were cultured on β-TCP ceramics, the ALP activity, as well as the Runx2 and osteocalcin (OCN) mRNA levels in the hPDLCs cultured on DBM were significantly enhanced both in the growth culture medium and the osteogenic culture medium. The organic elements of bone may exhibit greater osteogenic differentiation effects on hPDLCs than the inorganic elements.

Autonomous osteogenic differentiation of hASCs encapsulated in methacrylated gellan-gum hydrogels.

PubMed

Oliveira, Mariana B; Custódio, Catarina A; Gasperini, Luca; Reis, Rui L; Mano, João F

2016-09-01

Methacrylated gellan-gum (GG-MA) alone and combined with collagen type I (Coll) is suggested here for the first time as a cell-laden injectable biomaterial for bone regeneration. On-chip high-throughput studies allowed rapidly assessing the suitability of 15 biomaterials/media combinations for the osteodifferentiation of human adipose stem cells (hASCs). Hydrogels composed solely of GG-MA (GG100:0Coll) led hASCs from three different donors into the osteogenic lineage after 21days of cell culture, in the absence of any osteogenic or osteoconductive factors. Hydrogels containing more than 30% of Coll promoted increased cellular proliferation and led hASCs into osteogenic differentiation under basal conditions. Studies using isolated individual hydrogels - excluding eventual on-chip crosstalk - and standard biochemical assays corroborated such findings. The formation of focal adhesions of hASCs on GG100:0Coll hydrogels was verified. We hypothesize that the hydrogels osteogenic effect could be guided by mechanotransduction phenomena. Indeed, the hydrogels showed elastic modulus in ranges previously reported as osteoinductive and the inhibition of the actin-myosin contractility pathway impaired hASCs' osteodifferentiation. GG-MA hydrogels also did not promote hASCs' adipogenesis while used in basal conditions. Overall, GG-MA showed promising properties as an innovative and off-the shelf self-inducing osteogenic injectable biomaterial. Methacrylated gellan gum (GG-MA) is here suggested for the first time as a widely available polysaccharide to easily prepare hydrogels with cell adhesion properties and capability of inducing the autonomous osteogenic differentiation of human adipose-derived stem cells (hASCs). GG-MA was processed as stand-alone hydrogels or in different combinations with collage type I. All hydrogel formulations elicited the osteogenic differentiation of hASCs, independently of the addition of any osteoconductive or osteogenic stimuli, i.e. in basal/growth medium. Effective cellular adhesion to methacrylated gellan gum hydrogels in the absence of any cell-ligand peptide/protein was here proved for the first time. Moreover, we showed that the encapsulated hASCs underwent osteogenic differentiation due to a mechanotransduction phenomenon dependent on the actin-myosin contractility pathway. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

A novel osteogenic oxysterol compound for therapeutic development to promote bone growth: activation of hedgehog signaling and osteogenesis through smoothened binding.

PubMed

Montgomery, Scott R; Nargizyan, Taya; Meliton, Vicente; Nachtergaele, Sigrid; Rohatgi, Rajat; Stappenbeck, Frank; Jung, Michael E; Johnson, Jared S; Aghdasi, Bayan; Tian, Haijun; Weintraub, Gil; Inoue, Hirokazu; Atti, Elisa; Tetradis, Sotirios; Pereira, Renata C; Hokugo, Akishige; Alobaidaan, Raed; Tan, Yanlin; Hahn, Theodor J; Wang, Jeffrey C; Parhami, Farhad

2014-08-01

Osteogenic factors are often used in orthopedics to promote bone growth, improve fracture healing, and induce spine fusion. Osteogenic oxysterols are naturally occurring molecules that were shown to induce osteogenic differentiation in vitro and promote spine fusion in vivo. The purpose of this study was to identify an osteogenic oxysterol more suitable for clinical development than those previously reported, and evaluate its ability to promote osteogenesis in vitro and spine fusion in rats in vivo. Among more than 100 oxysterol analogues synthesized, Oxy133 induced significant expression of osteogenic markers Runx2, osterix (OSX), alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OCN) in C3H10T1/2 mouse embryonic fibroblasts and in M2-10B4 mouse marrow stromal cells. Oxy133-induced activation of an 8X-Gli luciferase reporter, its direct binding to Smoothened, and the inhibition of Oxy133-induced osteogenic effects by the Hedgehog (Hh) pathway inhibitor, cyclopamine, demonstrated the role of Hh pathway in mediating osteogenic responses to Oxy133. Oxy133 did not stimulate osteogenesis via BMP or Wnt signaling. Oxy133 induced the expression of OSX, BSP, and OCN, and stimulated robust mineralization in primary human mesenchymal stem cells. In vivo, bilateral spine fusion occurred through endochondral ossification and was observed in animals treated with Oxy133 at the fusion site on X-ray after 4 weeks and confirmed with manual assessment, micro-CT (µCT), and histology after 8 weeks, with equal efficiency to recombinant human bone morphogenetic protein-2 (rhBMP-2). Unlike rhBMP-2, Oxy133 did not induce adipogenesis in the fusion mass and resulted in denser bone evidenced by greater bone volume/tissue volume (BV/TV) ratio and smaller trabecular separation. Findings here suggest that Oxy133 has significant potential as an osteogenic molecule with greater ease of synthesis and improved time to fusion compared to previously studied oxysterols. Small molecule osteogenic oxysterols may serve as the next generation of bone anabolic agents for therapeutic development. © 2014 American Society for Bone and Mineral Research.

A Novel Osteogenic Oxysterol Compound for Therapeutic Development to Promote Bone Growth: Activation of Hedgehog Signaling and Osteogenesis through Smoothened Binding

PubMed Central

Montgomery, Scott R.; Nargizyan, Taya; Meliton, Vicente; Nachtergaele, Sigrid; Rohatgi, Rajat; Stappenbeck, Frank; Jung, Michael E.; Johnson, Jared S.; Aghdasi, Bayan; Tian, Haijun; Weintraub, Gil; Inoue, Hirokazu; Atti, Elisa; Tetradis, Sotirios; Pereira, Renata C; Hokugo, Akishige; Alobaidaan, Raed; Tan, Yanlin; Hahn, Theodor J.; Wang, Jeffrey C; Parhami, Farhad

2015-01-01

Osteogenic factors are often used in orthopedics to promote bone growth, improve fracture healing, and induce spine fusion. Osteogenic oxysterols are naturally occurring molecules that were shown to induce osteogenic differentiation in vitro and promote spine fusion in vivo. The purpose of this study was to identify an osteogenic oxysterol more suitable for clinical development than those previously reported, and evaluate its ability to promote osteogenesis in vitro and spine fusion in rats in vivo. Among more than 100 oxysterol analogues synthesized, Oxy133 induced significant expression of osteogenic markers Runx2, osterix (OSX), alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OCN) in C3H10T1/2 mouse embryonic fibroblasts and in M2-10B4 mouse marrow stromal cells. Oxy133-induced activation of an 8×-Gli luciferase reporter, its direct binding to Smoothened, and the inhibition of Oxy133-induced osteogenic effects by the Hedgehog (Hh) pathway inhibitor, cyclopamine, demonstrated the role of Hh pathway in mediating osteogenic responses to Oxy133. Oxy133 did not stimulate osteogenesis via BMP or Wnt signaling. Oxy133 induced the expression of OSX, BSP, and OCN and stimulated robust mineralization in primary human mesenchymal stem cells. In vivo, bilateral spine fusion occurred through endochondral ossification and was observed in animals treated with Oxy133 at the fusion site on xray after 4 weeks and confirmed with manual assessment, micro CT (μCT), and histology after 8 weeks, with equal efficiency to recombinant human bone morphogenetic protein-2 (rhBMP-2). Unlike rhBMP-2, Oxy133 did not induce adipogenesis in the fusion mass and resulted in denser bone evidenced by greater BV/TV ratio and smaller trabecular separation. Findings here suggest that Oxy133 has significant potential as an osteogenic molecule with greater ease of synthesis and improved time to fusion compared to previously studied oxysterols. Small molecule osteogenic oxysterols may serve as the next generation of bone anabolic agents for therapeutic development. PMID:24591126

Boron nitride nanotube-enhanced osteogenic differentiation of mesenchymal stem cells.

PubMed

Li, Xia; Wang, Xiupeng; Jiang, Xiangfen; Yamaguchi, Maho; Ito, Atsuo; Bando, Yoshio; Golberg, Dmitri

2016-02-01

The interaction between boron nitride nanotubes (BNNTs) layer and mesenchymal stem cells (MSCs) is evaluated for the first time in this study. BNNTs layer supports the attachment and growth of MSCs and exhibits good biocompatibility with MSCs. BNNTs show high protein adsorption ability, promote the proliferation of MSCs and increase the secretion of total protein by MSCs. Especially, BNNTs enhance the alkaline phosphatase (ALP) activity as an early marker of osteoblasts, ALP/total protein and osteocalcin (OCN) as a late marker of osteogenic differentiation, which shows that BNNTs can enhance osteogenesis of MSCs. The release of trace boron and the stress on cells exerted by BNNTs with a fiber structure may account for the enhanced differentiation of MSCs into osteoblasts. Therefore BNNTs are potentially useful for bone regeneration in orthopedic applications. © 2015 Wiley Periodicals, Inc.

Conditions Inducing Excessive O-GlcNAcylation Inhibit BMP2-Induced Osteogenic Differentiation of C2C12 Cells.

PubMed

Gu, Hanna; Song, Mina; Boonanantanasarn, Kanitsak; Baek, Kyunghwa; Woo, Kyung Mi; Ryoo, Hyun-Mo; Baek, Jeong-Hwa

2018-01-09

Hyperglycemic conditions in diabetic patients can affect various cellular functions, including the modulation of osteogenic differentiation. However, the molecular mechanisms by which hyperglycemia affects osteogenic differentiation are yet to be clarified. This study aimed to investigate whether the aberrant increase in protein O -linked-β- N -acetylglucosamine glycosylation ( O -GlcNAcylation) contributes to the suppression of osteogenic differentiation due to hyperglycemia. To induce osteogenic differentiation, C2C12 cells were cultured in the presence of recombinant human bone morphogenetic protein 2 (BMP2). Excessive protein O -GlcNAcylation was induced by treating C2C12 cells with high glucose, glucosamine, or N -acetylglucosamine concentrations or by O -GlcNAc transferase (OGT) overexpression. The effect of O -GlcNAcylation on osteoblast differentiation was then confirmed by examining the expression levels of osteogenic marker gene mRNAs, activity of alkaline phosphatase, and transcriptional activity of Runx2, a critical transcription factor for osteoblast differentiation and bone formation. Cell treatment with high glucose, glucosamine or N -acetylglucosamine increased O -GlcNAcylation of Runx2 and the total levels of O -GlcNAcylated proteins, which led to a decrease in the transcriptional activity of Runx2, expression levels of osteogenic marker genes (Runx2, osterix, alkaline phosphatase, and type I collagen), and activity of alkaline phosphatase. These inhibitory effects were rescued by lowering protein O -GlcNAcylation levels by adding STO45849, an OGT inhibitor, or by overexpressing β- N -acetylglucosaminidase. Our findings suggest that excessive protein O -GlcNAcylation contributes to high glucose-suppressed osteogenic differentiation.

Laser-modified titanium surfaces enhance the osteogenic differentiation of human mesenchymal stem cells.

PubMed

Bressel, Tatiana A B; de Queiroz, Jana Dara Freires; Gomes Moreira, Susana Margarida; da Fonseca, Jéssyca T; Filho, Edson A; Guastaldi, Antônio Carlos; Batistuzzo de Medeiros, Silvia Regina

2017-11-28

Titanium surfaces have been modified by various approaches with the aim of improving the stimulation of osseointegration. Laser beam (Yb-YAG) treatment is a controllable and flexible approach to modifying surfaces. It creates a complex surface topography with micro and nano-scaled patterns, and an oxide layer that can improve the osseointegration of implants, increasing their usefulness as bone implant materials. Laser beam irradiation at various fluences (132, 210, or 235 J/cm 2 ) was used to treat commercially pure titanium discs to create complex surface topographies. The titanium discs were investigated by scanning electron microscopy, X-ray diffraction, and measurement of contact angles. The surface generated at a fluence of 235 J/cm 2 was used in the biological assays. The behavior of mesenchymal stem cells from an umbilical cord vein was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, a mineralization assay, and an alkaline phosphatase activity assay and by carrying out a quantitative real-time polymerase chain reaction for osteogenic markers. CHO-k1 cells were also exposed to titanium discs in the MTT assay. The best titanium surface was that produced by laser beam irradiation at 235 J/cm 2 fluence. Cell proliferation analysis revealed that the CHO-k1 and mesenchymal stem cells behaved differently. The laser-processed titanium surface increased the proliferation of CHO-k1 cells, reduced the proliferation of mesenchymal stem cells, upregulated the expression of the osteogenic markers, and enhanced alkaline phosphatase activity. The laser-treated titanium surface modulated cellular behavior depending on the cell type, and stimulated osteogenic differentiation. This evidence supports the potential use of laser-processed titanium surfaces as bone implant materials, and their use in regenerative medicine could promote better outcomes.

Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation.

PubMed

Sharma, Sunita; Sapkota, Dipak; Xue, Ying; Sun, Yang; Finne-Wistrand, Anna; Bruland, Ove; Mustafa, Kamal

2016-01-01

Selection of appropriate osteoinductive growth factors, suitable delivery method and proper supportive scaffold are critical for a successful outcome in bone tissue engineering using bone marrow stromal cells (BMSC). This study examined the molecular and functional effect of a combination of adenoviral mediated expression of bone morphogenetic protein-2 (BMP2) in BMSC and recently developed and characterized, biodegradable Poly(L-lactide-co-є-caprolactone){poly(LLA-co-CL)}scaffolds in osteogenic molecular changes and ectopic bone formation by using in vitro and in vivo approaches. Pathway-focused custom PCR array, validation using TaqMan based quantitative RT-PCR (qRT-PCR) and ALP staining showed significant up-regulation of several osteogenic and angiogenic molecules, including ALPL and RUNX2 in ad-BMP2 BMSC group grown in poly(LLA-co-CL) scaffolds both at 3 and 14 days. Micro CT and histological analyses of the subcutaneously implanted scaffolds in NOD/SCID mice revealed significantly increased radiopaque areas, percentage bone volume and formation of vital bone in ad-BMP2 scaffolds as compared to the control groups both at 2 and 8 weeks. The increased bone formation in the ad-BMP2 group in vivo was paralleled at the molecular level with concomitant over-expression of a number of osteogenic and angiogenic genes including ALPL, RUNX2, SPP1, ANGPT1. The increased bone formation in ad-BMP2 explants was not found to be associated with enhanced endochondral activity as evidenced by qRT-PCR (SOX9 and FGF2) and Safranin O staining. Taken together, combination of adenoviral mediated BMP-2 expression in BMSC grown in the newly developed poly(LLA-co-CL) scaffolds induced expression of osteogenic markers and enhanced bone formation in vivo.

Effect of bone sialoprotein coating of ceramic and synthetic polymer materials on in vitro osteogenic cell differentiation and in vivo bone formation.

PubMed

Schaeren, Stefan; Jaquiéry, Claude; Wolf, Francine; Papadimitropoulos, Adam; Barbero, Andrea; Schultz-Thater, Elke; Heberer, Michael; Martin, Ivan

2010-03-15

In this study, we addressed whether Bone Sialoprotein (BSP) coating of various substrates could enhance the in vitro osteogenic differentiation and in vivo bone formation capacity of human Bone Marrow Stromal Cells (BMSC). Moreover, we tested whether synthetic polymer-based porous scaffolds, despite the absence of a mineral component, could support ectopic bone formation by human BMSC if coated with BSP. Adsorption of recombinant human BSP on tissue culture-treated polystyrene (TCTP), beta-tricalcium phosphate (Osteologic) or synthetic polymer (Polyactive) substrates was dose dependent, but did not consistently accelerate or enhance in vitro BMSC osteogenic differentiation, as assessed by the mRNA expression of osteoblast-related genes. Similarly, BSP coating of porous beta-tricalcium phosphate scaffolds (Skelite) did not improve the efficiency of bone tissue formation following loading with BMSC and ectopic implantation in nude mice. Finally, Polyactive foams seeded with BMSC did not form bone tissue in the same ectopic assay, even if coated with BSP. We conclude that BSP coating of a variety of substrates is not directly associated with an enhancement of osteoprogenitor cell differentiation in vitro or in vivo, and that presentation of BSP on polymeric materials is not sufficient to prime BMSC functional osteoblastic differentiation in vivo. (c) 2009 Wiley Periodicals, Inc.

A unique stylopod patterning mechanism by Shox2-controlled osteogenesis

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

Ye, Wenduo; Song, Yingnan; Huang, Zhen

Here, vertebrate appendage patterning is programmed by Hox-TALE factorbound regulatory elements. However, it remains unclear which cell lineages are commissioned by Hox-TALE factors to generate regional specific patterns and whether other Hox-TALE co-factors exist. In this study, we investigated the transcriptional mechanisms controlled by the Shox2 transcriptional regulator in limb patterning. Harnessing an osteogenic lineage-specific Shox2 inactivation approach we show that despite widespread Shox2 expression in multiple cell lineages, lack of the stylopod observed upon Shox2 deficiency is a specific result of Shox2 loss of function in the osteogenic lineage. ChIP-Seq revealed robust interaction of Shox2 with cis-regulatory enhancers clusteringmore » around skeletogenic genes that are also bound by Hox-TALE factors, supporting a lineage autonomous function of Shox2 in osteogenic lineage fate determination and skeleton patterning. Pbx ChIP-Seq further allowed the genome-wide identification of cis-regulatory modules exhibiting co-occupancy of Pbx, Meis and Shox2 transcriptional regulators. Integrative analysis of ChIP-Seq and RNA-Seq data and transgenic enhancer assays indicate that Shox2 patterns the stylopod as a repressor via interaction with enhancers active in the proximal limb mesenchyme and antagonizes the repressive function of TALE factors in osteogenesis.« less

Electrochemical synthesis of three-dimensional porous reduced graphene oxide film: Preparation and in vitro osteogenic activity evaluation.

PubMed

Tian, Zizhu; Huang, Lixun; Pei, Xibo; Chen, Junyu; Wang, Tong; Yang, Tao; Qin, Han; Sui, Lei; Wang, Jian

2017-07-01

In this study, three-dimensional reduced graphene oxide (3D-rGO) porous films were fabricated using a two-step electrochemical method, including an electrochemical deposition process for the self-assembly of GO and an electrochemical bubbling-based transfer. The morphology, physical properties, and phase composition of the 3D-rGO films were characterized, and the cellular bioactivities were evaluated using pre-osteoblasts (MC3T3-E1 cells). The attachment, proliferation and differentiation of the MC3T3-E1 cells on the 3D-rGO films was analyzed by scanning electron microscopy (SEM), Cell Counting Kit-8 (CCK-8) assays and live/dead cell staining, and alkaline phosphatase (ALP) activity assays, respectively. The expression of osteogenic-related genes in MC3T3-E1 cells was evaluated by reverse transcription-polymerase chain reaction (RT-PCR). The results showed that the 3D-rGO films supported cell viability and proliferation, as well as significantly enhanced ALP activity and osteogenic-related genes (ALP, OPN, Runx2) expressions. Our findings indicate the promising potential of the 3D-rGO porous films for bone tissue engineering. Copyright © 2017 Elsevier B.V. All rights reserved.

Influence of bone marrow-derived mesenchymal stem cells pre-implantation differentiation approach on periodontal regeneration in vivo.

PubMed

Cai, Xinjie; Yang, Fang; Yan, Xiangzhen; Yang, Wanxun; Yu, Na; Oortgiesen, Daniel A W; Wang, Yining; Jansen, John A; Walboomers, X Frank

2015-04-01

The implantation of bone marrow-derived mesenchymal stem cells (MSCs) has previously been shown successful to achieve periodontal regeneration. However, the preferred pre-implantation differentiation strategy (e.g. maintenance of stemness, osteogenic or chondrogenic induction) to obtain optimal periodontal regeneration is still unknown. This in vivo study explored which differentiation approach is most suitable for periodontal regeneration. Mesenchymal stem cells were obtained from Fischer rats and seeded onto poly(lactic-co-glycolic acid)/poly(ɛ-caprolactone) electrospun scaffolds, and then pre-cultured under different in vitro conditions: (i) retention of multilineage differentiation potential; (ii) osteogenic differentiation approach; and (iii) chondrogenic differentiation approach. Subsequently, the cell-scaffold constructs were implanted into experimental periodontal defects of Fischer rats, with empty scaffolds as controls. After 6 weeks of implantation, histomorphometrical analyses were applied to evaluate the regenerated periodontal tissues. The chondrogenic differentiation approach showed regeneration of alveolar bone and ligament tissues. The retention of multilineage differentiation potential supported only ligament regeneration, while the osteogenic differentiation approach boosted alveolar bone regeneration. Chondrogenic differentiation of MSCs before implantation is a useful strategy for regeneration of alveolar bone and periodontal ligament, in the currently used rat model. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A unique stylopod patterning mechanism by Shox2-controlled osteogenesis

DOE PAGES

Ye, Wenduo; Song, Yingnan; Huang, Zhen; ...

2016-06-10

Here, vertebrate appendage patterning is programmed by Hox-TALE factorbound regulatory elements. However, it remains unclear which cell lineages are commissioned by Hox-TALE factors to generate regional specific patterns and whether other Hox-TALE co-factors exist. In this study, we investigated the transcriptional mechanisms controlled by the Shox2 transcriptional regulator in limb patterning. Harnessing an osteogenic lineage-specific Shox2 inactivation approach we show that despite widespread Shox2 expression in multiple cell lineages, lack of the stylopod observed upon Shox2 deficiency is a specific result of Shox2 loss of function in the osteogenic lineage. ChIP-Seq revealed robust interaction of Shox2 with cis-regulatory enhancers clusteringmore » around skeletogenic genes that are also bound by Hox-TALE factors, supporting a lineage autonomous function of Shox2 in osteogenic lineage fate determination and skeleton patterning. Pbx ChIP-Seq further allowed the genome-wide identification of cis-regulatory modules exhibiting co-occupancy of Pbx, Meis and Shox2 transcriptional regulators. Integrative analysis of ChIP-Seq and RNA-Seq data and transgenic enhancer assays indicate that Shox2 patterns the stylopod as a repressor via interaction with enhancers active in the proximal limb mesenchyme and antagonizes the repressive function of TALE factors in osteogenesis.« less

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.

Low-power laser irradiation promotes the proliferation and osteogenic differentiation of human periodontal ligament cells via cyclic adenosine monophosphate

PubMed Central

Wu, Jyun-Yi; Chen, Chia-Hsin; Yeh, Li-Yin; Yeh, Ming-Long; Ting, Chun-Chan; Wang, Yan-Hsiung

2013-01-01

Retaining or improving periodontal ligament (PDL) function is crucial for restoring periodontal defects. The aim of this study was to evaluate the physiological effects of low-power laser irradiation (LPLI) on the proliferation and osteogenic differentiation of human PDL (hPDL) cells. Cultured hPDL cells were irradiated (660 nm) daily with doses of 0, 1, 2 or 4 J⋅cm−2. Cell proliferation was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and the effect of LPLI on osteogenic differentiation was assessed by Alizarin Red S staining and alkaline phosphatase (ALP) activity. Additionally, osteogenic marker gene expression was confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR). Our data showed that LPLI at a dose of 2 J⋅cm−2 significantly promoted hPDL cell proliferation at days 3 and 5. In addition, LPLI at energy doses of 2 and 4 J⋅cm−2 showed potential osteogenic capacity, as it stimulated ALP activity, calcium deposition, and osteogenic gene expression. We also showed that cyclic adenosine monophosphate (cAMP) is a critical regulator of the LPLI-mediated effects on hPDL cells. This study shows that LPLI can promote the proliferation and osteogenic differentiation of hPDL cells. These results suggest the potential use of LPLI in clinical applications for periodontal tissue regeneration. PMID:23788285

Osteogenic differentiation of dura mater stem cells cultured in vitro on three-dimensional porous scaffolds of poly(ε-caprolactone) fabricated via co-extrusion and gas foaming

PubMed Central

Aronin, C.E. Petrie; Cooper, J.A.; Sefcik, L.S.; Tholpady, S.S.; Ogle, R.C.; Botchwey, E.A.

2008-01-01

A novel scaffold fabrication method utilizing both polymer blend extrusion and gas foaming techniques to control pore size distribution is presented. Seventy five per cent of all pores produced using polymer blend extrusion alone were less than 50 μm. Introducing a gas technique provided better control of pore size distribution, expanding the range from 0-50 to 0-350 μm. Varying sintering time, annealing temperature and foaming pressure also helped reduced the percentage of pore sizes below 50 μm. Scaffolds chosen for in vitro cellular studies had a pore size distribution of 0-300 μm, average pore size 66 ± 17 μm, 0.54 ± 0.02% porosity and 98% interconnectivity, measured by micro computed tomography (microCT) analysis. The ability of the scaffolds to support osteogenic differentiation and cranial defect repair was evaluated by static and dynamic (0.035 ± 0.006 m s-1 terminal velocity) cultivation with dura mater stem cells (DSCs). In vitro studies showed minimal increases in proliferation over 28 days in culture in osteogenic media. Alkaline phosphatase expression remained constant throughout the study. Moderate increases in matrix deposition, as assessed by histochemical staining and microCT analysis, occurred at later time points, days 21 and 28. Although constructs cultured dynamically showed greater mineralization than static conditions, these trends were not significant. It remains unclear whether bioreactor culture of DSCs is advantageous for bone tissue engineering applications. However, these studies show that polycaprolactone (PCL) scaffolds alone, without the addition of other co-polymers or ceramics, support long-term attachment and mineralization of DSCs throughout the entire porous scaffold. PMID:18434267

Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion

PubMed Central

Kusuma, Gina D.; Brennecke, Shaun P.; O’Connor, Andrea J.; Kalionis, Bill

2017-01-01

Mesenchymal stem/stromal cells (MSCs) exhibit undesired phenotypic changes during ex vivo expansion, limiting production of the large quantities of high quality primary MSCs needed for both basic research and cell therapies. Primary MSCs retain many desired MSC properties including proliferative capacity and differentiation potential when expanded on decellularized extracellular matrix (dECM) prepared from primary MSCs. However, the need to use low passage number primary MSCs (passage 3 or lower) to produce the dECM drastically limits the utility and impact of this technology. Here, we report that primary MSCs expanded on dECM prepared from high passage number (passage 25) human telomerase reverse transcriptase (hTERT) transduced immortal MSC cell lines also exhibit increased proliferation and osteogenic differentiation. Two hTERT-transduced placenta-derived MSC cell lines, CMSC29 and DMSC23 [derived from placental chorionic villi (CMSCs) and decidua basalis (DMSCs), respectively], were used to prepare dECM-coated substrates. These dECM substrates showed structural and biochemical differences. Primary DMSCs cultured on dECM-DMSC23 showed a three-fold increase in cell number after 14 days expansion in culture and increased osteogenic differentiation compared with controls. Primary CMSCs cultured on the dECM-DMSC23 exhibited a two-fold increase in cell number and increased osteogenic differentiation. We conclude that immortal MSC cell lines derived from different parts of the placenta produce dECM with varying abilities for supporting increased primary MSC expansion while maintaining important primary MSC properties. Additionally, this is the first demonstration of using high passage number cells to produce dECM that can promote primary MSC expansion, and this advancement greatly increases the feasibility and applicability of dECM-based technologies. PMID:28152107

Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion.

PubMed

Kusuma, Gina D; Brennecke, Shaun P; O'Connor, Andrea J; Kalionis, Bill; Heath, Daniel E

2017-01-01

Mesenchymal stem/stromal cells (MSCs) exhibit undesired phenotypic changes during ex vivo expansion, limiting production of the large quantities of high quality primary MSCs needed for both basic research and cell therapies. Primary MSCs retain many desired MSC properties including proliferative capacity and differentiation potential when expanded on decellularized extracellular matrix (dECM) prepared from primary MSCs. However, the need to use low passage number primary MSCs (passage 3 or lower) to produce the dECM drastically limits the utility and impact of this technology. Here, we report that primary MSCs expanded on dECM prepared from high passage number (passage 25) human telomerase reverse transcriptase (hTERT) transduced immortal MSC cell lines also exhibit increased proliferation and osteogenic differentiation. Two hTERT-transduced placenta-derived MSC cell lines, CMSC29 and DMSC23 [derived from placental chorionic villi (CMSCs) and decidua basalis (DMSCs), respectively], were used to prepare dECM-coated substrates. These dECM substrates showed structural and biochemical differences. Primary DMSCs cultured on dECM-DMSC23 showed a three-fold increase in cell number after 14 days expansion in culture and increased osteogenic differentiation compared with controls. Primary CMSCs cultured on the dECM-DMSC23 exhibited a two-fold increase in cell number and increased osteogenic differentiation. We conclude that immortal MSC cell lines derived from different parts of the placenta produce dECM with varying abilities for supporting increased primary MSC expansion while maintaining important primary MSC properties. Additionally, this is the first demonstration of using high passage number cells to produce dECM that can promote primary MSC expansion, and this advancement greatly increases the feasibility and applicability of dECM-based technologies.

Hesperetin Alleviates the Inhibitory Effects of High Glucose on the Osteoblastic Differentiation of Periodontal Ligament Stem Cells

PubMed Central

Kim, So Yeon; Lee, Jin-Yong; Park, Yong-Duk; Kang, Kyung Lhi; Lee, Jeong-Chae; Heo, Jung Sun

2013-01-01

Hesperetin (3′,5,7-trihydroxy-4-methoxyflavanone) is a metabolite of hesperidin (hesperetin-7-O-rutinoside), which belongs to the flavanone subgroup and is found mainly in citrus fruits. Hesperetin has been reported to be an effective osteoinductive compound in various in vivo and in vitro models. However, how hesperetin effects osteogenic differentiation is not fully understood. In this study, we investigated the capacity of hesperetin to stimulate the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and to relieve the anti-osteogenic effect of high glucose. Osteogenesis of PDLSCs was assessed by measurement of alkaline phosphatase (ALP) activity, and evaluation of the mRNA expression of ALP, runt-related gene 2 (Runx2), osterix (OSX), and FRA1 as osteogenic transcription factors, as well as assessment of protein expression of osteopontin (OPN) and collagen type IA (COLIA). When PDLSCs were exposed to a high concentration (30 mM) of glucose, osteogenic activity decreased compared to control cells. Hesperetin significantly increased ALP activity at doses of 1, 10, and 100 µM. Pretreatment of cells with hesperetin alleviated the high-glucose-induced suppression of the osteogenic activity of PDLSCs. Hesperetin scavenged intracellular reactive oxygen species (ROS) produced under high glucose condition. Furthermore, hesperetin increased the activity of the PI3K/Akt and β-catenin pathways. Consistent with this, blockage of Akt or β-catenin diminished the protective effect of hesperetin against high glucose-inhibited osteogenic differentiation. Collectively, our results suggest that hesperetin alleviates the high glucose-mediated suppression of osteogenic differentiation in PDLSCs by regulating ROS levels and the PI3K/Akt and β-catenin signaling pathways. PMID:23840726

Canonical Wnt signaling differently modulates osteogenic differentiation of mesenchymal stem cells derived from bone marrow and from periodontal ligament under inflammatory conditions.

PubMed

Liu, Wenjia; Konermann, Anna; Guo, Tao; Jäger, Andreas; Zhang, Liqiang; Jin, Yan

2014-03-01

Cellular plasticity and complex functional requirements of the periodontal ligament (PDL) assume a local stem cell (SC) niche to maintain tissue homeostasis and repair. Here, pathological alterations caused by inflammatory insults might impact the regenerative capacities of these cells. As bone homeostasis is fundamentally controlled by Wnt-mediated signals, it was the aim of this study to characterize the SC-like capacities of cells derived from PDL and to investigate their involvement in bone pathophysiology especially regarding the canonical Wnt pathway. PDLSCs were investigated for their SC characteristics via analysis of cell surface marker expression, colony forming unit efficiency, proliferation, osteogenic differentiation and adipogenic differentiation, and compared to bone marrow derived mesenchymal SCs (BMMSCs). To determine the impact of both inflammation and the canonical Wnt pathway on osteogenic differentiation, cells were challenged with TNF-α, maintained with or without Wnt3a or DKK-1 under osteogenic induction conditions and investigated for p-IκBα, p-NF-κB, p-Akt, β-catenin, p-GSK-3β, ALP and Runx2. PDLSCs exhibit weaker adipogenic and osteogenic differentiation capacities compared to BMMSCs. TNF-α inhibited osteogenic differentiation of PDLSCs more than BMMSCs mainly through regulating canonical Wnt pathway. Blocking the canonical Wnt pathway by DKK-1 reconstituted osteogenic differentiation of PDLSCs under inflammatory conditions, whereas activation by Wnt3a increased osteogenic differentiation of BMMSCs. Our results suggest a diverse regulation of the inhibitory effect of TNF-α in BMMSCs and PDLSCs via canonical Wnt pathway modulation. These findings provide novel insights on PDLSC SC-like capacities and their involvement in bone pathophysiology under the impact of the canonical Wnt pathway. Copyright © 2013 Elsevier B.V. All rights reserved.

Mesenchymal stem cells with osteogenic potential in human maxillary sinus membrane: an in vitro study.

PubMed

Berbéri, Antoine; Al-Nemer, Fatima; Hamade, Eva; Noujeim, Ziad; Badran, Bassam; Zibara, Kazem

2017-06-01

The aim of our study is to prove and validate the existence of an osteogenic progenitor cell population within the human maxillary Schneiderian sinus membrane (hMSSM) and to demonstrate their potential for bone formation. Ten hMSSM samples of approximately 2 × 2 cm were obtained during a surgical nasal approach for treatment of chronic rhinosinusitis and were retained for this study. The derived cells were isolated, cultured, and assayed at passage 3 for their osteogenic potential using the expression of Alkaline phosphatase, alizarin red and Von Kossa staining, flow cytometry, and quantitative real-time polymerase chain reaction. hMSSM-derived cells were isolated, showed homogenous spindle-shaped fibroblast-like morphology, characteristic of mesenchymal progenitor cells (MPCs), and demonstrated very high expression of MPC markers such as STRO-1, CD44, CD90, CD105, and CD73 in all tested passages. In addition, von Kossa and Alizarin red staining showed significant mineralization, a typical feature of osteoblasts. Moreover, alkaline phosphatase (ALP) activity was significantly increased at days 7, 14, 21, and 28 of culture in hMSSM-derived cells grown in osteogenic medium, in comparison to controls. Furthermore, osteogenic differentiation significantly upregulated the transcriptional expression of osteogenic markers such as ALP, Runt-related transcription factor 2 (Runx-2), bone morphogenetic protein (BMP)-2, osteocalcin (OCN), osteonectin (ON), and osteopontin (OPN), confirming that hMSSM-derived cells are of osteoprogenitor origin. Finally, hMSSM-derived cells were also capable of producing OPN proteins upon culturing in an osteogenic medium. Our data showed that hMSSM holds mesenchymal osteoprogenitor cells capable of differentiating to the osteogenic lineage. hMSSM contains potentially multipotent postnatal stem cells providing a promising clinical application in preimplant and implant therapy.

Tunable osteogenic differentiation of hMPCs in tubular perfusion system bioreactor.

PubMed

Nguyen, Bao-Ngoc B; Ko, Henry; Fisher, John P

2016-08-01

The use of bioreactors for bone tissue engineering has been widely investigated. While the benefits of shear stress on osteogenic differentiation are well known, the underlying effects of dynamic culture on subpopulations within a bioreactor are less evident. In this work, we explore the influence of applied flow in the tubular perfusion system (TPS) bioreactor on the osteogenic differentiation of human mesenchymal progenitor cells (hMPCs), specifically analyzing the effects of axial position along the growth chamber. TPS bioreactor experiments conducted with unidirectional flow demonstrated enhanced expression of osteogenic markers in cells cultured downstream from the inlet flow. We utilized computational fluid dynamic modeling to confirm uniform shear stress distribution on the surface of the scaffolds and along the length of the growth chamber. The concept of paracrine signaling between cell populations was validated with the use of alternating flow, which diminished the differences in osteogenic differentiation between cells cultured at the inlet and outlet of the growth chamber. After the addition of controlled release of bone morphogenic protein-2 (BMP-2) into the system, osteogenic differentiation among subpopulations along the growth chamber was augmented, yet remained homogenous. These results allow for greater understanding of axial bioreactor cultures, their microenvironment, and how well-established parameters of osteogenic differentiation affect bone tissue development. With this work, we have demonstrated the capability of tuning osteogenic differentiation of hMPCs through the application of fluid flow and the addition of exogenous growth factors. Such precise control allows for the culture of distinct subpopulation within one dynamic system for the use of complex engineered tissue constructs. Biotechnol. Bioeng. 2016;113: 1805-1813. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

LncRNA PRNCR1 regulates osteogenic differentiation in osteolysis after hip replacement by targeting miR-211-5p.

PubMed

Gong, Zong-Ming; Tang, Zhen-Yu; Sun, Xiao-Liang

2018-05-11

Background Osteogenic differentiation and osteolysis after hip replacement are both associated with bone metabolism. Interaction between the long non-coding RNA (lncRNA) prostate cancer non-coding RNA 1 (PRNCR1) and miR-211-5p was analyzed to illuminate their roles in osteogenic differentiation and osteolysis. Methods The expression of PRNCR1, miR-211-5p and C-X-C chemokine receptor-4 (CXCR4) protein in tissues and mesenchymal stem cells (MSCs) were determined by qRT-PCR and western blot, separately. The osteogenic differentiation was assessed with Alkaline phosphatase (ALP) activity detection and ARS staining. The endogenous expressions of genes were modulated by recombinant plasmid and cell transfection. Combination condition and interaction between RNA and protein were determined with RIP and RNA pull-down assay, respectively. Interaction between miR-211-5p and CXCR4 was examined with Dual luciferase reporter assay. Results PRNCR1 and CXCR4 were up-regulated in wear particles around prosthesis and in MSCs incubated with Polymethylmethacrylate (PMMA), while miR-211-5p was down-regulated. Repression of PRNCR1 weakened the inhibitory effect of wear particles on osteogenic differentiation. PRNCR1 positively regulated CXCR4 through inhibiting miR-211-5p. Wear particles regulated CXCR4 level through miR-211-5p to affect osteogenic differentiation of MSCs. Wear particles regulated the miR-211-5p level through PRNCR1 to affect osteogenic differentiation of MSCs. Conclusion LncRNA PRNCR1 up-regulates CXCR4 through inhibiting miR-211-5p, which inhibits osteogenic differentiation and thereby leading to osteolysis after hip replacement. ©2018 The Author(s).

Mining for osteogenic surface topographies: In silico design to in vivo osseo-integration.

PubMed

Hulshof, Frits F B; Papenburg, Bernke; Vasilevich, Aliaksei; Hulsman, Marc; Zhao, Yiping; Levers, Marloes; Fekete, Natalie; de Boer, Meint; Yuan, Huipin; Singh, Shantanu; Beijer, Nick; Bray, Mark-Anthony; Logan, David J; Reinders, Marcel; Carpenter, Anne E; van Blitterswijk, Clemens; Stamatialis, Dimitrios; de Boer, Jan

2017-08-01

Stem cells respond to the physicochemical parameters of the substrate on which they grow. Quantitative material activity relationships - the relationships between substrate parameters and the phenotypes they induce - have so far poorly predicted the success of bioactive implant surfaces. In this report, we screened a library of randomly selected designed surface topographies for those inducing osteogenic differentiation of bone marrow-derived mesenchymal stem cells. Cell shape features, surface design parameters, and osteogenic marker expression were strongly correlated in vitro. Furthermore, the surfaces with the highest osteogenic potential in vitro also demonstrated their osteogenic effect in vivo: these indeed strongly enhanced bone bonding in a rabbit femur model. Our work shows that by giving stem cells specific physicochemical parameters through designed surface topographies, differentiation of these cells can be dictated. Copyright © 2017 Elsevier Ltd. All rights reserved.

Using an extreme bony prominence anatomical model to examine the influence of bed sheet materials and bed making methods on the distribution of pressure on the support surface.

PubMed

Iuchi, Terumi; Nakajima, Yukari; Fukuda, Moriyoshi; Matsuo, Junko; Okamoto, Hiroyuki; Sanada, Hiromi; Sugama, Junko

2014-05-01

Bed sheets generate high surface tension across the support surface and increase pressure to the body through a process known as the hammock effect. Using an anatomical model and a loading device characterized by extreme bony prominences, the present study compared pressure distributions on support surfaces across different bed making methods and bed sheet materials to determine the factors that influence pressure distribution. The model was placed on a pressure mapping system (CONFORMat; NITTA Corp., Osaka, Japan), and interface pressure was measured. Bed sheet elasticity and friction between the support surface and the bed sheets were also measured. For maximum interface pressure, the relative values of the following methods were higher than those of the control method, which did not use any bed sheets: cotton sheets with hospital corners (1.28, p = 0.02), polyester with no corners (1.29, p = 0.01), cotton with no corners (1.31, p = 0.003), and fitted polyester sheets (1.35, p = 0.002). Stepwise multiple regression analysis indicated that maximum interface pressure was negatively correlated with bed sheet elasticity (R(2) = 0.74). A statistically significant negative correlation was observed between maximum interface pressure and immersion depth, which was measured using the loading device (r = -0.40 and p = 0.04). We found that several combinations of bed making methods and bed sheet materials induced maximum interface pressures greater than those observed for the control method. Bed sheet materials influenced maximum interface pressure, and bed sheet elasticity was particularly important in reducing maximum interface pressure. Copyright © 2014 Tissue Viability Society. Published by Elsevier Ltd. All rights reserved.

Elevated waterproof access floor system and method of making the same

NASA Technical Reports Server (NTRS)

Cohen, Marc M. (Inventor)

1987-01-01

An elevated waterproof access floor system having subfloor channels or compartments for power lines, gas lines or the like is adapted such that it can be opened and subsequently resealed without destroying the waterproofing and without destroying its aesthetic appearance. A multiplicity of tiles are supported on a support grid, and a flooring sheet is supported on the tiles. Attachment means are provided to prevent lateral but not vertical movement of the flooring sheet with respect to the tiles so that the flooring sheet can be lifted off the tiles, but when the flooring sheet is supported on the tiles, no lateral slipping will occur. The flooring sheet is made of a heat resealable material, so that it can be cut away in sections, and the tiles therebelow lifted off, to provide access to subfloor compartments.

In vivo bone formation by human marrow stromal cells in biodegradable scaffolds that release dexamethasone and ascorbate-2-phosphate.

PubMed

Kim, Hyongbum; Suh, Hwal; Jo, Sangmee Ahn; Kim, Hyun Woo; Lee, Jung Min; Kim, Eun Hae; Reinwald, Yvonne; Park, Sang-Hyug; Min, Byoung-Hyun; Jo, Inho

2005-07-15

An unsolved problem with stem cell-based engineering of bone tissue is how to provide a microenvironment that promotes the osteogenic differentiation of multipotent stem cells. Previously, we fabricated porous poly(D,L-lactide-co-glycolide) (PLGA) scaffolds that released biologically active dexamethasone (Dex) and ascorbate-2-phosphate (AsP), and that acted as osteogenic scaffolds. To determine whether these osteogenic scaffolds can be used for bone formation in vivo, we seeded multipotent human marrow stromal cells (hMSCs) onto the scaffolds and implanted them subcutaneously into athymic mice. Higher alkaline phosphatase expression was observed in hMSCs in the osteogenic scaffolds compared with that of hMSCs in control scaffolds. Furthermore, there was more calcium deposition and stronger von Kossa staining in the osteogenic scaffolds, which suggested that there was enhanced mineralized bone formation. We failed to detect cartilage in the osteogenic scaffolds (negative Safranin O staining), which implied that there was intramembranous ossification. This is the first study to demonstrate the successful formation of mineralized bone tissue in vivo by hMSCs in PLGA scaffolds that release Dex and AsP.

The osteogenic differentiation of SSEA-4 sub-population of human adipose derived stem cells using silicate nanoplatelets.

PubMed

Mihaila, Silvia M; Gaharwar, Akhilesh K; Reis, Rui L; Khademhosseini, Ali; Marques, Alexandra P; Gomes, Manuela E

2014-11-01

How to surpass in vitro stem cell differentiation, reducing cell manipulation, and lead the in situ regeneration process after transplantation, remains to be unraveled in bone tissue engineering (bTE). Recently, we showed that the combination of human bone marrow stromal cells with bioactive silicate nanoplatelets (sNPs) promotes the osteogenic differentiation without the use of standard osteogenic inductors. Even more, using SSEA-4(+) cell-subpopulations (SSEA-4(+)hASCs) residing within the adipose tissue, as a single-cellular source to obtain relevant cell types for bone regeneration, was also proposed. Herein, sNPs were used to promote the osteogenic differentiation of SSEA-4(+)hASCs. The interactions between SSEA-4(+)hASCs and sNPs, namely the internalization pathway and effect on cells osteogenic differentiation, were evaluated. SNPs below 100 μg/mL showed high cytocompatibility and fast internalization via clathrin-mediated pathway. SNPs triggered an overexpression of osteogenic-related markers (RUNX2, osteopontin, osteocalcin) accompanied by increased alkaline phosphatase activity and deposition of a predominantly collagen-type I matrix. Consequently, a robust matrix mineralization was achieved, covering >90% of the culturing surface area. Overall, we demonstrated the high osteogenic differentiation potential of SSEA-4(+)hASCs, further enhanced by the addition of sNPs in a dose dependent manner. This strategy endorses the combination of an adipose-derived cell-subpopulation with inorganic compounds to achieve bone matrix-analogs with clinical relevance. Copyright © 2014 Elsevier Ltd. All rights reserved.

Electroactive BaTiO3 nanoparticle-functionalized fibrous scaffolds enhance osteogenic differentiation of mesenchymal stem cells

PubMed Central

Li, Yiping; Dai, Xiaohan; Bai, Yunyang; Liu, Yun; Wang, Yuehong; Liu, Ousheng; Yan, Fei; Tang, Zhangui; Zhang, Xuehui; Deng, Xuliang

2017-01-01

It has been proven that the surface topographic cues of fiber arrangement can induce osteogenic differentiation of mesenchymal stem cells. However, this effect alone is weak and insufficient to meet the needs of regenerative medicine. In this work, electroactivity concept was introduced to enhance the osteoinductivity of fibrous scaffolds. The randomly oriented and aligned electroactive fibrous scaffolds of poly-(l-lactic acid) (PLLA) with incorporation of ferroelectric ceramic BaTiO3 (BTO) nanoparticles (NPs) were fabricated by electrospinning. Physicochemical properties, including fiber morphology, microstructure, composition, thermal stability, surface roughness, and surface wettability, of these fibrous scaffolds were studied. The dielectric properties of the scaffolds were evaluated. The results showed that the randomly oriented BTO/PLLA composite fibrous scaffolds had the highest dielectric permittivity of 1.19, which is of the same order of magnitude as the natural bone. The combined effects of fiber orientation and electrical activity on the osteogenic responses of bone marrow mesenchymal stem cells (BM-MSCs) were specifically investigated. Randomly oriented composite fibrous scaffolds significantly promoted polygonal spreading and encouraged early osteogenic differentiation in BM-MSCs, whereas aligned composite fibrous scaffolds promoted cell elongation and discouraged osteogenic differentiation. These results evidenced that randomly fiber orientation and biomimetic electric activity have combining effects on osteogenic differentiation of BM-MSCs. Our findings indicate that coupling effects of multi-physical properties should be paid more attention to mimic the microenvironment for enhancing osteogenic differentiation of BM-MSCs. PMID:28603415

Enhanced osteogenic differentiation of rat bone marrow mesenchymal stem cells on titanium substrates by inhibiting Notch3.

PubMed

Wang, Huiming; Jiang, Zhiwei; Zhang, Jing; Xie, Zhijian; Wang, Ying; Yang, Guoli

2017-08-01

The role of the Notch pathway has already been identified as a crucial regulator of bone development. However, the Notch signaling pathway has gone largely unexplored during osseointegration. This study aims to investigate the role of Notch signaling on osteogenic differentiation of rat derived bone marrow mesenchymal stem cells (BMSCs) on sandblasted, large-grit, acid-etched (SLA) treated Ti disks. The involved target genes in Notch pathways were identified by in vitro microarray and bioinformatics analyses with or without osteogenic induction. Adhesion, proliferation, and osteogenic related assay were subsequently conducted with target gene shRNA treatment. We found that 11 genes in the Notch signaling pathway were differentially expressed after osteogenic induction on SLA-treated Ti disks, which included up-regulated genes (Notch2, Dll1, Dll3, Ncstn, Ncor2, and Hes5) and down-regulated genes (Notch3, Lfng, Mfng, Jag2 and Maml2). With Notch3 shRNA treatment, the adhesion and proliferation of BMSCs on SLA-treated Ti disks were inhibited. Moreover, the expression levels of alkaline phosphatase (ALP), osteocalcin (OCN), calcium deposition, BMP2 and Runx2 increased significantly compared with that observed in control groups, suggesting that the function of Notch3 was inhibitory in the osteogenic differentiation of BMSCs on SLA-treated titanium. Inhibition Notch3 can enhance osteogenic differentiation of BMSCs on SLA-treated Ti disks, which potentially provides a gene target for improving osseointegration. Copyright © 2017 Elsevier Ltd. All rights reserved.

Surface potential-governed cellular osteogenic differentiation on ferroelectric polyvinylidene fluoride trifluoroethylene films.

PubMed

Tang, Bolin; Zhang, Bo; Zhuang, Junjun; Wang, Qi; Dong, Lingqing; Cheng, Kui; Weng, Wenjian

2018-07-01

Surface potential of biomaterials can dramatically influence cellular osteogenic differentiation. In this work, a wide range of surface potential on ferroelectric polyvinylidene fluoride trifluoroethylene (P(VDF-TrFE)) films was designed to get insight into the interfacial interaction of cell-charged surface. The P(VDF-TrFE) films poled by contact electric poling at various electric fields obtained well stabilized surface potential, with wide range from -3 to 915 mV. The osteogenic differentiation level of cells cultured on the films was strongly dependent on surface potential and reached the optimum at 391 mV in this system. Binding specificity assay indicated that surface potential could effectively govern the binding state of the adsorbed fibronectin (FN) with integrin. Molecular dynamic (MD) simulation further revealed that surface potential brought a significant difference in the relative distance between RGD and synergy PHSRN sites of adsorbed FN, resulting in a distinct integrin-FN binding state. These results suggest that the full binding of integrin α5β1 with both RGD and PHSRN sites of FN possesses a strong ability to activate osteogenic signaling pathway. This work sheds light on the underlying mechanism of osteogenic differentiation behavior on charged material surfaces, and also provides a guidance for designing a reasonable charged surface to enhance osteogenic differentiation. The ferroelectric P(VDF-TrFE) films with steady and a wide range of surface potential were designed to understand underlying mechanism of cell-charged surface interaction. The results showed that the charged surface well favored upregulation of osteogenic differentiation of MC3T3-E1 cells, and more importantly, a highest level occurred on the film with a moderate surface potential. Experiments and molecular dynamics simulation demonstrated that the surface potential could govern fibronectin conformation and then the integrin-fibronectin binding. We propose that a full binding state of integrin α5β1 with fibronectin induces effective activation of integrin-mediated FAK/ERK signaling pathway to upregulate cellular osteogenic differentiation. This work provides a guidance for designing a reasonable charged surface to enhance osteogenic differentiation. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Topographical cues of direct metal laser sintering titanium surfaces facilitate osteogenic differentiation of bone marrow mesenchymal stem cells through epigenetic regulation.

PubMed

Zheng, Guoying; Guan, Binbin; Hu, Penghui; Qi, Xingying; Wang, Pingting; Kong, Yu; Liu, Zihao; Gao, Ping; Li, Rui; Zhang, Xu; Wu, Xudong; Sui, Lei

2018-04-27

To investigate the role of hierarchical micro/nanoscale topography of direct metal laser sintering (DMLS) titanium surfaces in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), as well as the possible underlying epigenetic mechanism. Three groups of titanium specimens were prepared, including DMLS group, sandblasted, large-grit, acid-etched (SLA) group and smooth titanium (Ti) group. BMSCs were cultured on discs followed by surface characterization. Cell adhesion and proliferation were examined by SEM and CCK-8 assay, while osteogenic-related gene expression was detected by real-time RT-PCR. Immunofluorescence, western blotting and in vivo study were also performed to evaluate the potential for osteogenic induction of materials. In addition, to investigate the underlying epigenetic mechanisms, immunofluorescence and western blotting were performed to evaluate the global level of H3K4me3 during osteogenesis. The H3K4me3 and H3K27me3 levels at the promoter area of the osteogenic gene Runx2 were detected by ChIP assay. The DMLS surface exhibits greater protein adsorption ability and shows better cell adhesion performance than SLA and Ti surfaces. Moreover, both in vitro and in vivo studies demonstrated that the DMLS surface is more favourable for the osteogenic differentiation of BMSCs than SLA and Ti surfaces. Accordingly, osteogenesis-associated gene expression in BMSCs is efficiently induced by a rapid H3K27 demethylation and increase in H3K4me3 levels at gene promoters upon osteogenic differentiation on DMLS titanium surface. Topographical cues of DMLS surfaces have greater potential for the induction of osteogenic differentiation of BMSCs than SLA and Ti surfaces both in vitro and in vivo. A potential epigenetic mechanism is that the appropriate topography allows rapid H3K27 demethylation and an increased H3K4me3 level at the promoter region of osteogenesis-associated genes during the osteogenic differentiation of BMSCs. © 2018 John Wiley & Sons Ltd.

Endosteal-like extracellular matrix expression on melt electrospun written scaffolds.

PubMed

Muerza-Cascante, Maria Lourdes; Shokoohmand, Ali; Khosrotehrani, Kiarash; Haylock, David; Dalton, Paul D; Hutmacher, Dietmar W; Loessner, Daniela

2017-04-01

Tissue engineering technology platforms constitute a unique opportunity to integrate cells and extracellular matrix (ECM) proteins into scaffolds and matrices that mimic the natural microenvironment in vitro. The development of tissue-engineered 3D models that mimic the endosteal microenvironment enables researchers to discover the causes and improve treatments for blood and immune-related diseases. The aim of this study was to establish a physiologically relevant in vitro model using 3D printed scaffolds to assess the contribution of human cells to the formation of a construct that mimics human endosteum. Melt electrospun written scaffolds were used to compare the suitability of primary human osteoblasts (hOBs) and placenta-derived mesenchymal stem cells (plMSCs) in (non-)osteogenic conditions and with different surface treatments. Using osteogenic conditions, hOBs secreted a dense ECM with enhanced deposition of endosteal proteins, such as fibronectin and vitronectin, and osteogenic markers, such as osteopontin and alkaline phosphatase, compared to plMSCs. The expression patterns of these proteins were reproducibly identified in hOBs derived from three individual donors. Calcium phosphate-coated scaffolds induced the expression of osteocalcin by hOBs when maintained in osteogenic conditions. The tissue-engineered endosteal microenvironment supported the growth and migration of primary human haematopoietic stem cells (HSCs) when compared to HSCs maintained using tissue culture plastic. This 3D testing platform represents an endosteal bone-like tissue and warrants future investigation for the maintenance and expansion of human HSCs. This work is motivated by the recent interest in melt electrospinning writing, a 3D printing technique used to produce porous scaffolds for biomedical applications in regenerative medicine. Our team has been among the pioneers in building a new class of melt electrospinning devices for scaffold-based tissue engineering. These scaffolds allow structural support for various cell types to invade and deposit their own ECM, mimicking a characteristic 3D microenvironment for experimental studies. We used melt electrospun written polycaprolactone scaffolds to develop an endosteal bone-like tissue that promotes the growth of HSCs. We combine tissue engineering concepts with cell biology and stem cell research to design a physiologically relevant niche that is of prime interest to the scientific community. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

BMP2-loaded hollow hydroxyapatite microspheres exhibit enhanced osteoinduction and osteogenicity in large bone defects.

PubMed

Xiong, Long; Zeng, Jianhua; Yao, Aihua; Tu, Qiquan; Li, Jingtang; Yan, Liang; Tang, Zhiming

2015-01-01

The regeneration of large bone defects is an osteoinductive, osteoconductive, and osteogenic process that often requires a bone graft for support. Limitations associated with naturally autogenic or allogenic bone grafts have demonstrated the need for synthetic substitutes. The present study investigates the feasibility of using novel hollow hydroxyapatite microspheres as an osteoconductive matrix and a carrier for controlled local delivery of bone morphogenetic protein 2 (BMP2), a potent osteogenic inducer of bone regeneration. Hollow hydroxyapatite microspheres (100±25 μm) with a core (60±18 μm) and a mesoporous shell (180±42 m(2)/g surface area) were prepared by a glass conversion technique and loaded with recombinant human BMP2 (1 μg/mg). There was a gentle burst release of BMP2 from microspheres into the surrounding phosphate-buffered saline in vitro within the initial 48 hours, and continued at a low rate for over 40 days. In comparison with hollow hydroxyapatite microspheres without BMP2 or soluble BMP2 without a carrier, BMP2-loaded hollow hydroxyapatite microspheres had a significantly enhanced capacity to reconstitute radial bone defects in rabbit, as shown by increased serum alkaline phosphatase; quick and complete new bone formation within 12 weeks; and great biomechanical flexural strength. These results indicate that BMP2-loaded hollow hydroxyapatite microspheres could be a potential new option for bone graft substitutes in bone regeneration.

Long Noncoding RNAs: New Players in the Osteogenic Differentiation of Bone Marrow- and Adipose-Derived Mesenchymal Stem Cells.

PubMed

Yang, Qiaolin; Jia, Lingfei; Li, Xiaobei; Guo, Runzhi; Huang, Yiping; Zheng, Yunfei; Li, Weiran

2018-06-01

Mesenchymal stem cells (MSCs) are an important population of multipotent stem cells that differentiate into multiple lineages and display great potential in bone regeneration and repair. Although the role of protein-coding genes in the osteogenic differentiation of MSCs has been extensively studied, the functions of noncoding RNAs in the osteogenic differentiation of MSCs are unclear. The recent application of next-generation sequencing to MSC transcriptomes has revealed that long noncoding RNAs (lncRNAs) are associated with the osteogenic differentiation of MSCs. LncRNAs are a class of non-coding transcripts of more than 200 nucleotides in length. Noncoding RNAs are thought to play a key role in osteoblast differentiation through various regulatory mechanisms including chromatin modification, transcription factor binding, competent endogenous mechanism, and other post-transcriptional mechanisms. Here, we review the roles of lncRNAs in the osteogenic differentiation of bone marrow- and adipose-derived stem cells and provide a theoretical foundation for future research.

Red algal extracts from Plocamium lyngbyanum and Ceramium secundatum stimulate osteogenic activities in vitro and bone growth in zebrafish larvae.

PubMed

Carson, Matthew A; Nelson, John; Cancela, M Leonor; Laizé, Vincent; Gavaia, Paulo J; Rae, Margaret; Heesch, Svenja; Verzin, Eugene; Maggs, Christine; Gilmore, Brendan F; Clarke, Susan A

2018-05-16

Through the current trend for bioprospecting, marine organisms - particularly algae - are becoming increasingly known for their osteogenic potential. Such organisms may provide novel treatment options for osteoporosis and other musculoskeletal conditions, helping to address their large healthcare burden and the limitations of current therapies. In this study, extracts from two red algae - Plocamium lyngbyanum and Ceramium secundatum - were tested in vitro and in vivo for their osteogenic potential. In vitro, the growth of human bone marrow stromal cells (hBMSCs) was significantly greater in the presence of the extracts, particularly with P. lyngbyanum treatment. Osteogenic differentiation was promoted more by C. secundatum (70 µg/ml), though P. lyngbyanum had greater in vitro mineralisation potential. Both species caused a marked and dose-dependent increase in the opercular bone area of zebrafish larvae. Our findings therefore indicate the presence of bioactive components in P. lyngbyanum and C. secundatum extracts, which can promote both in vitro and in vivo osteogenic activity.

Estrogen deficiency inhibits the odonto/osteogenic differentiation of dental pulp stem cells via activation of the NF-κB pathway.

PubMed

Wang, Yanping; Yan, Ming; Yu, Yan; Wu, Jintao; Yu, Jinhua; Fan, Zhipeng

2013-06-01

Various factors can affect the functions of dental pulp stem cells (DPSCs). However, little knowledge is available about the effects of estrogen deficiency on the differentiation of DPSCs. In this study, an estrogen-deficient rat model was constructed and multi-colony-derived DPSCs were obtained from the incisors of ovariectomized (OVX) or sham-operated rats. Odonto/osteogenic differentiation and the possible involvement of the nuclear factor kappa B (NF-κB) pathway in the OVX-DPSCs/Sham-DPSCs of these rats were then investigated. OVX-DPSCs presented decreased odonto/osteogenic capacity and an activated NF-κB pathway, as compared with Sham-DPSCs. When the cellular NF-κB pathway was specifically inhibited by BMS345541, the odonto/osteogenic potential in OVX-DPSCs was significantly upregulated. Thus, estrogen deficiency down-regulated the odonto/osteogenic differentiation of DPSCs by activating NF-κB signaling and inhibition of the NF-κB pathway effectively rescued the decreased differentiation potential of DPSCs.

Osteogenic capability of autologous rabbit adipose-derived stromal cells in repairing calvarial defects.

PubMed

Cheng, Shao-Wen; Lin, Zhong-Qin; Wang, Wei; Zhang, Wei; Kou, Dong-Quan; Ying, Xiao-Zhou; Chen, Qing-Yu; Shen, Yue; Cheng, Xiao-Jie; Peng, Lei; Lv, Chuan-Zhu

2011-01-01

To evaluate the in vitro and in vivo osteogenic capability of adipose-derived stromal cells (ASCs). ASCs were isolated from New Zealand white rabbits and determined by alkaline phosphatase (ALP) staining, von Kossa staining and alizarin red staining. Some specific markers of osteogenic differentiation, including ALP, osteocalcin (OCN), osteopontin (OPN) were examined by reverse transcription-polymerase chain reaction (RT-PCR). In vivo, demineralized bone matrix (DBM)-ASCs composites were implanted into the rabbit calvarial defects created at each side of the longitudinal midline. After 6 weeks, histologic properties of the transplants were analyzed. ASCs were successfully induced into osteogenesis. ALP staining, von Kossa staining and alizarin red staining showed positive results. The expressions of ALP, OCN and OPN were detected in ASCs after cultivation in osteogenic medium. Extensive new bone was observed in the defects transplanted with DBM-ASCs composites. ASCs have the potential to differentiate into osteogenic lineage and DBM-ASCs constructs are a promising method for regeneration in bone defects.

Semipermeable Capsules Wrapping a Multifunctional and Self-regulated Co-culture Microenvironment for Osteogenic Differentiation

NASA Astrophysics Data System (ADS)

Correia, Clara R.; Pirraco, Rogério P.; Cerqueira, Mariana T.; Marques, Alexandra P.; Reis, Rui L.; Mano, João F.

2016-02-01

A new concept of semipermeable reservoirs containing co-cultures of cells and supporting microparticles is presented, inspired by the multi-phenotypic cellular environment of bone. Based on the deconstruction of the “stem cell niche”, the developed capsules are designed to drive a self-regulated osteogenesis. PLLA microparticles functionalized with collagen I, and a co-culture of adipose stem (ASCs) and endothelial (ECs) cells are immobilized in spherical liquified capsules. The capsules are coated with multilayers of poly(L-lysine), alginate, and chitosan nano-assembled through layer-by-layer. Capsules encapsulating ASCs alone or in a co-culture with ECs are cultured in endothelial medium with or without osteogenic differentiation factors. Results show that osteogenesis is enhanced by the co-encapsulation, which occurs even in the absence of differentiation factors. These findings are supported by an increased ALP activity and matrix mineralization, osteopontin detection, and the up regulation of BMP-2, RUNX2 and BSP. The liquified co-capsules also act as a VEGF and BMP-2 cytokines release system. The proposed liquified capsules might be a valuable injectable self-regulated system for bone regeneration employing highly translational cell sources.

The efficacy of polycaprolactone/hydroxyapatite scaffold in combination with mesenchymal stem cells for bone tissue engineering.

PubMed

Chuenjitkuntaworn, Boontharika; Osathanon, Thanaphum; Nowwarote, Nunthawan; Supaphol, Pitt; Pavasant, Prasit

2016-01-01

Major drawbacks of using an autograft are the possibilities of insufficient bony source and patient's morbidity after operation. Bone tissue engineering technology, therefore, has been applied for repairing bony defects. Previous study showed that a novel fabricated 3D-Polycaprolactone/Hydroxyapatite (PCL/HAp) scaffold possessed a good biocompatibility for bone cells. This study aimed to determine the ability of PCL/HAp for supporting cell growth, gene expression, and osteogenic differentiation in three types of mesenchymal stem cells, including bone marrow-derived mesenchymal stem cells (BMSCs), dental pulp stem cells (DPSCs), and adiposed-derived mesenchymal stem cells (ADSCs). These were assessed by cell viability assay (MTT), reverse-transcription polymerase chain reaction (RT-PCR) analysis, alkaline phosphatase activity, and osteogenic differentiation by alizarin red-S staining. The results showed that PCL/HAp scaffold could support growth of all three types of mesenchymal stem cells. In addition, DPSCs with PCL/HAp showed the highest level of calcium deposition compared to other groups. In conclusion, DPSCs exhibited a better compatibility with these scaffolds compared to BMSCs and ADSCs. However, the PCL/HAp could be a good candidate scaffold for all tested mesenchymal stem cells in bone tissue engineering. © 2015 Wiley Periodicals, Inc.

Circulating osteogentic precursor cells in non-hereditary heterotopic ossification.

PubMed

Egan, Kevin P; Duque, Gustavo; Keenan, Mary Ann; Pignolo, Robert J

2018-04-01

Non-hereditary heterotopic ossification (NHHO) may occur after musculoskeletal trauma, central nervous system (CNS) injury, or surgery. We previously described circulating osteogenic precursor (COP) cells as a bone marrow-derived type 1 collagen + CD45 + subpopulation of mononuclear adherent cells that are able of producing extraskeletal ossification in a murine in vivo implantation assay. In the current study, we performed a tissue analysis of COP cells in NHHO secondary to defined conditions, including traumatic brain injury, spinal cord injury, cerebrovascular accident, trauma without neurologic injury, and joint arthroplasty. All bone specimens revealed the presence of COP cells at 2-14 cells per high power field. COP cells were localized to early fibroproliferative and neovascular lesions of NHHO with evidence for their circulatory status supported by their presence near blood vessels in examined lesions. This study provides the first systematic evaluation of COP cells as a contributory histopathological finding associated with multiple forms of NHHO. These data support that circulating, hematopoietic-derived cells with osteogenic potential can seed inflammatory sites, such as those subject to soft tissue injury, and due to their migratory nature, may likely be involved in seeding sites distant to CNS injury. Copyright © 2018 Elsevier Inc. All rights reserved.

Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells.

PubMed

Hofemeier, Arne D; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F W; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

2016-05-26

Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO4(3-) symmetric stretch vibrations at 959 cm(-1) assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue-implant-interfaces or disease diagnosis.

Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells

NASA Astrophysics Data System (ADS)

Hofemeier, Arne D.; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F. W.; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

2016-05-01

Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO43- symmetric stretch vibrations at 959 cm-1 assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue-implant-interfaces or disease diagnosis.

Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells

PubMed Central

Hofemeier, Arne D.; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F. W.; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

2016-01-01

Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO43− symmetric stretch vibrations at 959 cm−1 assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue–implant-interfaces or disease diagnosis. PMID:27225821

Nicotine Deteriorates the Osteogenic Differentiation of Periodontal Ligament Stem Cells through α7 Nicotinic Acetylcholine Receptor Regulating wnt Pathway

PubMed Central

Dong, Zhiwei; Liu, Fen; Zhang, Yu; Yu, Yang; Shang, Fengqing; Wu, Lizheng; Wang, Xiaojing; Jin, Yan

2013-01-01

Aims Cigarette smoking is one of the high risk factors of adult chronic periodontitis and nicotine is the well established toxic substance in cigarette. However, the mechanism of nicotine induced periodontitis is still unknown. Here we studied whether nicotine impaired the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) through activating α7 nicotinic acetylcholine receptor (α7 nAChR). Methods hPDLSCs with multi differentiation potential and surface makers for mesenchymal stem cells were harvested by limiting dilution technique. The level of mineralized nodule formation was assessed by alizarin red S staining. Expression level of ostegenic related genes and proteins were detected by real-time PCR and western blot analysis. The expression of α7 nAChR and its downstream signaling pathway were examined by western blot. The role of the receptor and related signaling pathway in nicotine impairing the osteogenic potential of hPDLSCs were also studied in different levels. Results Nicotine deteriorated the ostegenic differentiation of hPDLSCs in a dose dependent manner. Activation of α7 nAChR by nicotine treatment activated wnt/β-catenin signaling pathway, leading to osteogenic deficiency of hPDLSCs. Blockage of α7 nAChR and wnt pathway inhibitor treatment rescued nicotine induced osteogenic differentiation deficiency. Conclusions These data suggested that nicotine activated α7 nAChR expressed on PDLSCs and further activated wnt signaling downstream, thus deteriorating the osteogenic potential of PDLSCs. The impairment of osteogenic differentiation of PDLSCs by nicotine might lead to cigarette smoking related periodontitis. PMID:24376645

Naringin promotes osteogenic differentiation of bone marrow stromal cells by up-regulating Foxc2 expression via the IHH signaling pathway

PubMed Central

Lin, Fei-xiang; Du, Shi-xin; Liu, De-zhong; Hu, Qin-xiao; Yu, Guo-yong; Wu, Chu-cheng; Zheng, Gui-zhou; Xie, Da; Li, Xue-dong; Chang, Bo

2016-01-01

Naringin is an active compound extracted from Rhizoma Drynariae, and studies have revealed that naringin can promote proliferation and osteogenic differentiation of bone marrow stromal cells (BMSCs). In this study, we explored whether naringin could promote osteogenic differentiation of BMSCs by upregulating Foxc2 expression via the Indian hedgehog (IHH) signaling pathway. BMSCs were cultured in basal medium, basal medium with naringin, osteogenic induction medium, osteogenic induction medium with naringin and osteogenic induction medium with naringin in the presence of the IHH inhibitor cyclopamine (CPE). We examined cell proliferation by using a WST-8 assay, and differentiation by Alizarin Red S staining (for mineralization) and alkaline phosphatase (ALP) activity. In addition, we detected core-binding factor α1 (Cbfα1), osteocalcin (OCN), bone sialoprotein (BSP), peroxisome proliferation-activated receptor gamma 2 (PPARγ2) and Foxc2 expression by using RT-PCR. We also determined Foxc2 and IHH protein levels by western blotting. Naringin increased the mineralization of BMSCs, as shown by Alizarin red S assays, and induced ALP activity. In addition, naringin significantly increased the mRNA levels of Foxc2, Cbfα1, OCN, and BSP, while decreasing PPARγ2 mRNA levels. Furthermore, the IHH inhibitor CPE inhibited the osteogenesis-potentiating effects of naringin. Naringin increased Foxc2 and stimulated the activation of IHH, as evidenced by increased expression of proteins that were inhibited by CPE. Our findings indicate that naringin promotes osteogenic differentiation of BMSCs by up-regulating Foxc2 expression via the IHH signaling pathway. PMID:27904711

Naringin promotes osteogenic differentiation of bone marrow stromal cells by up-regulating Foxc2 expression via the IHH signaling pathway.

PubMed

Lin, Fei-Xiang; Du, Shi-Xin; Liu, De-Zhong; Hu, Qin-Xiao; Yu, Guo-Yong; Wu, Chu-Cheng; Zheng, Gui-Zhou; Xie, Da; Li, Xue-Dong; Chang, Bo

2016-01-01

Naringin is an active compound extracted from Rhizoma Drynariae, and studies have revealed that naringin can promote proliferation and osteogenic differentiation of bone marrow stromal cells (BMSCs). In this study, we explored whether naringin could promote osteogenic differentiation of BMSCs by upregulating Foxc2 expression via the Indian hedgehog (IHH) signaling pathway. BMSCs were cultured in basal medium, basal medium with naringin, osteogenic induction medium, osteogenic induction medium with naringin and osteogenic induction medium with naringin in the presence of the IHH inhibitor cyclopamine (CPE). We examined cell proliferation by using a WST-8 assay, and differentiation by Alizarin Red S staining (for mineralization) and alkaline phosphatase (ALP) activity. In addition, we detected core-binding factor α1 (Cbfα1), osteocalcin (OCN), bone sialoprotein (BSP), peroxisome proliferation-activated receptor gamma 2 (PPARγ2) and Foxc2 expression by using RT-PCR. We also determined Foxc2 and IHH protein levels by western blotting. Naringin increased the mineralization of BMSCs, as shown by Alizarin red S assays, and induced ALP activity. In addition, naringin significantly increased the mRNA levels of Foxc2, Cbfα1, OCN, and BSP, while decreasing PPARγ2 mRNA levels. Furthermore, the IHH inhibitor CPE inhibited the osteogenesis-potentiating effects of naringin. Naringin increased Foxc2 and stimulated the activation of IHH, as evidenced by increased expression of proteins that were inhibited by CPE. Our findings indicate that naringin promotes osteogenic differentiation of BMSCs by up-regulating Foxc2 expression via the IHH signaling pathway.

Long noncoding RNA MALAT1 promotes osterix expression to regulate osteogenic differentiation by targeting miRNA-143 in human bone marrow-derived mesenchymal stem cells.

PubMed

Gao, Yuan; Xiao, Fei; Wang, Chenglong; Wang, Chuandong; Cui, Penglei; Zhang, Xiaoling; Chen, Xiaodong

2018-05-09

Osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) is essential for the human bone formation, and emerging evidence shows that long non-coding RNAs (lncRNAs) play important roles in hBMSC osteogenic differentiation. MALAT1 is often regarded as a tumor-related lncRNA, but its function in mesenchymal stem cell differentiation remains to be defined. In this study, we aimed to investigate whether MALAT1 regulates Osterix (Osx) expression by sponging miR-143 to promote hBMSC osteogenic differentiation. Firstly, we found that the expression of MALAT1 was much lower in hBMSCs from osteoporosis patients and miR-143 was contrarily higher. In addition, MALAT1 expression increased, and miR-143 decreased when hBMSCs were treated with osteogenic induction. Then, we used short hairpin RNAs to knockdown MALAT1, and the results showed that hBMSC osteogenic differentiation decreased significantly, indicating that MALAT1 is a positive regulator of osteogenic differentiation in hBMSCs. Furthermore, by luciferase assays, we found that MALAT1 could directly bind to miR-143 and negatively regulate its expression. Similarly, miR-143 could directly bind to the target site on the Osx 3'-UTR and then inhibit Osx expression. Knockdown of MALAT1 decreased Osx expression, and co-transfection of miR-143 inhibitor could rescue Osx mRNA expression. While Osx expression was increased in MALAT1-overexpressing hBMSCs, it was reversed by the miR-143 mimics. Moreover, Osx silencing decreased ALP, OCN, and OPN mRNA expression induced by the miR-143 inhibitor. Altogether, our findings suggest that MALAT1 acts to regulate Osx expression through targeting miR-143; thus, it is considered as a positive regulator in hBMSC osteogenic differentiation. © 2018 Wiley Periodicals, Inc.

Substantial differences between human and ovine mesenchymal stem cells in response to osteogenic media: how to explain and how to manage?

PubMed

Kalaszczynska, Ilona; Ruminski, Slawomir; Platek, Anna E; Bissenik, Igor; Zakrzewski, Piotr; Noszczyk, Maria; Lewandowska-Szumiel, Malgorzata

2013-10-01

It is expected that use of adult multipotential mesenchymal stem cells (MSCs) for bone tissue engineering (TE) will lead to improvement of TE products. Prior to clinical application, biocompatibility of bone TE products need to be tested in vitro and in vivo. In orthopedic research, sheep are a well-accepted model due to similarities with humans and are assumed to be predictive of human outcomes. In this study we uncover differences between human and ovine bone marrow-derived MSCs (BMSCs) and adipose tissue-derived MSCs (ADSCs) in response to osteogenic media. Osteogenic differentiation of BMSCs and ADSCs was monitored by alkaline phosphatase (ALP) activity and calcium deposition. Mineralization of ovine BMSC was achieved in medium containing NaH2PO4 as a source of phosphate ions (Pi), but not in medium containing β-glycerophosphate (β-GP), which is most often used. In a detailed study we found no induction of ALP activity in ovine BMSCs and ADSCs upon osteogenic stimulation, which makes β-GP an unsuitable source of phosphate ions for ovine cells. Moreover, mineralization of human ADSCs was more efficient in osteogenic medium containing NaH2PO4. These results indicate major differences between ovine and human MSCs and suggest that standard in vitro osteogenic differentiation techniques may not be suitable for all types of cells used in cell-based therapies. Since mineralization is a widely accepted marker of the osteogenic differentiation and maturation of cells in culture, it may lead to potentially misleading results and should be taken into account at the stage of planning and interpreting preclinical observations performed in animal models. We also present a cell culture protocol for ovine ADSCs, which do not express ALP activity and do not mineralize under routine pro-osteogenic conditions in vitro. We plan to apply it in preclinical experiments of bone tissue-engineered products performed in an ovine model.

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

The differential effect of basic fibroblast growth factor and stromal cell‑derived factor‑1 pretreatment on bone morrow mesenchymal stem cells osteogenic differentiation potency.

PubMed

Wang, Ruolin; Liu, Wenhua; Du, Mi; Yang, Chengzhe; Li, Xuefen; Yang, Pishan

2018-03-01

In situ tissue engineering has become a novel strategy to repair periodontal/bone tissue defects. The choice of cytokines that promote the recruitment and proliferation, and potentiate and maintain the osteogenic differentiation ability of mesenchymal stem cells (MSCs) is the key point in this technique. Stromal cell‑derived factor‑1 (SDF‑1) and basic fibroblast growth factor (bFGF) have the ability to promote the recruitment, and proliferation of MSCs; however, the differential effect of SDF‑1 and bFGF pretreatment on MSC osteogenic differentiation potency remains to be explored. The present study comparatively observed osteogenic differentiation of bone morrow MSCs (BMMSCs) pretreated by bFGF or SDF‑1 in vitro. The gene and protein expression levels of alkaline phosphatase (ALP), runt related transcription factor 2 (Runx‑2) and bone sialoprotein (BSP) were detected using reverse transcription‑quantitative polymerase chain reaction and western blotting. The results showed that the expression of ALP mRNA on day 3, and BSP and Runx‑2 mRNA on day 7 in the bFGF pretreatment group was significantly higher than those in SDF‑1 pretreatment group. Expression levels of Runx‑2 mRNA, and ALP and Runx‑2 protein on day 3 in the SDF‑1 pretreatment group were higher than those in the bFGF pretreatment group. However, there was no significant difference in osteogenic differentiation ability on day 14 and 28 between the bFGF‑ or SDF‑1‑pretreatment groups and the control. In conclusion, bFGF and SDF‑1 pretreatment inhibits osteogenic differentiation of BMMSCs at the early stage, promotes it in the medium phase, and maintains it in the later stage during osteogenic induction, particularly at the mRNA level. Out of the two cytokines, bFGF appeared to have a greater effect on osteogenic differentiation.

Substantial Differences Between Human and Ovine Mesenchymal Stem Cells in Response to Osteogenic Media: How to Explain and How to Manage?

PubMed Central

Kalaszczynska, Ilona; Ruminski, Slawomir; Platek, Anna E.; Bissenik, Igor; Zakrzewski, Piotr; Noszczyk, Maria

2013-01-01

Abstract It is expected that use of adult multipotential mesenchymal stem cells (MSCs) for bone tissue engineering (TE) will lead to improvement of TE products. Prior to clinical application, biocompatibility of bone TE products need to be tested in vitro and in vivo. In orthopedic research, sheep are a well-accepted model due to similarities with humans and are assumed to be predictive of human outcomes. In this study we uncover differences between human and ovine bone marrow–derived MSCs (BMSCs) and adipose tissue–derived MSCs (ADSCs) in response to osteogenic media. Osteogenic differentiation of BMSCs and ADSCs was monitored by alkaline phosphatase (ALP) activity and calcium deposition. Mineralization of ovine BMSC was achieved in medium containing NaH2PO4 as a source of phosphate ions (Pi), but not in medium containing β-glycerophosphate (β-GP), which is most often used. In a detailed study we found no induction of ALP activity in ovine BMSCs and ADSCs upon osteogenic stimulation, which makes β-GP an unsuitable source of phosphate ions for ovine cells. Moreover, mineralization of human ADSCs was more efficient in osteogenic medium containing NaH2PO4. These results indicate major differences between ovine and human MSCs and suggest that standard in vitro osteogenic differentiation techniques may not be suitable for all types of cells used in cell-based therapies. Since mineralization is a widely accepted marker of the osteogenic differentiation and maturation of cells in culture, it may lead to potentially misleading results and should be taken into account at the stage of planning and interpreting preclinical observations performed in animal models. We also present a cell culture protocol for ovine ADSCs, which do not express ALP activity and do not mineralize under routine pro-osteogenic conditions in vitro. We plan to apply it in preclinical experiments of bone tissue–engineered products performed in an ovine model. PMID:24083091

Peri‐prosthetic tissue cells show osteogenic capacity to differentiate into the osteoblastic lineage

PubMed Central

Schoeman, Monique A.E.; Oostlander, Angela E.; Rooij, Karien Ede; Valstar, Edward R.

2017-01-01

ABSTRACT During the process of aseptic loosening of prostheses, particulate wear debris induces a continuous inflammatory‐like response resulting in the formation of a layer of fibrous peri‐prosthetic tissue at the bone‐prosthesis interface. The current treatment for loosening is revision surgery which is associated with a high‐morbidity rate, especially in old patients. Therefore, less invasive alternatives are necessary. One approach could be to re‐establish osseointegration of the prosthesis by inducing osteoblast differentiation in the peri‐prosthetic tissue. Therefore, the aim of this study was to investigate the capacity of peri‐prosthetic tissue cells to differentiate into the osteoblast lineage. Cells isolated from peri‐prosthetic tissue samples (n = 22)−obtained during revision surgeries−were cultured under normal and several osteogenic culture conditions. Osteogenic differentiation was assessed by measurement of Alkaline Phosphatse (ALP), mineralization of the matrix and expression of several osteogenic genes. Cells cultured in osteogenic medium showed a significant increase in ALP staining (p = 0.024), mineralization of the matrix (p < 0.001) and ALP gene expression (p = 0.014) compared to normal culture medium. Addition of bone morphogenetic proteins (BMPs), a specific GSK3β inhibitor (GIN) or a combination of BMP and GIN to osteogenic medium could not increase ALP staining, mineralization, and ALP gene expression. In one donor, addition of GIN was required to induce mineralization of the matrix. Overall, we observed a high‐inter‐donor variability in response to osteogenic stimuli. In conclusion, peri‐prosthetic tissue cells, cultured under osteogenic conditions, can produce alkaline phosphatase and mineralized matrix, and therefore show characteristics of differentiation into the osteoblastic lineage. © 2016 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 35:1732–1742, 2017. PMID:27714894

Transporter for Treated Sheet Materials

NASA Technical Reports Server (NTRS)

Pollack, M., H.

1983-01-01

Plastic spacers keep parts separated during transport or storage. Cart with rods and spacers holds sheets with delicate finishes for storage or transport. Sheets supported vertically by rods, or horizontally. Spacers keep sheets separated. Designed to eliminate time and expense of tapping, wrapping, and sometimes refinishing aluminum sheets with delicate anodized finished.

Family Support Programs and Incarcerated Parents: Overview of Family Support Programs.

ERIC Educational Resources Information Center

Family Resource Coalition, Chicago, IL.

This fact sheet begins with an overview of family support programs, which includes a discussion of the premises of family support programs and a list of typical program components. The remainder of the fact sheet focuses on support programs for incarcerated parents. The children of incarcerated parents, though innocent of any crime, are often…

Electrospun silk fibroin/poly(lactide-co-ε-caprolactone) nanofibrous scaffolds for bone regeneration

PubMed Central

Wang, Zi; Lin, Ming; Xie, Qing; Sun, Hao; Huang, Yazhuo; Zhang, DanDan; Yu, Zhang; Bi, Xiaoping; Chen, Junzhao; Wang, Jing; Shi, Wodong; Gu, Ping; Fan, Xianqun

2016-01-01

Background Tissue engineering has become a promising therapeutic approach for bone regeneration. Nanofibrous scaffolds have attracted great interest mainly due to their structural similarity to natural extracellular matrix (ECM). Poly(lactide-co-ε-caprolactone) (PLCL) has been successfully used in bone regeneration, but PLCL polymers are inert and lack natural cell recognition sites, and the surface of PLCL scaffold is hydrophobic. Silk fibroin (SF) is a kind of natural polymer with inherent bioactivity, and supports mesenchymal stem cell attachment, osteogenesis, and ECM deposition. Therefore, we fabricated hybrid nanofibrous scaffolds by adding different weight ratios of SF to PLCL in order to find a scaffold with improved properties for bone regeneration. Methods Hybrid nanofibrous scaffolds were fabricated by blending different weight ratios of SF with PLCL. Human adipose-derived stem cells (hADSCs) were seeded on SF/PLCL nanofibrous scaffolds of various ratios for a systematic evaluation of cell adhesion, proliferation, cytotoxicity, and osteogenic differentiation; the efficacy of the composite of hADSCs and scaffolds in repairing critical-sized calvarial defects in rats was investigated. Results The SF/PLCL (50/50) scaffold exhibited favorable tensile strength, surface roughness, and hydrophilicity, which facilitated cell adhesion and proliferation. Moreover, the SF/PLCL (50/50) scaffold promoted the osteogenic differentiation of hADSCs by elevating the expression levels of osteogenic marker genes such as BSP, Ocn, Col1A1, and OPN and enhanced ECM mineralization. In vivo assays showed that SF/PLCL (50/50) scaffold improved the repair of the critical-sized calvarial defect in rats, resulting in increased bone volume, higher trabecular number, enhanced bone mineral density, and increased new bone areas, compared with the pure PLCL scaffold. Conclusion The SF/PLCL (50/50) nanofibrous scaffold facilitated hADSC proliferation and osteogenic differentiation in vitro and further promoted new bone formation in vivo, suggesting that the SF/PLCL (50/50) nanofibrous scaffold holds great potential in bone tissue regeneration. PMID:27114708

Comparative Analysis of Mouse-Induced Pluripotent Stem Cells and Mesenchymal Stem Cells During Osteogenic Differentiation In Vitro

PubMed Central

Kayashima, Hiroki; Miura, Jiro; Uraguchi, Shinya; Wang, Fangfang; Okawa, Hiroko; Sasaki, Jun-Ichi; Saeki, Makio; Matsumoto, Takuya; Yatani, Hirofumi

2014-01-01

Induced pluripotent stem cells (iPSCs) can differentiate into mineralizing cells and are, therefore, expected to be useful for bone regenerative medicine; however, the characteristics of iPSC-derived osteogenic cells remain unclear. Here, we provide a direct in vitro comparison of the osteogenic differentiation process in mesenchymal stem cells (MSCs) and iPSCs from adult C57BL/6J mice. After 30 days of culture in osteogenic medium, both MSCs and iPSCs produced robustly mineralized bone nodules that contained abundant calcium phosphate with hydroxyapatite crystal formation. Mineral deposition was significantly higher in iPSC cultures than in MSC cultures. Scanning electron microscopy revealed budding matrix vesicles in early osteogenic iPSCs; subsequently, the vesicles propagated to exhibit robust mineralization without rich fibrous structures. Early osteogenic MSCs showed deposition of many matrix vesicles in abundant collagen fibrils that became solid mineralized structures. Both cell types demonstrated increased expression of osteogenic marker genes, such as runx2, osterix, dlx5, bone sialoprotein (BSP), and osteocalcin, during osteogenesis; however, real-time reverse transcription–polymerase chain reaction array analysis revealed that osteogenesis-related genes encoding mineralization-associated molecules, bone morphogenetic proteins, and extracellular matrix collagens were differentially expressed between iPSCs and MSCs. These data suggest that iPSCs are capable of differentiation into mature osteoblasts whose associated hydroxyapatite has a crystal structure similar to that of MSC-associated hydroxyapatite; however, the transcriptional differences between iPSCs and MSCs could result in differences in the mineral and matrix environments of the bone nodules. Determining the biological mechanisms underlying cell-specific differences in mineralization during in vitro iPSC osteogenesis may facilitate the development of clinically effective engineered bone. PMID:24625139

MiR-133a modulates osteogenic differentiation of vascular smooth muscle cells.

PubMed

Liao, Xiao-Bo; Zhang, Zhi-Yuan; Yuan, Ke; Liu, Yuan; Feng, Xiang; Cui, Rong-Rong; Hu, Ye-Rong; Yuan, Zhao-Shun; Gu, Lu; Li, Shi-Jun; Mao, Ding-An; Lu, Qiong; Zhou, Xin-Ming; de Jesus Perez, Vinicio A; Yuan, Ling-Qing

2013-09-01

Arterial calcification is a key pathologic component of vascular diseases such as atherosclerosis, coronary artery disease, and peripheral vascular disease. A hallmark of this pathological process is the phenotypic transition of vascular smooth muscle cells (VSMCs) to osteoblast-like cells. Several studies have demonstrated that microRNAs (miRNAs) regulate osteoblast differentiation, but it is unclear whether miRNAs also regulate VSMC-mediated arterial calcification. In the present study, we sought to characterize the role of miR-133a in regulating VSMC-mediated arterial calcification. Northern blotting analysis of VSMCs treated with β-glycerophosphate demonstrated that miR-133a was significantly decreased during osteogenic differentiation. Overexpression of miR-133a inhibited VSMC transdifferentiation into osteoblast-like cells as evidenced by a decrease in alkaline phosphatase activity, osteocalcin secretion, Runx2 expression, and mineralized nodule formation. Conversely, the knockdown of miR-133a using an miR-133a inhibitor promoted osteogenic differentiation of VSMCs by increasing alkaline phosphatase activity, osteocalcin secretion, and Runx2 expression. Runx2 was identified as a direct target of miR-133a by a cotransfection experiment in VSMCs with luciferase reporter plasmids containing wild-type or mutant 3'-untranslated region sequences of Runx2. Furthermore, the pro-osteogenic effects of miR-133a inhibitor were abrogated in Runx2-knockdown cells, and the inhibition of osteogenic differentiation by pre-miR-133a was reversed by overexpression of Runx2, providing functional evidence that the effects of miR-133a in osteogenic differentiation were mediated by targeting Runx2. These results demonstrate that miR-133a is a key negative regulator of the osteogenic differentiation of VSMCs.

Promotive Effect of Zinc Ions on the Vitality, Migration, and Osteogenic Differentiation of Human Dental Pulp Cells.

PubMed

An, Shaofeng; Gong, Qimei; Huang, Yihua

2017-01-01

Zinc is an essential trace element for proper cellular function and bone formation. However, its exact role in the osteogenic differentiation of human dental pulp cells (hDPCs) has not been fully clarified before. Here, we speculated that zinc may be effective to regulate their growth and osteogenic differentiation properties. To test this hypothesis, different concentrations (1 × 10 -5 , 4 × 10 -5 , and 8 × 10 -5  M) of zinc ions (Zn 2+ ) were added to the basic growth culture medium and osteogenic inductive medium. Cell viability and migration were measured by cell counting kit-8 (CCK-8) and transwell migration assay in the basic growth culture medium, respectively. The reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect the gene expression levels of selective osteogenic differentiation markers and zinc transporters. Alkaline phosphatase (ALP) activity analysis and alizarin red S staining were used to investigate the mineralization of hDPCs. Exposure of hDPCs to Zn 2+ stimulated their viability and migration capacity in a dose- and time-dependent manner. RT-qPCR assay revealed elevated expression levels of osteogenic differentiation-related genes and zinc transporters genes in various degrees. ALP activity was also increased with elevated Zn 2+ concentrations and extended culture periods, but enhanced matrix nodules formation were observed only in 4 × 10 -5 and 8 × 10 -5  M Zn 2+ groups. These findings suggest that specific concentrations of Zn 2+ could potentiate the vitality, migration, and osteogenic differentiation of hDPCs. We may combine optimum zinc element into pulp capping materials to improve their biological performance.

Effect of Adipose Tissue-Derived Osteogenic and Endothelial Cells on Bone Allograft Osteogenesis and Vascularization in Critical-Sized Calvarial Defects

DTIC Science & Technology

2012-05-10

1% peni - cillin/streptomycin, and 50 ng/mL recombinant rat VEGF-C (Promocell, Heidelberg, Germany). The media were changed every other day for 8...various animal models that have demonstrated an enhanced osteogenic effect after treating bone allografts with adipose tissue or bone marrow-derived... enhanced 1560 CORNEJO ET AL. performance of bone allografts using osteogenic differentiated adipose derived mesenchymal stem cells. Biomaterials 32, 8880

Surface topography of hydroxyapatite promotes osteogenic differentiation of human bone marrow mesenchymal stem cells.

PubMed

Yang, Wanlei; Han, Weiqi; He, Wei; Li, Jianlei; Wang, Jirong; Feng, Haotian; Qian, Yu

2016-03-01

Effective and safe induction of osteogenic differentiation is one of the key elements of bone tissue engineering. Surface topography of scaffold materials was recently found to promote osteogenic differentiation. Utilization of this topography may be a safer approach than traditional induction by growth factors or chemicals. The aim of this study is to investigate the enhancement of osteogenic differentiation by surface topography and its mechanism of action. Hydroxyapatite (HA) discs with average roughness (Ra) of surface topography ranging from 0.2 to 1.65 μm and mean distance between peaks (RSm) ranging from 89.7 to 18.6 μm were prepared, and human bone-marrow mesenchymal stem cells (hBMSCs) were cultured on these discs. Optimal osteogenic differentiation was observed on discs with surface topography characterized by Ra ranging from 0.77 to 1.09 μm and RSm ranging from 53.9 to 39.3 μm. On this surface configuration of HA, hBMSCs showed oriented attachment, F-actin arrangement, and a peak in the expression of Yes-associated protein (YAP) and PDZ binding motif (TAZ) (YAP/TAZ). These results indicated that the surface topography of HA promoted osteogenic differentiation of hBMSCs, possibly by increasing cell attachment and promoting the YAP/TAZ signaling pathway. Copyright © 2015 Elsevier B.V. All rights reserved.

Bmp 2 and bmp 7 induce odonto- and osteogenesis of human tooth germ stem cells.

PubMed

Taşlı, P Neslihan; Aydın, Safa; Yalvaç, Mehmet Emir; Sahin, Fikrettin

2014-03-01

Bone morphogenetic proteins (BMPs) initiate, promote, and maintain odontogenesis and osteogenesis. In this study, we studied the effect of bone morphogenic protein 2 (BMP 2) and bone morphogenic protein 7 (BMP 7) as differentiation inducers in tooth and bone regeneration. We compared the effect of BMP 2 and BMP 7 on odontogenic and osteogenic differentiation of human tooth germ stem cells (hTGSCs). Third molar-derived hTGSCs were characterized with mesenchymal stem cell surface markers by flow cytometry. BMP 2 and BMP 7 were transfected into hTGSCs and the cells were seeded onto six-well plates. One day after the transfection, hTGSCs were treated with odontogenic and osteogenic mediums for 14 days. For confirmation of odontogenic and osteogenic differentiation, mRNA levels of BMP2, BMP 7, collagen type 1 (COL1A), osteocalsin (OCN), and dentin sialophosphoprotein (DSPP) genes were measured by quantitative real-time PCR. In addition to this, immunocytochemistry was performed by odontogenic and osteogenic antibodies and mineralization obtained by von Kossa staining. Our results showed that the BMP 2 and BMP 7 both promoted odontogenic and osteogenic differentiation of hTGSCs. Data indicated that BMP 2 treatment and BMP 7 treatment induce odontogenic differentiation without affecting each other, whereas they induce osteogenic differentiation by triggering expression of each other. These findings provide a feasible tool for tooth and bone tissue engineering.

Oscillatory fluid flow induces the osteogenic lineage commitment of mesenchymal stem cells: The effect of shear stress magnitude, frequency, and duration.

PubMed

Stavenschi, Elena; Labour, Marie-Noelle; Hoey, David A

2017-04-11

A potent regulator of bone anabolism is physical loading. However, it is currently unclear whether physical stimuli such as fluid shear within the marrow cavity is sufficient to directly drive the osteogenic lineage commitment of resident mesenchymal stem cells (MSC). Therefore, the objective of the study is to employ a systematic analysis of oscillatory fluid flow (OFF) parameters predicted to occur in vivo on early MSC osteogenic responses and late stage lineage commitment. MSCs were exposed to OFF of 1Pa, 2Pa and 5Pa magnitudes at frequencies of 0.5Hz, 1Hz and 2Hz for 1h, 2h and 4h of stimulation. Our findings demonstrate that OFF elicits a positive osteogenic response in MSCs in a shear stress magnitude, frequency, and duration dependent manner that is gene specific. Based on the mRNA expression of osteogenic markers Cox2, Runx2 and Opn after short-term fluid flow stimulation, we identified that a regime of 2Pa shear magnitude and 2Hz frequency induces the most robust and reliable upregulation in osteogenic gene expression. Furthermore, long-term mechanical stimulation utilising this regime, elicits a significant increase in collagen and mineral deposition when compared to static control demonstrating that mechanical stimuli predicted within the marrow is sufficient to directly drive osteogenesis. Copyright © 2017. Published by Elsevier Ltd.

Wnt/β-catenin signaling enhances osteoblastogenic differentiation from human periodontal ligament fibroblasts.

PubMed

Heo, Jung Sun; Lee, Seung-Youp; Lee, Jeong-Chae

2010-11-01

Wnt/β-catenin signaling has been known to influence bone formation and homeostasis. In this study, we investigated the canonical Wnt signaling regulation of osteogenic differentiation from periodontal ligament (PDL) fibroblasts. Stimulating PDL fibroblasts with lithium chloride (LiCl), a canonical Wnt activator, significantly increased mineralized nodule and alkaline phosphatase (ALP) activity in a time- and dose-dependent manner. LiCl up-regulated protein expression of osteogenic transcription factors, including the runt-related gene 2, Msx2, and Osterix 2, in the PDL fibroblasts. Treatment of these cells with LiCl also increased the mRNA levels of ALP, FosB, and Fra1 in a dose-dependent manner. Blockage of canonical Wnt signaling by treating the cells with DKK1 inhibited Wnt1-stimulated mRNA expression of these osteogenic factors. Furthermore, pretreatment with DKK1 reduced the ALP activity and matrix mineralization stimulated by Wnt1. Collectively, these results suggest that canonical Wnt signaling leads to the differentiation of PDL fibroblasts into osteogenic lineage with the attendant stimulation of osteogenic transcription factors.

A paper-based scaffold for enhanced osteogenic differentiation of equine adipose-derived stem cells.

PubMed

Petersen, Gayle F; Hilbert, Bryan J; Trope, Gareth D; Kalle, Wouter H J; Strappe, Padraig M

2015-11-01

We investigated the applicability of single layer paper-based scaffolds for the three-dimensional (3D) growth and osteogenic differentiation of equine adipose-derived stem cells (EADSC), with comparison against conventional two-dimensional (2D) culture on polystyrene tissue culture vessels. Viable culture of EADSC was achieved using paper-based scaffolds, with EADSC grown and differentiated in 3D culture retaining high cell viability (>94 %), similarly to EADSC in 2D culture. Osteogenic differentiation of EADSC was significantly enhanced in 3D culture, with Alizarin Red S staining and quantification demonstrating increased mineralisation (p < 0.0001), and an associated increase in expression of the osteogenic-specific markers alkaline phosphatase (p < 0.0001), osteopontin (p < 0.0001), and runx2 (p < 0.01). Furthermore, scanning electron microscopy revealed a spherical morphology of EADSC in 3D culture, compared to a flat morphology of EADSC in 2D culture. Single layer paper-based scaffolds provide an enhanced environment for the in vitro 3D growth and osteogenic differentiation of EADSC, with high cell viability, and a spherical morphology.

Adrenaline inhibits osteogenesis via repressing miR-21 expression.

PubMed

Chen, Danying; Wang, Zuolin

2017-01-01

Sympathetic signaling is involved in bone homeostasis; however, the cellular and molecular mechanisms remain unknown. In this study, we found that the psychological stress mediator adrenaline inhibited osteogenic differentiation of human bone marrow-derived stem cells (hMSC) by reducing microRNA-21 (miR-21) expression. Briefly, adrenaline significantly inhibited the osteogenic differentiation of hMSCs, as observed with both Alizarin red staining and maker gene expression (RUNX2, OSX, OCN, and OPN). During this process, miR-21 was suppressed by adrenaline via inhibition of histone acetylation, as verified by H3K9Ac chromatin immunoprecipitation (ChIP) assay. MiR-21 was confirmed to promote hMSC osteogenic differentiation, and overexpression of miR-21 reversed the impeditive effect of adrenaline on hMSC osteogenic differentiation. Our results demonstrate that down-regulation of miR-21 is responsible for the adrenaline-mediated inhibition of hMSC osteogenic differentiation. These findings indicate a regulation of bone metabolism by psychological stress and also provide a molecular basis for psychological stress-associated bone diseases. © 2016 International Federation for Cell Biology.

Gravity, an Regulation Factor in BMSCs Differentiation to osteoblasts

NASA Astrophysics Data System (ADS)

Yan, Huang; Yinghui, Li; Fen, Yang; Zhongquan, Dai

PURPOSE Most studies of regulatory mechanisms of adult stem cell differentiation are concentrated in chemical factors but few efforts are put into physical factors Recent space life science studies indicate mechanical factors participate in the differentiation of cells The aim of this study is to investigate the effects of simulated microgravity or hypergravity on the osteogenic differentiation of rat bone marrow mesenchymal stem cells BMSCs METHODOLOGY The BMSCs at day 7 were added osteogenic inducer 10nM dexamethasone 10mM beta -glycerophosphate and 50 mu M asorbic acid-2-phosphate for 7 days and cultured under simulated microgravity or hypergravity 2g for 1 day 3 days 5 days or 7 days RESULTS After treating BMSCs with osteogenic inducer and hypergravity the cells expressed more ColIA1 Cbfa1 and ALP than in single steogenic inducer treatment Reversely the cells treated with osteogenic inducer and simulated microgravity expressed less ColIA1 Cbfa1 and ALP CONCLUSIONS Our study suggests that hypergravity promotes the osteogenic differentiation of BMSCs and simulated microgravity inhibits this process Gravity is an important regulation factor in BMSCs differentiation to osteoblasts

Addition of BMP-2 or BMP-6 to dexamethasone, ascorbic acid, and β-glycerophosphate may not enhance osteogenic differentiation of human periodontal ligament cells.

PubMed

Khanna-Jain, Rashi; Agata, Hideki; Vuorinen, Annukka; Sándor, George K B; Suuronen, Riitta; Miettinen, Susanna

2010-12-01

This study was designed to investigate the potential merits of the combined use of bone morphogenetic protein (BMP)-2 or BMP-6 and osteogenic supplements (OS) [dexamethasone, ascorbic acid (AA), and β-glycerophosphate] on osteogenic differentiation of periodontal ligament cells (PDLCs). Osteogenic differentiation was evaluated by quantitative alkaline phosphatase (ALP) assay, alizarin red staining, quantitative calcium assay, and the qRT-PCR analysis for the expression of collagen type I, runt-related transcription factor-2, osteopontin (OPN), and osteocalcin in PDLCs. Culture with BMP-2 or BMP-6+AA increased ALP activity of PDLCs, suggesting their osteo-inductive effects. However, longer duration of culture showed neither of the BMPs induced in vitro mineralization. In contrast, OS were able to increase ALP activity and OPN expressions, and also induced in vitro mineralization. The mineralization ability was not enhanced by the addition of BMP-2 or BMP-6. These findings suggest that the addition of BMP-2 or BMP-6 to OS may not enhance an osteogenic differentiation of hPDLCs.

Robot-based additive manufacturing for flexible die-modelling in incremental sheet forming

NASA Astrophysics Data System (ADS)

Rieger, Michael; Störkle, Denis Daniel; Thyssen, Lars; Kuhlenkötter, Bernd

2017-10-01

The paper describes the application concept of additive manufactured dies to support the robot-based incremental sheet metal forming process (`Roboforming') for the production of sheet metal components in small batch sizes. Compared to the dieless kinematic-based generation of a shape by means of two cooperating industrial robots, the supporting robot models a die on the back of the metal sheet by using the robot-based fused layer manufacturing process (FLM). This tool chain is software-defined and preserves the high geometrical form flexibility of Roboforming while flexibly generating support structures adapted to the final part's geometry. Test series serve to confirm the feasibility of the concept by investigating the process challenges of the adhesion to the sheet surface and the general stability as well as the influence on the geometric accuracy compared to the well-known forming strategies.

Active tissue stiffness modulation controls valve interstitial cell phenotype and osteogenic potential in 3D culture.

PubMed

Duan, Bin; Yin, Ziying; Hockaday Kang, Laura; Magin, Richard L; Butcher, Jonathan T

2016-05-01

Calcific aortic valve disease (CAVD) progression is a highly dynamic process whereby normally fibroblastic valve interstitial cells (VIC) undergo osteogenic differentiation, maladaptive extracellular matrix (ECM) composition, structural remodeling, and tissue matrix stiffening. However, how VIC with different phenotypes dynamically affect matrix properties and how the altered matrix further affects VIC phenotypes in response to physiological and pathological conditions have not yet been determined. In this study, we develop 3D hydrogels with tunable matrix stiffness to investigate the dynamic interplay between VIC phenotypes and matrix biomechanics. We find that VIC populated within hydrogels with valve leaflet like stiffness differentiate towards myofibroblasts in osteogenic media, but surprisingly undergo osteogenic differentiation when cultured within lower initial stiffness hydrogels. VIC differentiation progressively stiffens the hydrogel microenvironment, which further upregulates both early and late osteogenic markers. These findings identify a dynamic positive feedback loop that governs acceleration of VIC calcification. Temporal stiffening of pathologically lower stiffness matrix back to normal level, or blocking the mechanosensitive RhoA/ROCK signaling pathway, delays the osteogenic differentiation process. Therefore, direct ECM biomechanical modulation can affect VIC phenotypes towards and against osteogenic differentiation in 3D culture. These findings highlight the importance of the homeostatic maintenance of matrix stiffness to restrict pathological VIC differentiation. We implement 3D hydrogels with tunable matrix stiffness to investigate the dynamic interaction between valve interstitial cells (VIC, major cell population in heart valve) and matrix biomechanics. This work focuses on how human VIC responses to changing 3D culture environments. Our findings identify a dynamic positive feedback loop that governs acceleration of VIC calcification, which is the hallmark of calcific aortic valve disease. Temporal stiffening of pathologically lower stiffness matrix back to normal level, or blocking the mechanosensitive signaling pathway, delays VIC osteogenic differentiation. Our findings provide an improved understanding of VIC-matrix interactions to aid in interpretation of VIC calcification studies in vitro and suggest that ECM disruption resulting in local tissue stiffness decreases may promote calcific aortic valve disease. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Chemical and genetic blockade of HDACs enhances osteogenic differentiation of human adipose tissue-derived stem cells by oppositely affecting osteogenic and adipogenic transcription factors

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

Maroni, Paola; Brini, Anna Teresa; Dipartimento di Scienze Biomediche, Chirurgiche ed Odontoiatriche, Universita degli Studi di Milano, Milano

2012-11-16

Highlights: Black-Right-Pointing-Pointer Acetylation affected hASCs osteodifferentiation through Runx2-PPAR{gamma}. Black-Right-Pointing-Pointer HDACs knocking-down favoured the commitment effect of osteogenic medium. Black-Right-Pointing-Pointer HDACs silencing early activated Runx2 and ALP. Black-Right-Pointing-Pointer PPAR{gamma} reduction and calcium/collagen deposition occurred later. Black-Right-Pointing-Pointer Runx2/PPAR{gamma} target genes were modulated in line with HDACs role in osteo-commitment. -- Abstract: The human adipose-tissue derived stem/stromal cells (hASCs) are an interesting source for bone-tissue engineering applications. Our aim was to clarify in hASCs the role of acetylation in the control of Runt-related transcription factor 2 (Runx2) and Peroxisome proliferator activated receptor (PPAR) {gamma}. These key osteogenic and adipogenic transcription factors are oppositelymore » involved in osteo-differentiation. The hASCs, committed or not towards bone lineage with osteoinductive medium, were exposed to HDACs chemical blockade with Trichostatin A (TSA) or were genetically silenced for HDACs. Alkaline phosphatase (ALP) and collagen/calcium deposition, considered as early and late osteogenic markers, were evaluated concomitantly as index of osteo-differentiation. TSA pretreatment, useful experimental protocol to analyse pan-HDAC-chemical inhibition, and switch to osteogenic medium induced early-osteoblast maturation gene Runx2, while transiently decreased PPAR{gamma} and scarcely affected late-differentiation markers. Time-dependent effects were observed after knocking-down of HDAC1 and 3: Runx2 and ALP underwent early activation, followed by late-osteogenic markers increase and by PPAR{gamma}/ALP activity diminutions mostly after HDAC3 silencing. HDAC1 and 3 genetic blockade increased and decreased Runx2 and PPAR{gamma} target genes, respectively. Noteworthy, HDACs knocking-down favoured the commitment effect of osteogenic medium. Our results reveal a role for HDACs in orchestrating osteo-differentiation of hASCs at transcriptional level, and might provide new insights into the modulation of hASCs-based regenerative therapy.« less

Overexpression of Hif-1α in Mesenchymal Stem Cells Affects Cell-Autonomous Angiogenic and Osteogenic Parameters.

PubMed

Lampert, F M; Kütscher, C; Stark, G B; Finkenzeller, G

2016-03-01

Reconstruction of large bone defects still represents a major medical challenge. In recent years tissue engineering has developed techniques based on adult mesenchymal stem cells (MSCs) that could represent an attractive therapeutical option to treat large bone defects in the future. It has been demonstrated in various animal models that ex vivo expanded MSCs are capable of promoting the regeneration of skeletal defects after implantation. However, for the efficient regeneration of bone in tissue engineering applications, a rapid vascularization of implanted grafts is essential to ensure the survival of cells in the early post-implantational phase. A promising strategy to enhance vascularization of MSC-containing implants could consist of overexpression of the angiogenic master transcription factor Hypoxia-inducible factor 1 (Hif-1) in the MSCs in order to induce angiogenesis and support osteogenesis. In the present study, we overexpressed Hif-1α in MSCs by using recombinant adenoviruses and investigated cell-autonomous effects. Overexpression of Hif-1α enhanced proliferation, migration, cell survival and expression of pro-angiogenic genes. Other parameters such as expression of the osteogenic markers BMP-2 and RunX2 were decreased. Hif-1α overexpression had no effect on invasion, senescence and osteogenic differentiation of MSCs. Our experiments revealed multifarious effects of Hif-1α overexpression on cell-autonomous parameters. Therefore, Hif-1α overexpression may represent a therapeutic option to improve cellular functions of MSCs to treat critical sized bone defects. © 2015 Wiley Periodicals, Inc.

A synergistic approach to the design, fabrication and evaluation of 3D printed micro and nano featured scaffolds for vascularized bone tissue repair

PubMed Central

Holmes, Benjamin; Bulusu, Kartik; Plesniak, Michael; Zhang, Lijie Grace

2016-01-01

3D bioprinting has begun to show great promise in advancing the development of functional tissue/organ replacements. However, to realize the true potential of 3D bioprinted tissues for clinical use requires the fabrication of an interconnected and effective vascular network. Solving this challenge is critical, as human tissue relies on an adequate network of blood vessels to transport oxygen, nutrients, other chemicals, biological factors and waste, in and out of the tissue. Here, we have successfully designed and printed a series of novel 3D bone scaffolds with both bone formation supporting structures and highly interconnected 3D microvascular mimicking channels, for efficient and enhanced osteogenic bone regeneration as well as vascular cell growth. Using a chemical functionalization process, we have conjugated our samples with nano hydroxyapatite (nHA), for the creation of novel micro and nano featured devices for vascularized bone growth. We evaluated our scaffolds with mechanical testing, hydrodynamic measurements and in vitro human mesenchymal stem cell (hMSC) adhesion (4 h), proliferation (1, 3 and 5 d) and osteogenic differentiation (1, 2 and 3 weeks). These tests confirmed bone-like physical properties and vascular-like flow profiles, as well as demonstrated enhanced hMSC adhesion, proliferation and osteogenic differentiation. Additional in vitro experiments with human umbilical vein endothelial cells also demonstrated improved vascular cell growth, migration and organization on micro-nano featured scaffolds. PMID:26758780

BMP2-loaded hollow hydroxyapatite microspheres exhibit enhanced osteoinduction and osteogenicity in large bone defects

PubMed Central

Xiong, Long; Zeng, Jianhua; Yao, Aihua; Tu, Qiquan; Li, Jingtang; Yan, Liang; Tang, Zhiming

2015-01-01

The regeneration of large bone defects is an osteoinductive, osteoconductive, and osteogenic process that often requires a bone graft for support. Limitations associated with naturally autogenic or allogenic bone grafts have demonstrated the need for synthetic substitutes. The present study investigates the feasibility of using novel hollow hydroxyapatite microspheres as an osteoconductive matrix and a carrier for controlled local delivery of bone morphogenetic protein 2 (BMP2), a potent osteogenic inducer of bone regeneration. Hollow hydroxyapatite microspheres (100±25 μm) with a core (60±18 μm) and a mesoporous shell (180±42 m2/g surface area) were prepared by a glass conversion technique and loaded with recombinant human BMP2 (1 μg/mg). There was a gentle burst release of BMP2 from microspheres into the surrounding phosphate-buffered saline in vitro within the initial 48 hours, and continued at a low rate for over 40 days. In comparison with hollow hydroxyapatite microspheres without BMP2 or soluble BMP2 without a carrier, BMP2-loaded hollow hydroxyapatite microspheres had a significantly enhanced capacity to reconstitute radial bone defects in rabbit, as shown by increased serum alkaline phosphatase; quick and complete new bone formation within 12 weeks; and great biomechanical flexural strength. These results indicate that BMP2-loaded hollow hydroxyapatite microspheres could be a potential new option for bone graft substitutes in bone regeneration. PMID:25609957

The synergistic effect of nano-hydroxyapatite and dexamethasone in the fibrous delivery system of gelatin and poly(l-lactide) on the osteogenesis of mesenchymal stem cells.

PubMed

Amjadian, Sara; Seyedjafari, Ehsan; Zeynali, Bahman; Shabani, Iman

2016-06-30

Recently, electrospun nanofibrous scaffolds are vastly taken into consideration in the bone tissue engineering due to mimicking the natural structure of native tissue. In our study, surface features of nanofibers were modified through simultaneous electrospining of the synthetic and natural polymers using poly l-lactide (PLLA) and gelatin to fabricate the hybrid scaffold (PLLA/gelatin). Then, hydroxyapatite nanoparticles (nHA) were loaded in electrospun PLLA nanofibers (PLLA,nHA/gelatin) and also dexamethasone (DEX) was incorporated in these fibers (PLLA,nHA,DEX/gelatin) in the second experiment. Fabricated nanofibrous composite scaffolds were characterized via SEM, FTIR spectroscopy, contact angle, tensile strength measurements, DEX release profile and MTT assay. After seeding adipose derived mesenchymal stem cells, osteoinductivity and osteoconductivity of fabricated scaffolds were analyzed using common osteogenic markers such as alkaline phosphatase activity, calcium depositions and gene expression. These results confirmed that all properties of nanofibers were improved by modifications. Moreover, osteogenic differentiation of stem cells increased in PLLA,nHA/gelatin group in comparison with PLLA/gelatin. The sustained release of DEX was obtained from PLLA,nHA,DEX/gelatin which subsequently led to more osteogenic differentiation. Taken together, PLLA,nHA,DEX/gelatin showed significant potential to support the stem cell proliferation and ostogenic differentiation, and can be a good candidates for tissue engineering and regenerative medicine applications. Copyright © 2016 Elsevier B.V. All rights reserved.

A synergistic approach to the design, fabrication and evaluation of 3D printed micro and nano featured scaffolds for vascularized bone tissue repair

NASA Astrophysics Data System (ADS)

Holmes, Benjamin; Bulusu, Kartik; Plesniak, Michael; Zhang, Lijie Grace

2016-02-01

3D bioprinting has begun to show great promise in advancing the development of functional tissue/organ replacements. However, to realize the true potential of 3D bioprinted tissues for clinical use requires the fabrication of an interconnected and effective vascular network. Solving this challenge is critical, as human tissue relies on an adequate network of blood vessels to transport oxygen, nutrients, other chemicals, biological factors and waste, in and out of the tissue. Here, we have successfully designed and printed a series of novel 3D bone scaffolds with both bone formation supporting structures and highly interconnected 3D microvascular mimicking channels, for efficient and enhanced osteogenic bone regeneration as well as vascular cell growth. Using a chemical functionalization process, we have conjugated our samples with nano hydroxyapatite (nHA), for the creation of novel micro and nano featured devices for vascularized bone growth. We evaluated our scaffolds with mechanical testing, hydrodynamic measurements and in vitro human mesenchymal stem cell (hMSC) adhesion (4 h), proliferation (1, 3 and 5 d) and osteogenic differentiation (1, 2 and 3 weeks). These tests confirmed bone-like physical properties and vascular-like flow profiles, as well as demonstrated enhanced hMSC adhesion, proliferation and osteogenic differentiation. Additional in vitro experiments with human umbilical vein endothelial cells also demonstrated improved vascular cell growth, migration and organization on micro-nano featured scaffolds.

Human Mesenchymal Stem Cell Spheroids in Fibrin Hydrogels Exhibit Improved Cell Survival and Potential for Bone Healing

PubMed Central

Murphy, Kaitlin C.; Fang, Sophia Y.; Leach, J. Kent

2014-01-01

Mesenchymal stem cells (MSC) have great therapeutic potential for the repair of nonhealing bone defects due to their proliferative capacity, multilineage potential, trophic factor secretion, and lack of immunogenicity. However, a major barrier to the translation of cell-based therapies into clinical practice is ensuring their survival and function upon implantation into the defect site. We hypothesized that forming MSC into more physiologic 3-dimensional spheroids, rather than employing dissociated cells from 2-dimensional monolayer culture, would enhance their survival when exposed to a harsh microenvironment while maintaining their osteogenic potential. MSC spheroids were formed using the hanging drop method with increasing cell numbers. Compared to larger spheroids, the smallest spheroids which contained 15,000 cells exhibited increased metabolic activity, reduced apoptosis, and the most uniform distribution of proliferating cells. Spheroids were then entrapped in fibrin gels and cultured in serum-free media and 1% oxygen. Compared to identical numbers of dissociated MSC in fibrin gels, spheroids exhibited significantly reduced apoptosis and secreted up to 100-fold more VEGF. We also observed that fibrin gels containing spheroids and those containing an equivalent number of dissociated cells exhibited similar expression levels of early and late markers of osteogenic differentiation. These data demonstrate that MSC spheroids exhibit greater resistance to apoptosis and enhanced proangiogenic potential, while maintaining similar osteogenic potential to dissociated MSC entrapped in a clinically relevant biomaterial, supporting the use of MSC spheroids in cell-based approaches to bone repair. PMID:24781147

A synergistic approach to the design, fabrication and evaluation of 3D printed micro and nano featured scaffolds for vascularized bone tissue repair.

PubMed

Holmes, Benjamin; Bulusu, Kartik; Plesniak, Michael; Zhang, Lijie Grace

2016-02-12

3D bioprinting has begun to show great promise in advancing the development of functional tissue/organ replacements. However, to realize the true potential of 3D bioprinted tissues for clinical use requires the fabrication of an interconnected and effective vascular network. Solving this challenge is critical, as human tissue relies on an adequate network of blood vessels to transport oxygen, nutrients, other chemicals, biological factors and waste, in and out of the tissue. Here, we have successfully designed and printed a series of novel 3D bone scaffolds with both bone formation supporting structures and highly interconnected 3D microvascular mimicking channels, for efficient and enhanced osteogenic bone regeneration as well as vascular cell growth. Using a chemical functionalization process, we have conjugated our samples with nano hydroxyapatite (nHA), for the creation of novel micro and nano featured devices for vascularized bone growth. We evaluated our scaffolds with mechanical testing, hydrodynamic measurements and in vitro human mesenchymal stem cell (hMSC) adhesion (4 h), proliferation (1, 3 and 5 d) and osteogenic differentiation (1, 2 and 3 weeks). These tests confirmed bone-like physical properties and vascular-like flow profiles, as well as demonstrated enhanced hMSC adhesion, proliferation and osteogenic differentiation. Additional in vitro experiments with human umbilical vein endothelial cells also demonstrated improved vascular cell growth, migration and organization on micro-nano featured scaffolds.

Osteoblasts extracellular matrix induces vessel like structures through glycosylated collagen I

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

Palmieri, D.; Valli, M.; Viglio, S.

2010-03-10

Extracellular matrix (ECM) plays a fundamental role in angiogenesis affecting endothelial cells proliferation, migration and differentiation. Vessels-like network formation in vitro is a reliable test to study the inductive effects of ECM on angiogenesis. Here we utilized matrix deposed by osteoblasts as substrate where the molecular and structural complexity of the endogenous ECM is preserved, to test if it induces vessel-like network formation by endothelial cells in vitro. ECM is more similar to the physiological substrate in vivo than other substrates previously utilized for these studies in vitro. Osteogenic ECM, prepared in vitro from mature osteoblasts at the phase ofmore » maximal deposition and glycosylation of collagen I, induces EAhy926, HUVEC, and HDMEC endothelial cells to form vessels-like structures and promotes the activation of metalloproteinase-2 (MMP-2); the functionality of the p-38/MAPK signaling pathway is required. Osteogenic ECM also induces a transient increase of CXCL12 and a decrease of the receptor CXCR4. The induction of vessel-like networks is dependent from proper glycosylation of collagens and does not occur on osteogenic ECMs if deglycosylated by -galactosidase or on less glycosylated ECMs derived from preosteoblasts and normal fibroblasts, while is sustained on ECM from osteogenesis imperfecta fibroblasts only when their mutation is associated with over-glycosylation of collagen type I. These data support that post-translational glycosylation has a role in the induction in endothelial cells in vitro of molecules conductive to self-organization in vessels-like structures.« less

Isolation and Characterisation of Mesenchymal Stem Cells from Rat Bone Marrow and the Endosteal Niche: A Comparative Study

PubMed Central

Yusop, Norhayati; Battersby, Paul; Alraies, Amr; Moseley, Ryan

2018-01-01

Within bone, mesenchymal stromal cells (MSCs) exist within the bone marrow stroma (BM-MSC) and the endosteal niche, as cells lining compact bone (CB-MSCs). This study isolated and characterised heterogeneous MSC populations from each niche and subsequently investigated the effects of extensive cell expansion, analysing population doublings (PDs)/cellular senescence, colony-forming efficiencies (CFEs), MSC cell marker expression, and osteogenic/adipogenic differentiation. CB-MSCs and BM-MSCs demonstrated similar morphologies and PDs, reaching 100 PDs. Both populations exhibited consistent telomere lengths (12–17 kb), minimal senescence, and positive telomerase expression. CB-MSCs (PD15) had significantly lower CFEs than PD50. CB-MSCs and BM-MSCs both expressed MSC (CD73/CD90/CD105); embryonic (Nanog) and osteogenic markers (Runx2, osteocalcin) but no hematopoietic markers (CD45). CB-MSCs (PD15) strongly expressed Oct4 and p16INK4A. At early PDs, CB-MSCs possessed a strong osteogenic potency and low potency for adipogenesis, whilst BM-MSCs possessed greater overall bipotentiality for osteogenesis and adipogenesis. At PD50, CB-MSCs demonstrated reduced potency for both osteogenesis and adipogenesis, compared to BM-MSCs at equivalent PDs. This study demonstrates similarities in proliferative and mesenchymal cell characteristics between CB-MSCs and BM-MSCs, but contrasting multipotentiality. Such findings support further comparisons of human CB-MSCs and BM-MSCs, facilitating selection of optimal MSC populations for regenerative medicine purposes. PMID:29765418

Zirconium amine tris(phenolate): A more effective initiator for biomedical lactide.

PubMed

Jones, Matthew D; Wu, Xujun; Chaudhuri, Julian; Davidson, Matthew G; Ellis, Marianne J

2017-11-01

Here a zirconium amine tris(phenolate) is used as the initiator for the production of polylactide for biomedical applications, as a replacement for a tin initiator (usually tin octanoate). The ring opening polymerization (ROP) was carried out in the melt at 130°C. The zirconium-catalyzed PLA (PLA-Zr) required 30min, resulting in a polydispersity index (PDI) of 1.17, compared to 1h and PDI=1.77 for tin-catalyzed PLA (PLA-Sn). PLA-Zr and PLA-Sn supported osteosarcoma cell (MG63) culture to the same extent (cell number, morphology, extracellular matrix production and osteogenic function) until day 14 when the PLA-Zr showed increased cell number, overall extracellular matrix production and osteogenic function. To conclude, the reduction in reaction time, controllable microstructure and biologically benign nature of the zirconium amine tris(phenolate) initiator shows that it is a more effective initiator for ROP of polylactide for biomedical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Inhibitory effect of CT domain of CCN3/NOV on proliferation and differentiation of osteogenic mesenchymal stem cells, Kusa-A1

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

Katsuki, Yuko; Oral Pathology, Graduate School of Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549; Sakamoto, Kei

CCN3/NOV activates the Notch signal through the carboxyl terminal cysteine-rich (CT) domain. CCN3 transfection to Kusa-A1 inhibited osteogenic differentiation and cell proliferation, which is accompanied by upregulation of Hes/Hey, Notch downstream targets, and p21, a CDK inhibitor. Upregulation of Hes/Hey and p21 was abrogated by the deletion of CT domain. Anti-proliferative activity of CCN3 was also abrogated by CT domain deletion whereas anti-osteogenic activity was not completely abrogated. We found that CT domain-deleted CCN3 still possesses antagonistic effect on BMP-2. These results suggest that CCN3 employs Notch and BMP pathways in anti-osteogenic activity while it inhibits cell proliferation uniquely bymore » Notch/p21 pathway.« less

The Role of Magnesium Ion Substituted Biphasic Calcium Phosphate Spherical Micro-Scaffolds in Osteogenic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stem Cells.

PubMed

Kim, Dong-Hyun; Shin, Keun-Koo; Jung, Jin Sup; Chun, Ho Hwan; Park, Seong Soo; Lee, Jong Kook; Park, Hong-Chae; Yoon, Seog-Young

2015-08-01

This study was investigated the role of magnesium (Mg2+) ion substituted biphasic calcium phosphate (Mg-BCP) spherical micro-scaffolds in osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs). Mg-BCP micro-scaffolds with spherical morphology were successfully prepared using in situ co-precipitation and spray drying atomization process. The in vitro cell proliferation and differentiation of hAT-MSCs were determined up to day 14. After in vitro biological tests, Mg-BCP micro-scaffolds with hAT-MSCs showed more enhanced osteogenicity than pure hAT-MSCs as control group by unique biodegradation of TCP phase and influence of substituted Mg2+ ion in biphasic nanostructure. Therefore, these results suggest that Mg-BCP micro-scaffolds promote osteogenic differentiation of hAT-MSCs.

An Inhibitory Role of Osthole in Rat MSCs Osteogenic Differentiation and Proliferation via Wnt/β-Catenin and Erk1/2-MAPK Pathways.

PubMed

Hu, Hongyang; Chen, Min; Dai, Guangzu; Du, Guoqing; Wang, Xuezong; He, Jie; Zhao, Yongfang; Han, Dapeng; Cao, Yuelong; Zheng, Yuxin; Ding, Daofang

2016-01-01

Bone marrow-derived mesenchymal stem cells (MSCs) are responsible for new bone formation during adulthood. Accumulating evidences showed that Osthole promotes the osteogenic differentiation in primary osteoblasts. The aim of this study was to investigate whether Osthole exhibits a potential to stimulate the osteogenic differentiation of MSCs and the underlying mechanism. MSCs were treated with a gradient concentration of Osthole (6.25 µM, 12.5 µM, and 25 µM). Cell proliferation was assessed by western blotting with the proliferating cell nuclear antigen (PCNA) and Cyclin D1 antibodies, fluorescence activated cell sorting (FACS), and cell counting kit 8 (CCK8). MSCs were cultured in osteogenesis-induced medium for one or two weeks. The osteogenic differentiation of MSCs was estimated by Alkaline Phosphatase (ALP) staining, Alizarin red staining, Calcium influx, and quantitative PCR (qPCR). The underlying mechanism of Osthole-induced osteogenesis was further evaluated by western blotting with antibodies in Wnt/β-catenin, PI3K/Akt, BMPs/smad1/5/8, and MAPK signaling pathways. Osthole inhibited proliferation of rat MSCs in a dose-dependent manner. Osthole suppressed osteogenic differentiation of rat MSCs by down-regulating the activities of Wnt/β-catenin and Erk1/2-MAPK signaling. Osthole inhibits the proliferation and osteogenic differentiation of rat MSCs, which might be mediated through blocking the Wnt/β-catenin and Erk1/2-MAPK signaling pathways. © 2016 The Author(s) Published by S. Karger AG, Basel.

Osteogenic differentiation of human mesenchymal stem cells in mineralized alginate matrices.

PubMed

Westhrin, Marita; Xie, Minli; Olderøy, Magnus Ø; Sikorski, Pawel; Strand, Berit L; Standal, Therese

2015-01-01

Mineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic cells in such materials will potentially generate biological bone grafts that may even further augment bone healing. Here, we studied osteogenic differentiation of human mesenchymal stem cells (MSC) in an alginate hydrogel system where the cells were co-immobilized with alkaline phosphatase (ALP) for gradual mineralization of the microenvironment. MSC were embedded in unmodified alginate beads and alginate beads mineralized with ALP to generate a polymer/hydroxyapatite scaffold mimicking the composition of bone. The initial scaffold mineralization induced further mineralization of the beads with nanosized particles, and scanning electron micrographs demonstrated presence of collagen in the mineralized and unmineralized alginate beads cultured in osteogenic medium. Cells in both types of beads sustained high viability and metabolic activity for the duration of the study (21 days) as evaluated by live/dead staining and alamar blue assay. MSC in beads induced to differentiate in osteogenic direction expressed higher mRNA levels of osteoblast-specific genes (RUNX2, COL1AI, SP7, BGLAP) than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (SOST) and dental matrix protein-1 (DMP1), markers for late osteoblasts/osteocytes. In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture. Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering.

10(-7)  m 17β-oestradiol enhances odonto/osteogenic potency of human dental pulp stem cells by activation of the NF-κB pathway.

PubMed

Wang, Y; Zheng, Y; Wang, Z; Li, J; Wang, Z; Zhang, G; Yu, J

2013-12-01

Oestrogen has been proven to significantly enhance osteogenic potency, while oestrogen deficiency usually leads to impaired osteogenic differentiation of mesenchymal stem cells. However, little is known concerning direct effects of oestrogen on differentiation of human dental pulp stem cells (DPSCs). In this study, human DPSCs were isolated and treated with 10(-7)  m 17β-oestradiol (E2). Alkaline phosphatase (ALP) assay and alizarin red staining were performed. Alkaline phosphatase and alizarin red showed that E2 treatment significantly enhanced ALP activity and mineralization ability of DPSCs, but had no effect on cell proliferation. Real-time RT-PCR and western blot assay demonstrated that odonto/osteogenic markers (ALP, RUNX2/RUNX2, OSX/OSX, OCN/OCN and DSPP/DSP) were significantly upregulated in the cells after E2 treatment. Moreover, phosphorylation of cytoplasmic IκBα/P65 and expression of nuclear P65 were enhanced in a time-dependent manner following E2 treatment, suggesting activation of NF-κB signaling. Conversely, inhibition of the NF-κB pathway suppressed E2-mediated upregulation of odonto/osteogenic markers, indicating that the NF-κB pathway was pivotal for E2-mediated differentiation. These findings provide evidence that 10(-7)  m 17β-oestradiol promoted odonto/osteogenic differentiation of human DPSCs via activation of the NF-κB signaling pathway. © 2013 The Authors. Cell Proliferation published by John Wiley & Sons Ltd.

10−7 m 17β-oestradiol enhances odonto/osteogenic potency of human dental pulp stem cells by activation of the NF-κB pathway

PubMed Central

Wang, Y; Zheng, Y; Wang, Z; Li, J; Wang, Z; Zhang, G; Yu, J

2013-01-01

Objectives Oestrogen has been proven to significantly enhance osteogenic potency, while oestrogen deficiency usually leads to impaired osteogenic differentiation of mesenchymal stem cells. However, little is known concerning direct effects of oestrogen on differentiation of human dental pulp stem cells (DPSCs). Materials and methods In this study, human DPSCs were isolated and treated with 10−7 m 17β-oestradiol (E2). Alkaline phosphatase (ALP) assay and alizarin red staining were performed. Results Alkaline phosphatase and alizarin red showed that E2 treatment significantly enhanced ALP activity and mineralization ability of DPSCs, but had no effect on cell proliferation. Real-time RT-PCR and western blot assay demonstrated that odonto/osteogenic markers (ALP, RUNX2/RUNX2, OSX/OSX, OCN/OCN and DSPP/DSP) were significantly upregulated in the cells after E2 treatment. Moreover, phosphorylation of cytoplasmic IκBα/P65 and expression of nuclear P65 were enhanced in a time-dependent manner following E2 treatment, suggesting activation of NF-κB signaling. Conversely, inhibition of the NF-κB pathway suppressed E2-mediated upregulation of odonto/osteogenic markers, indicating that the NF-κB pathway was pivotal for E2-mediated differentiation. Conclusion These findings provide evidence that 10−7 m 17β-oestradiol promoted odonto/osteogenic differentiation of human DPSCs via activation of the NF-κB signaling pathway. PMID:24152244

Osteogenic Differentiation of Human Mesenchymal Stem Cells in Mineralized Alginate Matrices

PubMed Central

Westhrin, Marita; Xie, Minli; Olderøy, Magnus Ø.; Sikorski, Pawel

2015-01-01

Mineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic cells in such materials will potentially generate biological bone grafts that may even further augment bone healing. Here, we studied osteogenic differentiation of human mesenchymal stem cells (MSC) in an alginate hydrogel system where the cells were co-immobilized with alkaline phosphatase (ALP) for gradual mineralization of the microenvironment. MSC were embedded in unmodified alginate beads and alginate beads mineralized with ALP to generate a polymer/hydroxyapatite scaffold mimicking the composition of bone. The initial scaffold mineralization induced further mineralization of the beads with nanosized particles, and scanning electron micrographs demonstrated presence of collagen in the mineralized and unmineralized alginate beads cultured in osteogenic medium. Cells in both types of beads sustained high viability and metabolic activity for the duration of the study (21 days) as evaluated by live/dead staining and alamar blue assay. MSC in beads induced to differentiate in osteogenic direction expressed higher mRNA levels of osteoblast-specific genes (RUNX2, COL1AI, SP7, BGLAP) than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (SOST) and dental matrix protein-1 (DMP1), markers for late osteoblasts/osteocytes. In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture. Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering. PMID:25769043

Cytokines TNF-α, IL-6, IL-17F, and IL-4 Differentially Affect Osteogenic Differentiation of Human Adipose Stem Cells

PubMed Central

Bravenboer, Nathalie

2016-01-01

During the initial stages of bone repair, proinflammatory cytokines are released within the injury site, quickly followed by a shift to anti-inflammatory cytokines. The effect of pro- and anti-inflammatory cytokines on osteogenic differentiation of mesenchymal stem cells is controversial. Here, we investigated the effect of the proinflammatory cytokines TNF-α, IL-6, IL-8, and IL-17F and the anti-inflammatory cytokine IL-4 on proliferation and osteogenic differentiation of human adipose stem cells (hASCs). hASCs were treated with TNF-α, IL-6, IL-8, IL-17F, or IL-4 (10 ng/mL) for 72 h mimicking bone repair. TNF-α reduced collagen type I gene expression but increased hASC proliferation and ALP activity. IL-6 also strongly enhanced ALP activity (18-fold), as well as bone nodule formation by hASCs. IL-8 did not affect proliferation or osteogenic gene expression but reduced bone nodule formation. IL-17F decreased hASC proliferation but enhanced ALP activity. IL-4 enhanced osteocalcin gene expression and ALP activity but reduced RUNX2 gene expression and bone nodule formation. In conclusion, all cytokines studied have both enhancing and reducing effects on osteogenic differentiation of hASCs, even when applied for 72 h only. Some cytokines, specifically IL-6, may be suitable to induce osteogenic differentiation of mesenchymal stem cells as a strategy for enhancing bone repair. PMID:27667999

Thermally stabilized heliostat

DOEpatents

Anderson, Alfred J.

1983-01-01

An improvement in a heliostat having a main support structure and pivoting and tilting motors and gears and a mirror module for reflecting solar energy onto a collector, the improvement being characterized by an internal support structure within each mirror module and front and back sheets attached to the internal support structure, the front and back sheets having the same coefficient of thermal expansion such that no curvature is induced by temperature change, and a layer of adhesive adhering the mirror to the front sheet. The adhesive is water repellent and has adequate set strength to support the mirror but has sufficient shear tolerance to permit the differential expansion of the mirror and the front sheet without inducing stresses or currature effect. The adhesive also serves to dampen fluttering of the mirror and to protect the mirror backside against the adverse effects of weather. Also disclosed are specific details of the preferred embodiment.

Osteogenically differentiated mesenchymal stem cells and ceramics for bone tissue engineering.

PubMed

Ohgushi, Hajime

2014-02-01

In the human body, cells having self-renewal and multi-differentiation capabilities reside in many tissues and are called adult stem cells. In bone marrow tissue, two types of stem cells are well known: hematopoietic stem cells and mesenchymal stem cells (MSCs). Though the number of MSCs in bone marrow tissue is very low, it can be increased by in vitro culture of the marrow, and culture-expanded MSCs are available for various tissue regeneration. The culture-expanded MSCs can further differentiate into osteogenic cells such as bone forming osteoblasts by culturing the MSCs in an osteogenic medium. This paper discusses osteogenically differentiated MSCs derived from the bone marrow of patients. Importantly, the differentiation can be achieved on ceramic surfaces which demonstrate mineralized bone matrix formation as well as appearance of osteogenic cells. The cell/matrix/ceramic constructs could show immediate in vivo bone formation and are available for bone reconstruction surgery. Currently, MSCs are clinically available for the regeneration of various tissues due to their high proliferation/differentiation capabilities. However, the capabilities are still limited and thus technologies to improve or recover the inherent capabilities of MSCs are needed.

RSPO3-LGR4 Regulates Osteogenic Differentiation Of Human Adipose-Derived Stem Cells Via ERK/FGF Signalling.

PubMed

Zhang, Min; Zhang, Ping; Liu, Yunsong; Lv, Longwei; Zhang, Xiao; Liu, Hao; Zhou, Yongsheng

2017-02-21

The four R-spondins (RSPOs) and their three related receptors, LGR4, 5 and 6, have emerged as a major ligand-receptor system with critical roles in development and stem cell survival. However, the exact roles of the RSPO-LGR system in osteogenesis remain largely unknown. In the present study, we showed that RSPO3-shRNA increased the osteogenic potential of human adipose-derived stem cells (hASCs) significantly. Mechanistically, we demonstrated that RSPO3 is a negative regulator of ERK/FGF signalling. We confirmed that inhibition of the ERK1/2 signalling pathway blocked osteogenic differentiation in hASCs, and the increased osteogenic capacity observed after RSPO3 knockdown in hASCs was reversed by inhibition of ERK signalling. Further, silencing of LGR4 inhibited the activity of ERK signalling and osteogenic differentiation of hASCs. Most importantly, we found that loss of LGR4 abrogated RSPO3-regulated osteogenesis and RSPO3-induced ERK1/2 signalling inhibition. Collectively, our data show that ERK signalling works downstream of LGR4 and RSPO3 regulates osteoblastic differentiation of hASCs possibly via the LGR4-ERK signalling.

RSPO3-LGR4 Regulates Osteogenic Differentiation Of Human Adipose-Derived Stem Cells Via ERK/FGF Signalling

PubMed Central

Zhang, Min; Zhang, Ping; Liu, Yunsong; Lv, Longwei; Zhang, Xiao; Liu, Hao; Zhou, Yongsheng

2017-01-01

The four R-spondins (RSPOs) and their three related receptors, LGR4, 5 and 6, have emerged as a major ligand-receptor system with critical roles in development and stem cell survival. However, the exact roles of the RSPO-LGR system in osteogenesis remain largely unknown. In the present study, we showed that RSPO3-shRNA increased the osteogenic potential of human adipose-derived stem cells (hASCs) significantly. Mechanistically, we demonstrated that RSPO3 is a negative regulator of ERK/FGF signalling. We confirmed that inhibition of the ERK1/2 signalling pathway blocked osteogenic differentiation in hASCs, and the increased osteogenic capacity observed after RSPO3 knockdown in hASCs was reversed by inhibition of ERK signalling. Further, silencing of LGR4 inhibited the activity of ERK signalling and osteogenic differentiation of hASCs. Most importantly, we found that loss of LGR4 abrogated RSPO3-regulated osteogenesis and RSPO3-induced ERK1/2 signalling inhibition. Collectively, our data show that ERK signalling works downstream of LGR4 and RSPO3 regulates osteoblastic differentiation of hASCs possibly via the LGR4-ERK signalling. PMID:28220828

Development of Biodegradable Poly(citrate)-Polyhedral Oligomeric Silsesquioxanes Hybrid Elastomers with High Mechanical Properties and Osteogenic Differentiation Activity.

PubMed

Du, Yuzhang; Yu, Meng; Chen, Xiaofeng; Ma, Peter X; Lei, Bo

2016-02-10

Biodegradable elastomeric biomaterials have attracted much attention in tissue engineering due to their biomimetic viscoelastic behavior and biocompatibility. However, the low mechanical stability at hydrated state, fast biodegradation in vivo, and poor osteogenic activity greatly limited bioelastomers applications in bone tissue regeneration. Herein, we develop a series of poly(octanediol citrate)-polyhedral oligomeric silsesquioxanes (POC-POSS) hybrids with highly tunable elastomeric behavior (hydrated state) and biodegradation and osteoblasts biocompatibility through a facile one-pot thermal polymerization strategy. POC-POSS hybrids show significantly improved stiffness and ductility in either dry or hydrated conditions, as well as good antibiodegradation ability (20-50% weight loss in 3 months). POC-POSS hybrids exhibit significantly enhanced osteogenic differentiation through upregulating alkaline phosphatase (ALP) activity, calcium deposition, and expression of osteogenic markers (ALPL, BGLAP, and Runx2). The high mechanical stability at hydrated state and enhanced osteogenic activity make POC-POSS hybrid elastomers promising as scaffolds and nanoscale vehicles for bone tissue regeneration and drug delivery. This study may also provide a new strategy (controlling the stiffness under hydrated condition) to design advanced hybrid biomaterials with high mechanical properties under physiological condition for tissue regeneration applications.

In vitro proliferation of human osteogenic cells in presence of different commercial bone substitute materials combined with enamel matrix derivatives

PubMed Central

2009-01-01

Background Cellular reactions to alloplastic bone substitute materials (BSM) are a subject of interest in basic research. In regenerative dentistry, these bone grafting materials are routinely combined with enamel matrix derivatives (EMD) in order to additionally enhance tissue regeneration. Materials and methods The aim of this study was to evaluate the proliferative activity of human osteogenic cells after incubation over a period of seven days with commercial BSM of various origin and chemical composition. Special focus was placed on the potential additional benefit of EMD on cellular proliferation. Results Except for PerioGlas®, osteogenic cell proliferation was significantly promoted by the investigated BSM. The application of EMD alone also resulted in significantly increased cellular proliferation. However, a combination of BSM and EMD resulted in only a moderate additional enhancement of osteogenic cell proliferation. Conclusion The application of most BSM, as well as the exclusive application of EMD demonstrated a positive impact on the proliferation of human osteogenic cells in vitro. In order to increase the benefit from substrate combination (BSM + EMD), further studies on the interactions between BSM and EMD are needed. PMID:19909545

Graphene oxide nanoflakes incorporated gelatin-hydroxyapatite scaffolds enhance osteogenic differentiation of human mesenchymal stem cells

NASA Astrophysics Data System (ADS)

Nair, Manitha; Nancy, D.; Krishnan, Amit G.; Anjusree, G. S.; Vadukumpully, Sajini; Nair, Shantikumar V.

2015-04-01

In this study, graphene oxide (GO) nanoflakes (0.5 and 1 wt%) were incorporated into a gelatin-hydroxyapatite (GHA) matrix through a freeze drying technique and its effect to enhance mechanical strength and osteogenic differentiation was studied. The GHA matrix with GO demonstrated less brittleness in comparison to GHA scaffolds. There was no significant difference in mechanical strength between GOGHA0.5 and GOGHA1.0 scaffolds. When the scaffolds were immersed in phosphate buffered saline (to mimic physiologic condition) for 60 days, around 50-60% of GO was released in sustained and linear manner and the concentration was within the toxicity limit as reported earlier. Further, GOGHA0.5 scaffolds were continued for cell culture experiments, wherein the scaffold induced osteogenic differentiation of human adipose derived mesenchymal stem cells without providing supplements like dexamethasone, L-ascorbic acid and β glycerophosphate in the medium. The level of osteogenic differentiation of stem cells was comparable to those cultured on GHA scaffolds with osteogenic supplements. Thus biocompatible, biodegradable and porous GO reinforced gelatin-HA 3D scaffolds may serve as a suitable candidate in promoting bone regeneration in orthopaedics.

High levels of β-catenin signaling reduce osteogenic differentiation of stem cells in inflammatory microenvironments through inhibition of the noncanonical Wnt pathway.

PubMed

Liu, Na; Shi, Songtao; Deng, Manjing; Tang, Liang; Zhang, Guangjing; Liu, Ning; Ding, Bofu; Liu, Wenjia; Liu, Yali; Shi, Haigang; Liu, Luchuan; Jin, Yan

2011-09-01

Periodontal ligament stem cells (PDLSCs), a new population of mesenchymal stem cells (MSCs), have been isolated from the periodontal ligament (PDL). The capacity of multipotency and self-renewal makes them an excellent cell source for bone regeneration and repair. However, their bone-regeneration ability could be awakened in inflammatory microenvironments, which may be the result of changes in their differentiation potential. Recently, genetic evidences has shown that the Wnt pathway plays an important role in bone homeostasis. In this study we have determined the specific role of β-catenin in osteogenic differentiation of PDLSCs obtained from inflammatory microenvironments (P-PDLSCs). The inflammatory microenvironment, while inhibiting osteogenic differentiation potential, promotes proliferation of MSCs. A higher the level of β-catenin in P-PDLSCs than in H-PDLSCs (PDLSCs obtained from a healthy microenvironment) resulted in the same disparity in canonical Wnt signaling pathway activation between each cell type. Here we show that activation of β-catenin suppresses the noncanonical Wnt/Ca(2+) pathway, leading to increased proliferation but reduced osteogenic differentiation of P-PDLSCs. Downregulation of the levels of β-catenin by treatment with dickkopf-1 (DKK-1) leads to activation of the noncanonical Wnt/Ca(2+) pathway, which, in turn, results in the promotion of osteogenic differentiation in P-PDLSCs. Interestingly, β-catenin can affect both the canonical Wnt/β-catenin pathway and the noncanonical Wnt/Ca(2+) pathway. Our data indicate that β-catenin plays a central role in regulating osteogenic differentiation of MSCs in inflammatory microenvironments. Given the important role of Wnt signaling in osteogenic differentiation, it is possible that agents that can modify this pathway may be of value in bone regeneration by MSCs in chronic inflammatory microenvironments. Copyright © 2011 American Society for Bone and Mineral Research.

Baghdadite ceramics modulate the cross talk between human adipose stem cells and osteoblasts for bone regeneration.

PubMed

Lu, Zufu; Wang, Guocheng; Roohani-Esfahani, Iman; Dunstan, Colin R; Zreiqat, Hala

2014-03-01

Understanding interactions among the three elements (cells, scaffolds, and bioactive factors) is critical for successful tissue engineering. This study was aimed to investigate how scaffolds would affect osteogenic gene expression in human adipose tissue-derived stem cells (ASCs) or human primary osteoblasts (HOBs), and their cross talk. Either ASCs or HOBs were seeded on Baghdadite (Ca3ZrSi2O9) and hydroxyapatite/tricalcium phosphate (HA/TCP) scaffolds, and osteogenic gene expression was assessed. To further evaluate how substrate affected HOB and ASC cross talk, an indirect co-culture system with semipermeable inserts placed on the culture plate was set up to co-culture ASCs or HOBs, which were grown in monolayer or seeded on Baghdadite or HA/TCP scaffolds, and osteogenic differentiation of the cells was assessed. We found that Baghdadite scaffolds induced a significantly greater increase in RUNX2, osteopontin, bone sialoprotein, and osteocalcin gene expression in HOBs in comparison to HA/TCP scaffolds; Baghdadite scaffolds also significantly induced RUNX2 and osteopontin, but not bone sialoprotein and osteocalcin gene expression in ASCs. In the co-culture system, the HOBs on Baghdadite scaffolds more markedly promoted osteogenic gene expression in ASCs compared to HOBs in monolayer or the HOBs on HA/TCP scaffolds. In addition, the ASCs seeded on Baghdadite scaffolds more markedly promoted osteogenic gene expression in HOBs than did the ASCs on HA/TCP scaffolds. BMP-2 expression in ASCs or HOBs was increased when they were seeded on Baghdadite scaffolds, and adding Noggin into the co-culture medium largely abrogated Baghdadite scaffold-modulated ASC-HOB cross talk. In summary, Baghdadite scaffolds not only promote the osteogenic differentiation of HOBs or ASCs but also modulate the cross talk between ASCs and HOBs, in part via increasing BMP2 expression, thereby promoting their osteogenic differentiation.

Low-intensity pulsed ultrasound stimulation facilitates in vitro osteogenic differentiation of human adipose-derived stem cells via up-regulation of heat shock protein (HSP)70, HSP90, and bone morphogenetic protein (BMP) signaling pathway.

PubMed

Zhang, Zhonglei; Ma, Yalin; Guo, Shaowen; He, Yi; Bai, Gang; Zhang, Wenjun

2018-05-29

Low-intensity pulsed ultrasound (LIPUS) has positive effects on osteogenic differentiation. However, the effect of LIPUS on osteogenic differentiation of human adipose-derived stem cells (hASCs) is unclear. In the present study, we investigated whether LIPUS could promote the proliferation and osteogenic differentiation of hASCs. hASCs were isolated and osteogenically induced with LIPUS stimulation at 20 and 30 mW cm -2 for 30 min day -1 Cell proliferation and osteogenic differentiation potential of hASCs were respectively analyzed by cell counting kit-8 assay, Alizarin Red S staining, real-time polymerase chain reaction, and Western blotting. The results indicated that LIPUS stimulation did not significantly affect the proliferation of hASCs, but significantly increased their alkaline phosphatase activity on day 6 of culture and markedly promoted the formation of mineralized nodules on day 21 of culture. The mRNA expression levels of runt-related transcription factor, osteopontin, and osteocalcin were significantly up-regulated by LIPUS stimulation. LIPUS stimulation did not affect the expression of heat shock protein (HSP) 27, HSP40, bone morphogenetic protein (BMP)-6 and BMP-9, but significantly up-regulated the protein levels of HSP70, HSP90, BMP-2, and BMP-7 in the hASCs. Further studies found that LIPUS increased the mRNA levels of Smad 1 and Smad 5, elevated the phosphorylation of Smad 1/5, and suppressed the expression of BMP antagonist Noggin. These findings indicated that LIPUS stimulation enhanced osteogenic differentiation of hASCs possibly through the up-regulation of HSP70 and HSP90 expression and activation of BMP signaling pathway. Therefore, LIPUS might have the potential to promote the repair of bone defect. © 2018 The Author(s).

Effect of boron on osteogenic differentiation of human bone marrow stromal cells.

PubMed

Ying, Xiaozhou; Cheng, Shaowen; Wang, Wei; Lin, Zhongqin; Chen, Qingyu; Zhang, Wei; Kou, Dongquan; Shen, Yue; Cheng, Xiaojie; Rompis, Ferdinand An; Peng, Lei; Zhu Lu, Chuan

2011-12-01

Bone marrow stromal cells (BMSCs) have been well established as an ideal source of cell-based therapy for bone tissue engineering applications. Boron (B) is a notable trace element in humans; so far, the effects of boron on the osteogenic differentiation of BMSCs have not been reported. The aim of this study was to evaluate the effects of boron (0, 1, 10,100, and 1,000 ng/ml) on osteogenic differentiation of human BMSCs. In this study, BMSCs proliferation was analyzed by cell counting kit-8 (CCK8) assay, and cell osteogenic differentiation was evaluated by alkaline phosphatase (ALP) activity assay, Von Kossa staining, and real-time PCR. The results indicated that the proliferation of BMSCs was no different from the control group when added with B at the concentration of 1, 10, and 100 ng/ml respectively (P > 0.05); in contrast, 1,000 ng/ml B inhibited the proliferation of BMSCs at days 4, 7, and 14 (P < 0.05). By ALP staining, we discovered that BMSCs treated with 10 and 100 ng/ml B presented a higher ALP activity compared with control (P < 0.05). By real-time PCR, we detected the messenger RNA expression of ALP, osteocalcin, collagen type I, and bone morphogenetic proteins 7 were also increased in 10 and 100 ng/ml B treatment groups (P < 0.05). The calcium depositions were increased in 1 and 10 ng/ml B treatment groups (P < 0.05). Taken all together, it was the first time to report that B could increase osteogenic effect by stimulating osteogenic differentiation-related marker gene synthesis during the proliferation and differentiation phase in human BMSCs and could be a promising approach for enhancing osteogenic capacity of cell-based construction in bone tissue engineering.

Alendronate-Eluting Biphasic Calcium Phosphate (BCP) Scaffolds Stimulate Osteogenic Differentiation

PubMed Central

Kim, Sung Eun; Lee, Deok-Won; Kang, Eun Young; Jeong, Won Jae; Lee, Boram; Jeong, Myeong Seon; Kim, Hak Jun; Park, Kyeongsoon; Song, Hae-Ryong

2015-01-01

Biphasic calcium phosphate (BCP) scaffolds have been widely used in orthopedic and dental fields as osteoconductive bone substitutes. However, BCP scaffolds are not satisfactory for the stimulation of osteogenic differentiation and maturation. To enhance osteogenic differentiation, we prepared alendronate- (ALN-) eluting BCP scaffolds. The coating of ALN on BCP scaffolds was confirmed by scanning electron microscopy (FE-SEM), energy-dispersive X-ray spectroscopy (EDS), and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). An in vitro release study showed that release of ALN from ALN-eluting BCP scaffolds was sustained for up to 28 days. In vitro results revealed that MG-63 cells grown on ALN-eluting BCP scaffolds exhibited increased ALP activity and calcium deposition and upregulated gene expression of Runx2, ALP, OCN, and OPN compared with the BCP scaffold alone. Therefore, this study suggests that ALN-eluting BCP scaffolds have the potential to effectively stimulate osteogenic differentiation. PMID:26221587

Detection of Osteogenic Differentiation by Differential Mineralized Matrix Production in Mesenchymal Stromal Cells by Raman Spectroscopy

PubMed Central

Chen, He-Guei; Chiang, Hui-Hua Kenny; Lee, Oscar Kuang-Sheng

2013-01-01

Mesenchymal stromal cells (MSCs) hold great potential in skeletal tissue engineering and regenerative medicine. However, conventional methods that are used in molecular biology to evaluate osteogenic differentiation of MSCs require a relatively large amount of cells. Cell lysis and cell fixation are also required and all these steps are time-consuming. Therefore, it is imperative to develop a facile technique which can provide real-time information with high sensitivity and selectivity to detect the osteogenic maturation of MSCs. In this study, we use Raman spectroscopy as a biosensor to monitor the production of mineralized matrices during osteogenic induction of MSCs. In summary, Raman spectroscopy is an excellent biosensor to detect the extent of maturation level during MSCs-osteoblast differentiation with a non-disruptive, real-time and label free manner. We expect that this study will promote further investigation of stem cell research and clinical applications. PMID:23734254

Data book: Space station/base food system study. Book 3: Study selection rationale sheets

NASA Technical Reports Server (NTRS)

1970-01-01

The supporting rationale sheets are presented which were utilized in the selection and support of the concepts considered in the final phase of the study. Each concept, conceived to fulfill a specific function of the food system, was assessed in terms of the eight critical factors depicted on the rationale sheet. When weighted and totaled, the resulting selection factor was used as a guide in making the final decision.

DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic program in calcific aortic valve disease.

PubMed

Mkannez, Ghada; Gagné-Ouellet, Valérie; Nsaibia, Mohamed Jalloul; Boulanger, Marie-Chloé; Rosa, Mickael; Argaud, Deborah; Hadji, Fayez; Gaudreault, Nathalie; Rhéaume, Gabrielle; Bouchard, Luigi; Bossé, Yohan; Mathieu, Patrick

2018-05-02

Calcific aortic valve disease (CAVD) is characterized by the osteogenic transition of valve interstitial cells (VICs). In CAVD, lysophosphatidic acid (LysoPA), a lipid mediator with potent osteogenic activity, is produced in the aortic valve (AV) and is degraded by membrane-associated phospholipid phosphatases (PLPPs). We thus hypothesized that a dysregulation of PLPPs could participate to the osteogenic reprograming of VICs during CAVD. The expression of PLPPs was examined in human control and mineralized AVs and comprehensive analyses were performed to document the gene regulation and impact of PLPPs on the osteogenic transition of VICs. We found that PLPP3 gene and enzymatic activity were downregulated in mineralized AVs. Multidimensional gene profiling in 21 human AVs showed that expression of PLPP3 was inversely correlated with the level of 5-methylcytosine (5meC) located in an intronic mammalian interspersed repeat element (MIR). Bisulfite pyrosequencing in a larger series of 67 AVs confirmed that 5meC in intron 1 was increased by 2.2-fold in CAVD compared to control AVs. In isolated cells, epigenome editing with CRISPR-Cas9 system containing a deficient Cas9 fused with DNA methyltransferase (dCas9-DNMT) was used to increase 5meC in the intronic enhancer and showed that it reduced significantly the expression of PLPP3. Knockdown experiments showed that lower expression of PLPP3 in VICs promotes an osteogenic program. DNA methylation of a MIR-based enhancer downregulates the expression of PLPP3 and promotes the mineralization of the AV.

Application of a Parallelizable Perfusion Bioreactor for Physiologic 3D Cell Culture.

PubMed

Egger, Dominik; Spitz, Sarah; Fischer, Monica; Handschuh, Stephan; Glösmann, Martin; Friemert, Benedikt; Egerbacher, Monika; Kasper, Cornelia

2017-01-01

It is crucial but challenging to keep physiologic conditions during the cultivation of 3D cell scaffold constructs for the optimization of 3D cell culture processes. Therefore, we demonstrate the benefits of a recently developed miniaturized perfusion bioreactor together with a specialized incubator system that allows for the cultivation of multiple samples while screening different conditions. Hence, a decellularized bone matrix was tested towards its suitability for 3D osteogenic differentiation under flow perfusion conditions. Subsequently, physiologic shear stress and hydrostatic pressure (HP) conditions were optimized for osteogenic differentiation of human mesenchymal stem cells (MSCs). X-ray computed microtomography and scanning electron microscopy (SEM) revealed a closed cell layer covering the entire matrix. Osteogenic differentiation assessed by alkaline phosphatase activity and SEM was found to be increased in all dynamic conditions. Furthermore, screening of different fluid shear stress (FSS) conditions revealed 1.5 mL/min (equivalent to ∼10 mPa shear stress) to be optimal. However, no distinct effect of HP compared to flow perfusion without HP on osteogenic differentiation was observed. Notably, throughout all experiments, cells cultivated under FSS or HP conditions displayed increased osteogenic differentiation, which underlines the importance of physiologic conditions. In conclusion, the bioreactor system was used for biomaterial testing and to develop and optimize a 3D cell culture process for the osteogenic differentiation of MSCs. Due to its versatility and higher throughput efficiency, we hypothesize that this bioreactor/incubator system will advance the development and optimization of a variety of 3D cell culture processes. © 2017 S. Karger AG, Basel.

[The effect of Toll-like receptor 4 in nicotine suppressing the osteogenic potential of periodontal ligament stem cells].

PubMed

Luan, Yan; Deqin, Yang

2017-08-01

Objective To explore the impact of nicotine on proliferation and osteogenic capability of periodontal ligament stem cells (PDLSCs), and the role of Toll-like receptor 4 (TLR4) in nicotine, suppressing the osteogenic capability of PDLSCs. Methods PDLSCs were cultured in vitro, and the flow cytometer was used to identify the surface antigen markers of PDLSCs. WST-1 was used to detect the proliferation ability of PDLSCs, which were stimulated by different concentrations of nicotine. Alizarin red staining was used to observe the formation of mineralized nodules after PDLSCs stimulation with different concentrations of nicotine. Real-time polymerase chain reaction (RT-PCR) and Western blot were used to detect the change in osteogenic potential of PDLSCs stimulated by nicotine, after TAK-242, and with the inhibitor of TLR4. Results PDLSCs expressed mesenchymal stem cell-associated markers CD90 and CD105. When the concentration of nicotine was 10⁻⁴ mol·L⁻¹, the PDLSC proliferation could be suppressed after 3 d compared with the control group (P<0.05). The amount of mineralized nodules reduced after osteogenic differentiation at 21 d by alizarin red staining. RT-PCR and Western blot showed the expression levels of alkaline phosphatase (ALP), and osteocalcin (OCN), and the Runt-related transcription factor-2 (Runx-2) were lower than in the control group when nicotine suppressed the PDLSCs (P<0.05). This effect was attenuated after TAK-242 was added. Conclusion Nicotine suppresses the proliferation and osteogenic capability of PDLSCs, which may be regulated by TLR4.

Characterization and comparison of osteoblasts derived from mouse embryonic stem cells and induced pluripotent stem cells.

PubMed

Ma, Ming-San; Kannan, Vishnu; de Vries, Anneriek E; Czepiel, Marcin; Wesseling, Evelyn M; Balasubramaniyan, Veerakumar; Kuijer, Roel; Vissink, Arjan; Copray, Sjef C V M; Raghoebar, Gerry M

2017-01-01

New developments in stem cell biology offer alternatives for the reconstruction of critical-sized bone defects. One of these developments is the use of induced pluripotent stem (iPS) cells. These stem cells are similar to embryonic stem (ES) cells, but can be generated from adult somatic cells and therefore do not raise ethical concerns. Proper characterization of iPS-derived osteoblasts is important for future development of safe clinical applications of these cells. For this reason, we differentiated mouse ES and iPS cells toward osteoblasts using osteogenic medium and compared their functionality. Immunocytochemical analysis showed significant expression of bone markers (osteocalcin and collagen type I) in osteoblasts differentiated from ES and iPS cells on days 7 and 30. An in vitro mineralization assay confirmed the functionality of osteogenically differentiated ES and iPS cells. Gene expression arrays focusing on osteogenic differentiation were performed in order to compare the gene expression pattern in both differentiated and undifferentiated ES cells and iPS cells. We observed a significant upregulation of osteogenesis-related genes such as Runx2, osteopontin, collagen type I, Tnfsf11, Csf1, and alkaline phosphatase upon osteogenic differentiation of the ES and iPS cells. We further validated the expression of key osteogenic genes Runx2, osteopontin, osteocalcin, collagen type I, and osterix in both differentiated and undifferentiated ES and iPS cells by means of quantified real-time polymerase chain reaction. We conclude that ES and iPS cells are similar in their osteogenic differentiation capacities, as well as in their gene expression patterns.

Gelatin-Derived Graphene–Silicate Hybrid Materials Are Biocompatible and Synergistically Promote BMP9-Induced Osteogenic Differentiation of Mesenchymal Stem Cells

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

Zou, Yulong; Qazvini, Nader Taheri; Zane, Kylie

Graphene-based materials are used in many fields but have found only limited applications in biomedicine, including bone tissue engineering. Here, we demonstrate that novel hybrid materials consisting of gelatin-derived graphene and silicate nanosheets of Laponite (GL) are biocompatible and promote osteogenic differentiation of mesenchymal stem cells (MSCs). Homogeneous cell attachment, long-term proliferation, and osteogenic differentiation of MSCs on a GL-scaffold were confirmed using optical microscopy and scanning electron microscopy. GL-powders made by pulverizing the GL-scaffold were shown to promote bone morphogenetic protein (BMP9)-induced osteogenic differentiation. GL-powders increased the alkaline phosphatase (ALP) activity in immortalized mouse embryonic fibroblasts but decreased themore » ALP activity in more-differentiated immortalized mouse adipose-derived cells. Note, however, that GL-powders promoted BMP9-induced calcium mineral deposits in both MSC lines, as assessed using qualitative and quantitative alizarin red assays. Furthermore, the expression of chondro-osteogenic regulator markers such as Runx2, Sox9, osteopontin, and osteocalcin was upregulated by the GL-powder, independent of BMP9 stimulation; although the powder synergistically upregulated the BMP9-induced Osterix expression, the adipogenic marker PPAR gamma was unaffected. Furthermore, in vivo stem cell implantation experiments demonstrated that GL-powder could significantly enhance the BMP9-induced ectopic bone formation from MSCs. Collectively, our results strongly suggest that the GL hybrid materials promote BMP9-induced osteogenic differentiation of MSCs and hold promise for the development of bone tissue engineering platforms.« less

Effects of naringin on the proliferation and osteogenic differentiation of human amniotic fluid-derived stem cells.

PubMed

Liu, Meimei; Li, Yan; Yang, Shang-Tian

2017-01-01

Human amniotic fluid-derived stem cells (hAFSCs) are a novel cell source for generating osteogenic cells to treat bone diseases. Effective induction of osteogenic differentiation from hAFSCs is critical to fulfil their therapeutic potential. In this study, naringin, the main active compound of Rhizoma drynariae (a Chinese herbal medicine), was used to stimulate the proliferation and osteogenic differentiation of hAFSCs. The results showed that naringin enhanced the proliferation and alkaline phosphatase activity (ALP) of hAFSCs in a dose-dependent manner in the range 1-100 µg/ml, while an inhibition effect was observed at 200 µg/ml. Consistently, the calcium content also increased with naringin concentration up to 100 µg/ml. The enhanced osteogenic differentiation of hAFSCs by naringin was further confirmed by the dose-dependent upregulation of marker genes, including osteopontin (OPN) and Collagen I from RT-PCR analysis. The increased osteoprotegerin (OPG) expression and minimal expression of receptor activator of nuclear factor-κB ligand (RANKL) suggested that naringin also inhibited osteoclastogenesis of hAFSCs. In addition, the gene expressions of bone morphogenetic protein 4 (BMP4), runt-related transcription factor 2 (RUNX2), β-catenin and Cyclin D1 also increased significantly, indicating that naringin promotes the osteogenesis of hAFSCs via the BMP and Wnt-β-catenin signalling pathways. These results suggested that naringin can be used to upregulate the osteogenic differentiation of hAFSCs, which could provide an attractive and promising treatment for bone disorders. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

The role of non-thermal atmospheric pressure biocompatible plasma in the differentiation of osteoblastic precursor cells, MC3T3-E1.

PubMed

Han, Ihn; Choi, Eun Ha

2017-05-30

Non-thermal atmospheric pressure plasma is ionized matter, composed of highly reactive species that include positive ions, negative ions, free radicals, neutral atoms, and molecules. Recent reports have suggested that non-thermal biocompatible plasma (NBP) can selectively kill a variety of cancer cells, and promote stem cell differentiation. However as of yet, the regulation of proliferation and differentiation potential of NBP has been poorly understood.Here, we investigated the effects of NBP on the osteogenic differentiation of precursor cell lines of osteoblasts, MC3T3 E1 and SaOS-2. For in vitro osteogenic differentiation, precursor cell lines were treated with NBP, and cultured with osteogenic induction medium. After 10 days of treatment, the NBP was shown to be effective in osteogenic differentiation in MC3T3 E1 cells by von Kossa and Alizarin Red S staining assay. Real-time PCR was then performed to investigate the expression of osteogenic specific genes, Runx2, OCN, COL1, ALP and osterix in MC3T3 E1 cells after treatment with NBP for 4 days. Furthermore, analysis of the protein expression showed that NBP treatment significantly reduced PI3K/AKT signaling and MAPK family signaling. However, p38 controlled phosphorylation of transcription factor forkhead box O1 (FoxO1) that related to cell differentiation with increased phosphorylated p38. These results suggest that non-thermal atmospheric pressure plasma can induce osteogenic differentiation, and enhance bone formation.

Fact Sheet: Environmental Pathway Models-Ground-Water Modeling in Support of Remedial Decision Making at Sites Contaminated with Radioactive Material

EPA Pesticide Factsheets

This fact sheet was designed to be used by technical staff responsible for identifying and implementing flow and transport models to support cleanup decisions at hazardous and radioactive waste sites.

Pulsed magnetic therapy increases osteogenic differentiation of mesenchymal stem cells only if they are pre-committed.

PubMed

Ferroni, Letizia; Tocco, Ilaria; De Pieri, Andrea; Menarin, Martina; Fermi, Enrico; Piattelli, Adriano; Gardin, Chiara; Zavan, Barbara

2016-05-01

Pulsed electromagnetic field (PEMF) therapy has been documented to be an effective, non-invasive, safe treatment method for a variety of clinical conditions, especially in settings of recalcitrant healing. The underlying mechanisms on the different biological components of tissue regeneration are still to be elucidated. The aim of the present study was to characterize the effects of extremely low frequency (ELF)-PEMFs on commitment of mesenchymal stem cell (MSCs) culture system, through the determination of gene expression pattern and cellular morphology. Human MSCs derived from adipose tissue (ADSCs) were cultured in presence of adipogenic, osteogenic, neural, or glial differentiative medium and basal medium, then exposed to ELF-PEMFs daily stimulation for 21days. Control cultures were performed without ELF-PEMFs stimulation for all cell populations. Effects on commitment were evaluated after 21days of cultures. The results suggested ELF-PEMFs does not influence ADSCs commitment and does not promote adipogenic, osteogenic, neural or glial differentiation. However, ELF-PEMFs treatment on ADSCs cultured in osteogenic differentiative medium markedly increased osteogenesis. We concluded that PEMFs affect the osteogenic differentiation of ADSCs only if they are pre-commitment and that this therapy can be an appropriate candidate for treatment of conditions requiring an acceleration of repairing process. Copyright © 2016 Elsevier Inc. All rights reserved.

Osteocalcin Mediates Biomineralization during Osteogenic Maturation in Human Mesenchymal Stromal Cells

PubMed Central

Tsao, Yu-Tzu; Huang, Yi-Jeng; Wu, Hao-Hsiang; Liu, Yu-An; Liu, Yi-Shiuan; Lee, Oscar K.

2017-01-01

There is a growing interest in cell therapies using mesenchymal stromal cells (MSCs) for repairing bone defects. MSCs have the ability to differentiate into osteoprogenitors and osteoblasts as well as to form calcified bone matrix. However, the molecular mechanisms governing mineralization during osteogenic differentiation remain unclear. Non-collagenous proteins in the extracellular matrix are believed to control different aspects of the mineralization. Since osteocalcin is the most abundant non-collagenous bone matrix protein, the purpose of this study is to investigate the roles of osteocalcin in mineral species production during osteogenesis of MSCs. Using Raman spectroscopy, we found that the maturation of mineral species was affected by osteocalcin expression level. After osteocalcin was knocked down, the mineral species maturation was delayed and total hydroxyapatite was lower than the control group. In addition, the expression of osteogenic marker genes, including RUNX2, alkaline phosphatase, type I collagen, and osteonectin, was downregulated during osteogenic differentiation compared to the control group; whereas gene expression of osterix was upregulated after the knockdown. Together, osteocalcin plays an essential role for the maturation of mineral species and modulates osteogenic differentiation of MSCs. The results offer new insights into the enhancement of new bone formation, such as for the treatments of osteoporosis and fracture healing. PMID:28106724

Reduced graphene oxide-coated hydroxyapatite composites stimulate spontaneous osteogenic differentiation of human mesenchymal stem cells

NASA Astrophysics Data System (ADS)

Lee, Jong Ho; Shin, Yong Cheol; Jin, Oh Seong; Kang, Seok Hee; Hwang, Yu-Shik; Park, Jong-Chul; Hong, Suck Won; Han, Dong-Wook

2015-07-01

Human mesenchymal stem cells (hMSCs) have great potential as cell sources for bone tissue engineering and regeneration, but the control and induction of their specific differentiation into bone cells remain challenging. Graphene-based nanomaterials are considered attractive candidates for biomedical applications such as scaffolds in tissue engineering, substrates for SC differentiation and components of implantable devices, due to their biocompatible and bioactive properties. Despite the potential biomedical applications of graphene and its derivatives, only limited information is available regarding their osteogenic activity. This study concentrates upon the effects of reduced graphene oxide (rGO)-coated hydroxyapatite (HAp) composites on osteogenic differentiation of hMSCs. The average particle sizes of HAp and rGO were 1270 +/- 476 nm and 438 +/- 180 nm, respectively. When coated on HAp particulates, rGO synergistically enhanced spontaneous osteogenic differentiation of hMSCs, without hampering their proliferation. This result was confirmed by determining alkaline phosphatase activity and mineralization of calcium and phosphate as early and late stage markers of osteogenic differentiation. It is suggested that rGO-coated HAp composites can be effectively utilized as dental and orthopedic bone fillers since these graphene-based particulate materials have potent effects on stimulating the spontaneous differentiation of MSCs and show superior bioactivity and osteoinductive potential.Human mesenchymal stem cells (hMSCs) have great potential as cell sources for bone tissue engineering and regeneration, but the control and induction of their specific differentiation into bone cells remain challenging. Graphene-based nanomaterials are considered attractive candidates for biomedical applications such as scaffolds in tissue engineering, substrates for SC differentiation and components of implantable devices, due to their biocompatible and bioactive properties. Despite the potential biomedical applications of graphene and its derivatives, only limited information is available regarding their osteogenic activity. This study concentrates upon the effects of reduced graphene oxide (rGO)-coated hydroxyapatite (HAp) composites on osteogenic differentiation of hMSCs. The average particle sizes of HAp and rGO were 1270 +/- 476 nm and 438 +/- 180 nm, respectively. When coated on HAp particulates, rGO synergistically enhanced spontaneous osteogenic differentiation of hMSCs, without hampering their proliferation. This result was confirmed by determining alkaline phosphatase activity and mineralization of calcium and phosphate as early and late stage markers of osteogenic differentiation. It is suggested that rGO-coated HAp composites can be effectively utilized as dental and orthopedic bone fillers since these graphene-based particulate materials have potent effects on stimulating the spontaneous differentiation of MSCs and show superior bioactivity and osteoinductive potential. Electronic supplementary information (ESI) available: Additional figures. See DOI: 10.1039/c5nr01580d

BMP2 induced osteogenic differentiation of human umbilical cord stem cells in a peptide-based hydrogel scaffold

NASA Astrophysics Data System (ADS)

Lakshmana, Shruthi M.

Craniofacial tissue loss due to traumatic injuries and congenital defects is a major clinical problem around the world. Cleft palate is the second most common congenital malformation in the United States occurring with an incidence of 1 in 700. Some of the problems associated with this defect are feeding difficulties, speech abnormalities and dentofacial anomalies. Current treatment protocol offers repeated surgeries with extended healing time. Our long-term goal is to regenerate bone in the palatal region using tissue-engineering approaches. Bone tissue engineering utilizes osteogenic cells, osteoconductive scaffolds and osteoinductive signals. Mesenchymal stem cells derived from human umbilical cord (HUMSCs) are highly proliferative with the ability to differentiate into osteogenic precursor cells. The primary objective of the study was to characterize HUMSCs and culture them in a 3D hydrogel scaffold and investigate their osteogenic potential. PuraMatrix(TM) is an injectable 3D nanofiber scaffold capable of self-assembly when exposed to physiologic conditions. Our second objective was to investigate the effect of Bone Morphogenic Protein 2 (BMP2) in enhancing the osteogenic differentiation of HUMSCs encapsulated in PuraMatrix(TM). We isolated cells isolated from Wharton's Jelly region of the umbilical cord obtained from NDRI (New York, NY). Isolated cells satisfied the minimal criteria for mesenchymal stem cells (MSCs) as defined by International Society of Cell Therapy in terms of plastic adherence, fibroblastic phenotype, surface marker expression and osteogenic differentiation. Flow Cytometry analysis showed that cells were positive for CD73, CD90 and CD105 while negative for hematopoietic marker CD34. Alkaline phosphatase activity (ALP) of HUMSCs showed peak activity at 2 weeks (p<0.05). Cells were encapsulated in 0.2% PuraMatrix(TM) at cell densities of 10x104, 20x104, 40x10 4 and 80x104. Cell viability with WST and proliferation with Live-Dead cell assays showed viable cells at all cell concentrations (p<0.05). A two- fold upregulation of ALP gene was seen for cells encapsulated in PuraMatrix(TM) with osteogenic medium compared to cells in culture medium (p<0.05). HUMSCs encapsulated in PuraMatrix(TM) were treated with BMP2 at doses of 50ng/ml, 100ng/ml and 200ng/ml. A significant upregulation of ALP gene in BMP2 treated cells was seen compared to HUMSCs treated in osteogenic medium (p<0.05). Peak osteogenic activity was noted at BMP2 dose of 100ng/ml (p<0.05). We have developed a composite system of HUMSCs, PuraMatrix(TM) and BMP2 for repair of bone defects that is injectable precluding additional surgeries.

The botanical molecule p-hydroxycinnamic acid as a new osteogenic agent: insight into the treatment of cancer bone metastases.

PubMed

Yamaguchi, Masayoshi

2016-10-01

Bone homeostasis is maintained through a balance between osteoblastic bone formation and osteoclastic bone resorption. Bone loss with aging is induced by decreasing in osteoblastic bone formation and increasing in osteoclastic bone resorption, thereby leading to osteoporosis. Osteoporosis with its accompanying decrease in bone mass is widely recognized as a major public heath problem. Pharmacologic and nutritional factors may play a role in the prevention and treatment of bone loss with aging. p-Hydroxycinnamic acid (HCA), which stimulates bone mineralization in mouse bone tissues in vitro, has been found to be present in the leafstalk of wasabi (Wasabi japonica MATSUM) among various food and plants. Other phenolic acids including cinnamic acid, ferulic acid, caffeic acid and 3,4-dimethoxycinnamic acid did not have osteogenic effects. HCA was demonstrated to stimulate osteoblastic bone formation and suppresses osteoclastic bone resorption in vitro by antagonizing activation of the nuclear factor kappa B. Oral administration of HCA was found to exhibit restorative effects on bone loss induced by ovariectomy and diabetic states, supporting a role in the treatment of osteoporosis. Moreover, HCA was demonstrated to prevent the suppressed osteoblastic mineralization and the enhanced osteoclastogenesis in mouse bone marrow cells cocultured with bone metastatic MDA-MB-231 human breast cancer cells in vitro. The botanical molecule HCA, as a new osteogenic agent, is suggested to play a role in the treatment of cancer bone metastases. This review will discuss an advanced recent finding that HCA may be a useful agent to treat bone metabolic disorder.

Periodontal ligament cellular structures engineered with electrospun poly(DL-lactide-co-glycolide) nanofibrous membrane scaffolds.

PubMed

Inanç, Bülend; Arslan, Y Emre; Seker, Sükran; Elçin, A Eser; Elçin, Y Murat

2009-07-01

Periodontal tissue engineering is expected to overcome the limitations associated with the existing regenerative techniques for the treatment of periodontal defects involving alveolar bone, cementum, and periodontal ligament. Cell-based tissue engineering approaches involve the utilization of in vitro expanded cells with regenerative capacity and their delivery to the appropriate sites via biomaterial scaffolds. The aim of this study was to establish living periodontal ligament cell-containing structures on electrospun poly(DL-lactic-co-glycolic acid) (PLGA) nanofiber membrane scaffolds, assess their viability and characteristics, and engineer multilayered structures amenable to easy handling. Human periodontal ligament (hPDL) cells were expanded in explant culture and then characterized morphologically and immunohistochemically. PLGA nanofiber membranes were prepared by the electrospinning process; mechanical tensile properties were determined, surface topography, nanofiber size, and porosity status were investigated with SEM. Cells were seeded on the membranes at approximately 50,000 cell/cm(2) and cultured for 21 days either in expansion or in osteogenic induction medium. Cell adhesion and viability were demonstrated using SEM and MTT, respectively, and osteogenic differentiation was determined with IHC and immunohistomorphometric evaluation of osteopontin, osteocalcin, and bone sialoprotein marker expression. At days 3, 6, 9, and 12 additional cell/membrane layers were deposited on the existing ones and multilayered hybrid structures were established. Results indicate the feasibility of periodontal ligament cell-containing tissue-like structures engineering with PDL cells and electrospun nanofiber PLGA scaffolds supporting cell adhesion, viability and osteogenic differentiation properties of cells in hybrid structures amenable to macroscopic handling.

Learning Support Centers and International Tutor Training Program Certification: An Interview with Rick A. Sheets

ERIC Educational Resources Information Center

Walker, Luann

2016-01-01

This article presents an interview with Rick A. Sheets, who has been working in learning assistance, faculty training, and technology support for over 30 years. He collaborated with Frank Christ as the co-founder and webmaster of the Learning Support Centers in Higher Education (LSCHE) website, a resource established in 1996 for learning center…

Hypervelocity impact shield

NASA Technical Reports Server (NTRS)

Cour-Palais, Burton G. (Inventor); Crews, Jeanne Lee (Inventor)

1991-01-01

A hypervelocity impact shield and method for protecting a wall structure, such as a spacecraft wall, from impact with particles of debris having densities of about 2.7 g/cu cm and impact velocities up to 16 km/s are disclosed. The shield comprises a stack of ultra thin sheets of impactor disrupting material supported and arranged by support means in spaced relationship to one another and mounted to cover the wall in a position for intercepting the particles. The sheets are of a number and spacing such that the impacting particle and the resulting particulates of the impacting particle and sheet material are successively impact-shocked to a thermal state of total melt and/or vaporization to a degree as precludes perforation of the wall. The ratio of individual sheet thickness to the theoretical diameter of particles of debris which may be of spherical form is in the range of 0.03 to 0.05. The spacing between adjacent sheets is such that the debris cloud plume of liquid and vapor resulting from an impacting particle penetrating a sheet does not puncture the next adjacent sheet prior to the arrival thereat of fragment particulates of sheet material and the debris particle produced by a previous impact.

Priming integrin α5 promotes human mesenchymal stromal cell osteoblast differentiation and osteogenesis

PubMed Central

Hamidouche, Zahia; Fromigué, Olivia; Ringe, Jochen; Häupl, Thomas; Vaudin, Pascal; Pagès, Jean-Christophe; Srouji, Samer; Livne, Erella; Marie, Pierre J.

2009-01-01

Adult human mesenchymal stromal cells (hMSCs) have the potential to differentiate into chondrogenic, adipogenic, or osteogenic lineages, providing a potential source for tissue regeneration. An important issue for efficient bone regeneration is to identify factors that can be targeted to promote the osteogenic potential of hMSCs. Using transcriptome analysis, we found that integrin α5 (ITGA5) expression is up-regulated during dexamethasone-induced osteoblast differentiation of hMSCs. Gain-of-function studies showed that ITGA5 promotes the expression of osteoblast phenotypic markers and in vitro osteogenesis of hMSCs. Down-regulation of endogenous ITGA5 using specific shRNAs blunted osteoblast marker gene expression and osteogenic differentiation. Molecular analyses showed that the enhanced osteoblast differentiation induced by ITGA5 was mediated by activation of focal adhesion kinase/ERK1/2-MAPKs and PI3K signaling pathways. Remarkably, activation of endogenous ITGA5 using agonists such as a specific antibody that primes the integrin or a peptide that specifically activates ITGA5 was sufficient to enhance ERK1/2-MAPKs and PI3K signaling and to promote osteoblast differentiation and osteogenic capacity of hMSCs. Importantly, we demonstrated that hMSCs engineered to overexpress ITGA5 exhibited a marked increase in their osteogenic potential in vivo. Taken together, these findings not only reveal that ITGA5 is required for osteoblast differentiation of adult hMSCs but also provide a targeted strategy using ITGA5 agonists to promote the osteogenic capacity of hMSCs. This may be used for tissue regeneration in bone disorders where the recruitment or capacity of hMSCs is compromised. PMID:19843692

Priming integrin alpha5 promotes human mesenchymal stromal cell osteoblast differentiation and osteogenesis.

PubMed

Hamidouche, Zahia; Fromigué, Olivia; Ringe, Jochen; Häupl, Thomas; Vaudin, Pascal; Pagès, Jean-Christophe; Srouji, Samer; Livne, Erella; Marie, Pierre J

2009-11-03

Adult human mesenchymal stromal cells (hMSCs) have the potential to differentiate into chondrogenic, adipogenic, or osteogenic lineages, providing a potential source for tissue regeneration. An important issue for efficient bone regeneration is to identify factors that can be targeted to promote the osteogenic potential of hMSCs. Using transcriptome analysis, we found that integrin alpha5 (ITGA5) expression is up-regulated during dexamethasone-induced osteoblast differentiation of hMSCs. Gain-of-function studies showed that ITGA5 promotes the expression of osteoblast phenotypic markers and in vitro osteogenesis of hMSCs. Down-regulation of endogenous ITGA5 using specific shRNAs blunted osteoblast marker gene expression and osteogenic differentiation. Molecular analyses showed that the enhanced osteoblast differentiation induced by ITGA5 was mediated by activation of focal adhesion kinase/ERK1/2-MAPKs and PI3K signaling pathways. Remarkably, activation of endogenous ITGA5 using agonists such as a specific antibody that primes the integrin or a peptide that specifically activates ITGA5 was sufficient to enhance ERK1/2-MAPKs and PI3K signaling and to promote osteoblast differentiation and osteogenic capacity of hMSCs. Importantly, we demonstrated that hMSCs engineered to overexpress ITGA5 exhibited a marked increase in their osteogenic potential in vivo. Taken together, these findings not only reveal that ITGA5 is required for osteoblast differentiation of adult hMSCs but also provide a targeted strategy using ITGA5 agonists to promote the osteogenic capacity of hMSCs. This may be used for tissue regeneration in bone disorders where the recruitment or capacity of hMSCs is compromised.

Mesenchymal Stem Cells Promote the Osteogenesis in Collagen-Induced Arthritic Mice through the Inhibition of TNF-α

PubMed Central

Liu, Chang; Tang, Xiaojun; Feng, Ruihai; Yao, Genhong; Chen, Weiwei; Li, Wenchao; Liang, Jun; Feng, Xuebing

2018-01-01

Objective To investigate the effects of umbilical cord mesenchymal stem cell (UC-MSC) transplantation on joint damage and osteoporosis in collagen-induced arthritis (CIA) mice and to explore the mechanisms by which UC-MSCs modulate the osteogenic differentiation. Methods CIA mice were divided into the following treated groups: UC-MSC transplantation group, antitumor necrosis factor- (TNF-) α group, and zoledronic acid (ZA) group. Microcomputed tomography (micro-CT) was used to analyze the bone morphology parameters. Osteogenic differentiation of treated CIA mice was determined. Bone marrow mesenchymal stem cells (BM-MSCs) from CIA mice were treated with TNF-α in vitro to explore their effects on osteogenesis. Results The arthritis score was significantly reduced in the UC-MSC transplantation and anti-TNF-α-treated CIA groups, compared with control mice (P < 0.001). Micro-CT showed that CIA mice developed osteoporosis at 12 weeks after immunization. The bone morphology parameters were partially improved in UC-MSC-treated CIA mice. Impaired osteogenic differentiation functions were indicated by decreased ALP activity (P < 0.001) and reduced mRNA and protein levels of osteogenic marker genes (P < 0.05) in CIA mice compared with DBA/1 mice. UC-MSC treatment significantly upregulated the impaired osteogenic differentiation ability in CIA mice. Meanwhile, the serum TNF-α level was decreased significantly in the UC-MSC group. The osteogenesis was reduced with the addition of TNF-α in vitro. Conclusion This study demonstrated that UC-MSC transplantation not only significantly improved the joint damage but also played a beneficial role in osteoporosis in CIA mice. Mechanistically, the improved osteogenic differentiation of CIA under UC-MSC treatment may be achieved by inhibition of TNF-α. PMID:29853911

Bioreactor strategy in bone tissue engineering: pre-culture and osteogenic differentiation under two flow configurations.

PubMed

Kim, Junho; Ma, Teng

2012-11-01

Since robust osteogenic differentiation and mineralization are integral to the engineering of bone constructs, understanding the impact of the cellular microenvironments on human mesenchymal stem cell (hMSCs) osteogenic differentiation is crucial to optimize bioreactor strategy. Two perfusion flow conditions were utilized in order to understand the impact of the flow configuration on hMSC construct development during both pre-culture (PC) in growth media and its subsequent osteogenic induction (OI). The media in the in-house perfusion bioreactor was controlled to perfuse either around (termed parallel flow [PF]) the construct surfaces or penetrate through the construct (termed transverse flow [TF]) for 7 days of the PC followed by 7 days of the OI. The flow configuration during the PC not only changed growth kinetics but also influenced cell distribution and potency of osteogenic differentiation and mineralization during the subsequent OI. While shear stress resulted from the TF stimulated cell proliferation during PC, the convective removal of de novo extracellular matrix (ECM) proteins and growth factors (GFs) reduced cell proliferation on OI. In contrast, the effective retention of de novo ECM proteins and GFs in the PC constructs under the PF maintained cell proliferation under the OI but resulted in localized cell aggregations, which influenced their osteogenic differentiation. The results revealed the contrasting roles of the convective flow as a mechanical stimulus, the redistribution of the cells and macromolecules in 3D constructs, and their divergent impacts on cellular events, leading to bone construct formation. The results suggest that the modulation of the flow configuration in the perfusion bioreactor is an effective strategy that regulates the construct properties and maximizes the functional outcome.

Correlation between ECM guidance and actin polymerization on osteogenic differentiation of human adipose-derived stem cells

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

Keller, Vivian; Deiwick, Andrea; Pflaum, Michael

The correlation between extracellular matrix (ECM) components, cell shape, and stem cell guidance can shed light in understanding and mimicking the functionality of stem cell niches for various applications. This interplay on osteogenic guidance of human adipose-derived stem cells (hASCs) was focus of this study. Proliferation and osteogenic markers like alkaline phosphatase activity and calcium mineralization were slightly increased by the ECM components laminin (LA), collagen I (COL), and fibronectin (FIB); with control medium no differentiation occurred. ECM guided differentiation was rather dependent on osterix than on Runx2 pathway. FIB significantly enhanced cell elongation even in presence of actin polymerizationmore » blockers cytochalasin D (CytoD) and ROCK inhibitor Y-27632, which generally caused more rounded cells. Except for the COL surface, both inhibitors increased the extent of osterix, while the Runx2 pathway was more sensitive to the culture condition. Both inhibitors did not affect hASC proliferation. CytoD enabled osteogenic differentiation independently from the ECM, while it was rather blocked via Y-27632 treatment; on FIB the general highest extent of differentiation occurred. Taken together, the ECM effect on hASCs occurs indirectly and selectively via a dominant role of FIB: it sustains osteogenic differentiation in case of a tension-dependent control of actin polymerization. - Highlights: • Interplay of ECM and cell shape guides osteogenic differentiation of hASCs. • ECM components only present a promotive but not stimulative effect. • No direct correlation between ECM-enhanced cell elongation and differentiation. • Suppression of differentiation depends on a specific actin polymerization blocking. • Fibronectin sustains cell elongation and differentiation in case of blocking actin.« less

Distinct Effects of RGD-glycoproteins on Integrin-Mediated Adhesion and Osteogenic Differentiation of Human Mesenchymal Stem Cells

PubMed Central

Schwab, Elisabeth H.; Halbig, Maria; Glenske, Kristina; Wagner, Alena-Svenja; Wenisch, Sabine; Cavalcanti-Adam, Elisabetta A.

2013-01-01

The detailed interactions of mesenchymal stem cells (MSCs) with their extracellular matrix (ECM) and the resulting effects on MSC differentiation are still largely unknown. Integrins are the main mediators of cell-ECM interaction. In this study, we investigated the adhesion of human MSCs to fibronectin, vitronectin and osteopontin, three ECM glycoproteins which contain an integrin-binding sequence, the RGD motif. We then assayed MSCs for their osteogenic commitment in the presence of the different ECM proteins. As early as 2 hours after seeding, human MSCs displayed increased adhesion when plated on fibronectin, whereas no significant difference was observed when adhering either to vitronectin or osteopontin. Over a 10-day observation period, cell proliferation was increased when cells were cultured on fibronectin and osteopontin, albeit after 5 days in culture. The adhesive role of fibronectin was further confirmed by measurements of cell area, which was significantly increased on this type of substrate. However, integrin-mediated clusters, namely focal adhesions, were larger and more mature in MSCs adhering to vitronectin and osteopontin. Adhesion to fibronectin induced elevated expression of α5-integrin, which was further upregulated under osteogenic conditions also for vitronectin and osteopontin. In contrast, during osteogenic differentiation the expression level of β3-integrin was decreased in MSCs adhering to the different ECM proteins. When MSCs were cultured under osteogenic conditions, their commitment to the osteoblast lineage and their ability to form a mineralized matrix in vitro was increased in presence of fibronectin and osteopontin. Taken together these results indicate a distinct role of ECM proteins in regulating cell adhesion, lineage commitment and phenotype of MSCs, which is due to the modulation of the expression of specific integrin subunits during growth or osteogenic differentiation. PMID:24324361

Distinct effects of RGD-glycoproteins on Integrin-mediated adhesion and osteogenic differentiation of human mesenchymal stem cells.

PubMed

Schwab, Elisabeth H; Halbig, Maria; Glenske, Kristina; Wagner, Alena-Svenja; Wenisch, Sabine; Cavalcanti-Adam, Elisabetta A

2013-01-01

The detailed interactions of mesenchymal stem cells (MSCs) with their extracellular matrix (ECM) and the resulting effects on MSC differentiation are still largely unknown. Integrins are the main mediators of cell-ECM interaction. In this study, we investigated the adhesion of human MSCs to fibronectin, vitronectin and osteopontin, three ECM glycoproteins which contain an integrin-binding sequence, the RGD motif. We then assayed MSCs for their osteogenic commitment in the presence of the different ECM proteins. As early as 2 hours after seeding, human MSCs displayed increased adhesion when plated on fibronectin, whereas no significant difference was observed when adhering either to vitronectin or osteopontin. Over a 10-day observation period, cell proliferation was increased when cells were cultured on fibronectin and osteopontin, albeit after 5 days in culture. The adhesive role of fibronectin was further confirmed by measurements of cell area, which was significantly increased on this type of substrate. However, integrin-mediated clusters, namely focal adhesions, were larger and more mature in MSCs adhering to vitronectin and osteopontin. Adhesion to fibronectin induced elevated expression of α₅-integrin, which was further upregulated under osteogenic conditions also for vitronectin and osteopontin. In contrast, during osteogenic differentiation the expression level of β₃-integrin was decreased in MSCs adhering to the different ECM proteins. When MSCs were cultured under osteogenic conditions, their commitment to the osteoblast lineage and their ability to form a mineralized matrix in vitro was increased in presence of fibronectin and osteopontin. Taken together these results indicate a distinct role of ECM proteins in regulating cell adhesion, lineage commitment and phenotype of MSCs, which is due to the modulation of the expression of specific integrin subunits during growth or osteogenic differentiation.

BMP-2 Derived Peptide and Dexamethasone Incorporated Mesoporous Silica Nanoparticles for Enhanced Osteogenic Differentiation of Bone Mesenchymal Stem Cells.

PubMed

Zhou, Xiaojun; Feng, Wei; Qiu, Kexin; Chen, Liang; Wang, Weizhong; Nie, Wei; Mo, Xiumei; He, Chuanglong

2015-07-29

Bone morphogenetic protein-2 (BMP-2), a growth factor that induces osteoblast differentiation and promotes bone regeneration, has been extensively investigated in bone tissue engineering. The peptides of bioactive domains, corresponding to residues 73-92 of BMP-2 become an alternative to reduce adverse side effects caused by the use of high doses of BMP-2 protein. In this study, BMP-2 peptide functionalized mesoporous silica nanoparticles (MSNs-pep) were synthesized by covalently grafting BMP-2 peptide on the surface of nanoparticles via an aminosilane linker, and dexamethasone (DEX) was then loaded into the channel of MSNs to construct nanoparticulate osteogenic delivery systems (DEX@MSNs-pep). The in vitro cell viability of MSNs-pep was tested with bone mesenchymal stem cells (BMSCs) exposure to different particle concentrations, revealing that the functionalized MSNs had better cytocompatibility than their bare counterparts, and the cellular uptake efficiency of MSNs-pep was remarkably larger than that of bare MSNs. The in vitro results also show that the MSNs-pep promoted osteogenic differentiation of BMSCs in terms of the levels of alkaline phosphatase (ALP) activity, calcium deposition, and expression of bone-related protein. Moreover, the osteogenic differentiation of BMSCs can be further enhanced by incorporating of DEX into MSNs-pep. After intramuscular implantation in rats for 3 weeks, the computed tomography (CT) images and histological examination indicate that this nanoparticulate osteogenic delivery system induces effective osteoblast differentiation and bone regeneration in vivo. Collectively, the BMP-2 peptide and DEX incorporated MSNs can act synergistically to enhance osteogenic differentiation of BMSCs, which have potential applications in bone tissue engineering.

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.

Cementogenic genes in human periodontal ligament stem cells are downregulated in response to osteogenic stimulation while upregulated by vitamin C treatment

PubMed Central

Gauthier, Philippe; Yu, Zongdong; Tran, Quynh T.; Bhatti, Fazal-Ur-Rehman; Zhu, Xiaofei

2016-01-01

Regeneration of periodontal tissues, particularly cementum, is key to regaining periodontal attachment and health. Human periodontal ligament stem cells (hPDLSCs) have been shown to be a good cell source to regenerate periodontal tissues. However, their subpopulations and the differentiation induction in relation to cementogenic lineages is unclear. Thus, we aim to examine the expression of cementum-associated genes in PDLSC subpopulations and determine the effect of broadly used osteogenic stimulus or vitamin C (VC) on the expression of cementogenic and osteogenic genes in PDLSCs. Our real-time quantitative polymerase chain reaction (qPCR) analysis showed that cementogenic marker cementum attachment protein (CAP) expressed only slightly higher in STRO-1+/CD146+, STRO-1−/CD146+ and STRO-1−/CD146− subpopulations than in the original cell pool, while cementum protein 1 (CEMP1) expression in these subpopulations was not different from the original pool. Notably, under the stimulation with osteogenic differentiation medium, CAP and CEMP1 were down-regulated while osteogenic markers bone sialoprotein (BSP) and osteocalcin (OCN) were upregulated. Both CAP and CEMP1 were upregulated by VC treatment. Transplantation of VC-treated PDLSCs into immunocompromised mice resulted in forming significantly more ectopic cementum- and bone-like mineral tissues in vivo. Immunohistochemical analysis of the ectopic growth showed that CAP and CEMP1 were mainly expressed in the mineral tissue and in some cells of the fibrous tissues. We conclude that osteogenic stimulation is not inductive but appears to be inhibitory of cementogenic pathways, whereas VC induces cementogenic lineage commitment by PDLSCs and may be a useful stimulus for cementogenesis in periodontal regeneration. PMID:27757536

Cementogenic genes in human periodontal ligament stem cells are downregulated in response to osteogenic stimulation while upregulated by vitamin C treatment.

PubMed

Gauthier, Philippe; Yu, Zongdong; Tran, Quynh T; Bhatti, Fazal-Ur-Rehman; Zhu, Xiaofei; Huang, George T-J

2017-04-01

Regeneration of periodontal tissues, particularly cementum, is key to regaining periodontal attachment and health. Human periodontal ligament stem cells (hPDLSCs) have been shown to be a good cell source to regenerate periodontal tissues. However, their subpopulations and the differentiation induction in relation to cementogenic lineages is unclear. Thus, we aim to examine the expression of cementum-associated genes in PDLSC subpopulations and determine the effect of broadly used osteogenic stimulus or vitamin C (VC) on the expression of cementogenic and osteogenic genes in PDLSCs. Our real-time quantitative polymerase chain reaction (qPCR) analysis showed that cementogenic marker cementum attachment protein (CAP) expressed only slightly higher in STRO-1 + /CD146 + , STRO-1 - /CD146 + and STRO-1 - /CD146 - subpopulations than in the original cell pool, while cementum protein 1 (CEMP1) expression in these subpopulations was not different from the original pool. Notably, under the stimulation with osteogenic differentiation medium, CAP and CEMP1 were downregulated while osteogenic markers bone sialoprotein (BSP) and osteocalcin (OCN) were upregulated. Both CAP and CEMP1 were upregulated by VC treatment. Transplantation of VC-treated PDLSCs into immunocompromised mice resulted in forming significantly more ectopic cementum- and bone-like mineral tissues in vivo. Immunohistochemical analysis of the ectopic growth showed that CAP and CEMP1 were mainly expressed in the mineral tissue and in some cells of the fibrous tissues. We conclude that osteogenic stimulation is not inductive but appears to be inhibitory of cementogenic pathways, whereas VC induces cementogenic lineage commitment by PDLSCs and may be a useful stimulus for cementogenesis in periodontal regeneration.

Heparin-binding EGF-like growth factor and miR-1192 exert opposite effect on Runx2-induced osteogenic differentiation.

PubMed

Yu, S; Geng, Q; Ma, J; Sun, F; Yu, Y; Pan, Q; Hong, A

2013-10-17

Osteoblast differentiation is a pivotal event in bone formation. Runt-related transcription factor-2 (Runx2) is an essential factor required for osteoblast differentiation and bone formation. However, the underlying mechanism of Runx2-regulated osteogenic differentiation is still unclear. Here, we explored the corresponding mechanism using the C2C12/Runx2(Dox) subline, which expresses Runx2 in response to doxycycline (Dox). We found that Runx2-induced osteogenic differentiation of C2C12 cells results in a sustained decrease in the expression of heparin-binding EGF-like growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family. Forced expression of HB-EGF or treatment with HB-EGF is capable of reducing the expression of alkaline phosphatase (ALP), a defined marker of early osteoblast differentiation. HB-EGF-mediated inhibition of ALP depends upon activation of the EGFR and the downstream extracellular signal-regulated kinase, c-Jun N-terminal kinase mitogen-activated protein kinase pathways as well as phosphatidylinositol 3-kinase/Akt pathway. Runx2 specifically binds to the Hbegf promoter, suggesting that Hbegf transcription is directly inhibited by Runx2. Runx2 can upregulate miR-1192, which enhances Runx2-induced osteogenic differentiation. Moreover, miR-1192 directly targets Hbegf through translational inhibition, suggesting enhancement of Runx2-induced osteogenic differentiation by miR-1192 through the downregulation of HB-EGF. Taken together, our results suggest that Runx2 induces osteogenic differentiation of C2C12 cells by inactivating HB-EGF-EGFR signaling through the downregulation of HB-EGF via both transcriptional and post-transcriptional mechanisms.

Heparin-binding EGF-like growth factor and miR-1192 exert opposite effect on Runx2-induced osteogenic differentiation

PubMed Central

Yu, S; Geng, Q; Ma, J; Sun, F; Yu, Y; Pan, Q; Hong, A

2013-01-01

Osteoblast differentiation is a pivotal event in bone formation. Runt-related transcription factor-2 (Runx2) is an essential factor required for osteoblast differentiation and bone formation. However, the underlying mechanism of Runx2-regulated osteogenic differentiation is still unclear. Here, we explored the corresponding mechanism using the C2C12/Runx2Dox subline, which expresses Runx2 in response to doxycycline (Dox). We found that Runx2-induced osteogenic differentiation of C2C12 cells results in a sustained decrease in the expression of heparin-binding EGF-like growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family. Forced expression of HB-EGF or treatment with HB-EGF is capable of reducing the expression of alkaline phosphatase (ALP), a defined marker of early osteoblast differentiation. HB-EGF-mediated inhibition of ALP depends upon activation of the EGFR and the downstream extracellular signal-regulated kinase, c-Jun N-terminal kinase mitogen-activated protein kinase pathways as well as phosphatidylinositol 3-kinase/Akt pathway. Runx2 specifically binds to the Hbegf promoter, suggesting that Hbegf transcription is directly inhibited by Runx2. Runx2 can upregulate miR-1192, which enhances Runx2-induced osteogenic differentiation. Moreover, miR-1192 directly targets Hbegf through translational inhibition, suggesting enhancement of Runx2-induced osteogenic differentiation by miR-1192 through the downregulation of HB-EGF. Taken together, our results suggest that Runx2 induces osteogenic differentiation of C2C12 cells by inactivating HB-EGF-EGFR signaling through the downregulation of HB-EGF via both transcriptional and post-transcriptional mechanisms. PMID:24136232

Osteogenic Performance of Donor-Matched Human Adipose and Bone Marrow Mesenchymal Cells Under Dynamic Culture

PubMed Central

Wu, Wei; Le, Andrew V.; Mendez, Julio J.; Chang, Julie; Niklason, Laura E.

2015-01-01

Adipose-derived mesenchymal cells (ACs) and bone marrow-derived mesenchymal cells (BMCs) have been widely used for bone regeneration and can be seeded on a variety of rigid scaffolds. However, to date, a direct comparison of mesenchymal cells (MC) harvested from different tissues from the same donor and cultured in identical osteogenic conditions has not been investigated. Indeed, it is unclear whether marrow-derived or fat-derived MC possess intrinsic differences in bone-forming capabilities, since within-patient comparisons have not been previously done. This study aims at comparing ACs and BMCs from three donors ranging in age from neonatal to adult. Matched cells from each donor were studied in three distinct bioreactor settings, to determine the best method to create a viable osseous engineered construct. Human ACs and BMCs were isolated from each donor, cultured, and seeded on decellularized porcine bone (DCB) constructs. The constructs were then subjected to either static or dynamic (stirring or perfusion) bioreactor culture conditions for 7–21 days. Afterward, the constructs were analyzed for cell adhesion and distribution and osteogenic differentiation. ACs demonstrated higher seeding efficiency than BMCs. However, static and dynamic culture significantly increased BMCs proliferation more than ACs. In all conditions, BMCs demonstrated stronger osteogenic activity as compared with ACs, through higher alkaline phosphatase activity and gene expression for various bony markers. Conversely, ACs expressed more collagen I, which is a nonspecific matrix molecule in most connective tissues. Overall, dynamic bioreactor culture conditions enhanced osteogenic gene expression in both ACs and BMCs. Scaffolds seeded with BMCs in dynamic stirring culture conditions exhibit the greatest osteogenic proliferation and function in vitro, proving that marrow-derived MC have superior bone-forming potential as compared with adipose-derived cells. PMID:25668104

The transcription factor cyclic adenosine 3',5'-monophosphate response element-binding protein enhances the odonto/osteogenic differentiation of stem cells from the apical papilla.

PubMed

Su, S; Zhu, Y; Li, S; Liang, Y; Zhang, J

2017-09-01

To investigate the role of cAMP response element-binding protein (CREB) in the regulation of odonto/osteogenic differentiation of stem cells from the apical papilla (SCAPs). Stem cells from the apical papilla were obtained from human impacted third molars (n = 15). Isolated SCAPs were transfected with CREB overexpressing/silenced lentivirus. Transfected cells were stained with alizarin red to investigate mineralized nodule formation. The expression of the mineralization-related genes, alkaline phosphatase (ALP), collagen type I (Col I), runt-related transcription factor 2 (RUNX2), osterix (OSX) and osteocalcin (OCN), was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Protein expression of the odontogenic-related marker dentine sialoprotein (DSP) and the osteogenic-related marker RUNX2 was measured by Western blotting analysis. One-way analysis of variance (anova) and Student's t-test were used for statistical analysis (a = 0.05). The overexpression of CREB enhanced mineralized nodule formation and up-regulated (P < 0.05) the mRNA levels of odonto/osteogenic-related markers, including ALP, Col I, RUNX2, OSX and OCN, and also increased (P < 0.05) the protein expression of DSP and RUNX2. In contrast, the silencing of CREB inhibited (P < 0.05) the mineralization capacity of the SCAPs and decreased (P < 0.05) the expression of odonto/osteogenic-related markers. Up-regulation of CREB expression promoted odonto/osteogenic differentiation of SCAPs and provided a potential method for the regeneration of the dentine-pulp complex. © 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Ectopic Hard Tissue Formation by Odonto/Osteogenically In Vitro Differentiated Human Deciduous Teeth Pulp Stem Cells.

PubMed

Kim, Seunghye; Song, Je Seon; Jeon, Mijeong; Shin, Dong Min; Kim, Seong-Oh; Lee, Jae Ho

2015-07-01

There have been many attempts to use the pulp tissue from human deciduous teeth for dentin or bone regeneration. The objective of this study was to determine the effects of odonto/osteogenic in vitro differentiation of deciduous teeth pulp stem cells (DTSCs) on their in vivo hard tissue-forming potential. DTSCs were isolated from extracted deciduous teeth using the outgrowth method. These cells were exposed to odonto/osteogenic stimuli for 4 and 8 days (Day 4 and Day 8 groups, respectively), while cells in the control group were cultured in normal medium. The in vitro differentiated DTSCs and the control DTSCs were transplanted subcutaneously into immunocompromised mice with macroporous biphasic calcium phosphate and sacrificed at 8 weeks post-implantation. The effect of odonto/osteogenic in vitro differentiation was evaluated using alkaline phosphatase (ALP) staining and quantitative reverse transcription polymerase chain reaction (RT-PCR). The in vivo effect was evaluated by qualitative RT-PCR, assessment of ALP activity, histologic analysis, and immunohistochemical staining. The amount of hard tissue was greater in Day 4 group than Day 8 group (p = 0.014). However, Day 8 group generated lamellar bone-like structure, which was immunonegative to anti-human dentin sialoprotein with significantly low expression level of DSPP compared with the control group (p = 0.008). This study demonstrates that odonto/osteogenic in vitro differentiation of DTSCs enhances the formation of bone-like tissue, instead of dentin-like tissue, when transplanted subcutaneously using MBCP as a carrier. The odonto/osteogenic in vitro differentiation of DTSCs may be an effective modification that enhances in vivo bone formation by DTSCs.

17beta-estradiol promotes the odonto/osteogenic differentiation of stem cells from apical papilla via mitogen-activated protein kinase pathway.

PubMed

Li, Yao; Yan, Ming; Wang, Zilu; Zheng, Yangyu; Li, Junjun; Ma, Shu; Liu, Genxia; Yu, Jinhua

2014-11-17

Estrogen plays an important role in the osteogenic differentiation of mesenchymal stem cells, while stem cells from apical papilla (SCAP) can contribute to the formation of dentin/bone-like tissues. To date, the effects of estrogen on the differentiation of SCAP remain unclear. SCAP was isolated and treated with 10⁻⁷ M 17beta-estradiol (E2). The odonto/osteogenic potency and the involvement of mitogen-activated protein kinase (MAPK) signaling pathway were subsequently investigated by using methyl-thiazolyl-tetrazolium (MTT) assay, and other methods. MTT and flow cytometry results demonstrated that E2 treatment had no effect on the proliferation of SCAP in vitro, while alkaline phosphatase (ALP) assay and alizarin red staining showed that E2 can significantly promote ALP activity and mineralization ability in SCAP. Real-time reverse transcription polymerase chain reaction (RT-PCR) and western blot assay revealed that the odonto/osteogenic markers (ALP, DMP1/DMP1, DSPP/DSP, RUNX2/RUNX2, OSX/OSX and OCN/OCN) were significantly upregulated in E2-treated SCAP. In addition, the expression of phosphor-p38 and phosphor-JNK in these stem cells was enhanced by E2 treatment, as was the expression of the nuclear downstream transcription factors including phosphor-Sp1, phosphor-Elk-1, phosphor-c-Jun and phosphor-c-Fos, indicating the activation of MAPK signaling pathway during the odonto/osteogenic differentiation of E2-treated SCAP. Conversely, the differentiation of E2-treated SCAP was inhibited in the presence of MAPK specific inhibitors. The ondonto/osteogenic differentiation of SCAP is enhanced by 10⁻⁷ M 17beta-estradiol via the activation of MAPK signaling pathway.

Impact of bacteria and bacterial components on osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells

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

Fiedler, Tomas, E-mail: tomas.fiedler@med.uni-rostock.de; Salamon, Achim; Adam, Stefanie

Adult mesenchymal stem cells (MSC) are present in several tissues, e.g. bone marrow, heart muscle, brain and subcutaneous adipose tissue. In invasive infections MSC get in contact with bacteria and bacterial components. Not much is known about how bacterial pathogens interact with MSC and how contact to bacteria influences MSC viability and differentiation potential. In this study we investigated the impact of three different wound infection relevant bacteria, Escherichia coli, Staphylococcus aureus, and Streptococcus pyogenes, and the cell wall components lipopolysaccharide (LPS; Gram-negative bacteria) and lipoteichoic acid (LTA; Gram-positive bacteria) on viability, proliferation, and osteogenic as well as adipogenic differentiationmore » of human adipose tissue-derived mesenchymal stem cells (adMSC). We show that all three tested species were able to attach to and internalize into adMSC. The heat-inactivated Gram-negative E. coli as well as LPS were able to induce proliferation and osteogenic differentiation but reduce adipogenic differentiation of adMSC. Conspicuously, the heat-inactivated Gram-positive species showed the same effects on proliferation and adipogenic differentiation, while its cell wall component LTA exhibited no significant impact on adMSC. Therefore, our data demonstrate that osteogenic and adipogenic differentiation of adMSC is influenced in an oppositional fashion by bacterial antigens and that MSC-governed regeneration is not necessarily reduced under infectious conditions. - Highlights: • Staphylococcus aureus, Streptococcus pyogenes and Escherichia coli bind to and internalize into adMSC. • Heat-inactivated cells of these bacterial species trigger proliferation of adMSC. • Heat-inactivated E. coli and LPS induce osteogenic differentiation of adMSC. • Heat-inactivated E. coli and LPS reduce adipogenic differentiation of adMSC. • LTA does not influence adipogenic or osteogenic differentiation of adMSC.« less

MiR-133 is Involved in Estrogen Deficiency-Induced Osteoporosis through Modulating Osteogenic Differentiation of Mesenchymal Stem Cells.

PubMed

Lv, Hao; Sun, Yujie; Zhang, Yuchen

2015-05-27

MiR-133 expression is dysregulated in postmenopausal osteoporosis. However, its role in postmenopausal osteoporosis is still not well understood. In the current study, we explore how estrogen deficiency affects miR-133 expression and how miR-133 is involved in osteogenic differentiation of mesenchymal stem cells (MSCs). qRT-PCR analysis was performed to assess miR-133 expression in MSCs isolated from bone marrow of an ovariectomized (OVX) animal model and postmenopausal osteoporosis patients (PMOP) and their corresponding controls. The binding between miR-133 and predicted target SLC39A1 was verified using dual luciferase assay and Western blot analysis. The effect of miR-133 and SLC39A1 on osteogenic differentiation of MSCs was assessed through measuring alkaline phosphatase (ALP), mineralization nodules, and osteoblast-specific genes Runx2 and Osterix expression. miR-133 expression is significantly enhanced as a result of estrogen deficiency. Its overexpression is negatively correlated to osteogenic differentiation of hMSCs. SLC39A1 showed an inverse expression trend to miR-133 during the differentiation. miR-133 can directly target 3'UTR of SLC39A1 and thereby modulate its expression in hMSCs. The miR-133-SLC39A1 axis might play an important role in osteogenic differentiation of hMSCs. SLC39A1 can promote ALP activity and formation of mineralization nodules. In addition, SLC39A1 expression level is also positively correlated with RUNX2 and Osterix. Estrogen deficiency is associated with miR-133 overexpression. MiR-133 can induce postmenopausal osteoporosis by weakening osteogenic differentiation of hMSCs, at least partly through repressing SLC39A1 expression.

MiR-133 is Involved in Estrogen Deficiency-Induced Osteoporosis through Modulating Osteogenic Differentiation of Mesenchymal Stem Cells

PubMed Central

Lv, Hao; Sun, Yujie; Zhang, Yuchen

2015-01-01

Background MiR-133 expression is dysregulated in postmenopausal osteoporosis. However, its role in postmenopausal osteoporosis is still not well understood. In the current study, we explore how estrogen deficiency affects miR-133 expression and how miR-133 is involved in osteogenic differentiation of mesenchymal stem cells (MSCs). Material/Methods qRT-PCR analysis was performed to assess miR-133 expression in MSCs isolated from bone marrow of an ovariectomized (OVX) animal model and postmenopausal osteoporosis patients (PMOP) and their corresponding controls. The binding between miR-133 and predicted target SLC39A1 was verified using dual luciferase assay and Western blot analysis. The effect of miR-133 and SLC39A1 on osteogenic differentiation of MSCs was assessed through measuring alkaline phosphatase (ALP), mineralization nodules, and osteoblast-specific genes Runx2 and Osterix expression. Results miR-133 expression is significantly enhanced as a result of estrogen deficiency. Its overexpression is negatively correlated to osteogenic differentiation of hMSCs. SLC39A1 showed an inverse expression trend to miR-133 during the differentiation. miR-133 can directly target 3′UTR of SLC39A1 and thereby modulate its expression in hMSCs. The miR-133-SLC39A1 axis might play an important role in osteogenic differentiation of hMSCs. SLC39A1 can promote ALP activity and formation of mineralization nodules. In addition, SLC39A1 expression level is also positively correlated with RUNX2 and Osterix. Conclusions Estrogen deficiency is associated with miR-133 overexpression. MiR-133 can induce postmenopausal osteoporosis by weakening osteogenic differentiation of hMSCs, at least partly through repressing SLC39A1 expression. PMID:26013661

Interleukin-37 suppresses the osteogenic responses of human aortic valve interstitial cells in vitro and alleviates valve lesions in mice.

PubMed

Zeng, Qingchun; Song, Rui; Fullerton, David A; Ao, Lihua; Zhai, Yufeng; Li, Suzhao; Ballak, Dov B; Cleveland, Joseph C; Reece, T Brett; McKinsey, Timothy A; Xu, Dingli; Dinarello, Charles A; Meng, Xianzhong

2017-02-14

Calcific aortic valve disease is a chronic inflammatory process, and aortic valve interstitial cells (AVICs) from diseased aortic valves express greater levels of osteogenic factors in response to proinflammatory stimulation. Here, we report that lower cellular levels of IL-37 in AVICs of diseased human aortic valves likely account for augmented expression of bone morphogenetic protein-2 (BMP-2) and alkaline phosphatase (ALP) following stimulation of Toll-like receptor (TLR) 2 or 4. Treatment of diseased AVICs with recombinant human IL-37 suppresses the levels of BMP-2 and ALP as well as calcium deposit formation. In mice, aortic valve thickening is observed when exposed to a TLR4 agonist or a high fat diet for a prolonged period; however, mice expressing human IL-37 exhibit significantly lower BMP-2 levels and less aortic valve thickening when subjected to the same regimens. A high fat diet in mice results in oxidized low-density lipoprotein (oxLDL) deposition in aortic valve leaflets. Moreover, the osteogenic responses in human AVICs induced by oxLDL are suppressed by recombinant IL-37. Mechanistically, reduced osteogenic responses to oxLDL in human AVICs are associated with the ability of IL-37 to inhibit NF-κB and ERK1/2. These findings suggest that augmented expression of osteogenic factors in AVICs of diseased aortic valves from humans is at least partly due to a relative IL-37 deficiency. Because recombinant IL-37 suppresses the osteogenic responses in human AVICs and alleviates aortic valve lesions in mice exposed to high fat diet or a proinflammatory stimulus, IL-37 has therapeutic potential for progressive calcific aortic valve disease.

Interleukin-37 suppresses the osteogenic responses of human aortic valve interstitial cells in vitro and alleviates valve lesions in mice

PubMed Central

Zeng, Qingchun; Song, Rui; Fullerton, David A.; Ao, Lihua; Zhai, Yufeng; Li, Suzhao; Ballak, Dov B.; Cleveland, Joseph C.; Reece, T. Brett; McKinsey, Timothy A.; Xu, Dingli; Dinarello, Charles A.; Meng, Xianzhong

2017-01-01

Calcific aortic valve disease is a chronic inflammatory process, and aortic valve interstitial cells (AVICs) from diseased aortic valves express greater levels of osteogenic factors in response to proinflammatory stimulation. Here, we report that lower cellular levels of IL-37 in AVICs of diseased human aortic valves likely account for augmented expression of bone morphogenetic protein-2 (BMP-2) and alkaline phosphatase (ALP) following stimulation of Toll-like receptor (TLR) 2 or 4. Treatment of diseased AVICs with recombinant human IL-37 suppresses the levels of BMP-2 and ALP as well as calcium deposit formation. In mice, aortic valve thickening is observed when exposed to a TLR4 agonist or a high fat diet for a prolonged period; however, mice expressing human IL-37 exhibit significantly lower BMP-2 levels and less aortic valve thickening when subjected to the same regimens. A high fat diet in mice results in oxidized low-density lipoprotein (oxLDL) deposition in aortic valve leaflets. Moreover, the osteogenic responses in human AVICs induced by oxLDL are suppressed by recombinant IL-37. Mechanistically, reduced osteogenic responses to oxLDL in human AVICs are associated with the ability of IL-37 to inhibit NF-κB and ERK1/2. These findings suggest that augmented expression of osteogenic factors in AVICs of diseased aortic valves from humans is at least partly due to a relative IL-37 deficiency. Because recombinant IL-37 suppresses the osteogenic responses in human AVICs and alleviates aortic valve lesions in mice exposed to high fat diet or a proinflammatory stimulus, IL-37 has therapeutic potential for progressive calcific aortic valve disease. PMID:28137840

Rest Intervals Reduce the Number of Loading Bouts Required to Enhance Bone Formation

PubMed Central

Srinivasan, Sundar; Ausk, Brandon J.; Bain, Steven D.; Gardiner, Edith M.; Kwon, Ronald Y.; Gross, Ted S.

2015-01-01

Purpose As our society becomes increasingly sedentary, compliance with exercise regimens that require numerous high-energy activities each week become less likely. Alternatively, given an osteogenic exercise intervention that required minimal effort, it is reasonable to presume that participation would be enhanced. Insertion of brief rest-intervals between each cycle of mechanical loading holds potential to achieve this result as substantial osteoblast function is activated by many fewer loading repetitions within each loading bout. Here, we examined the complementary hypothesis that the number of bouts/wk of rest-inserted loading could be reduced from 3/wk without loss of osteogenic efficacy. Methods We conducted a series of 3 wk in vivo experiments that non-invasively exposed the right tibiae of mice to either cyclic (1 Hz) or rest-inserted loading interventions and quantified osteoblast function via dynamic histomorphometry. Results While reducing loading bouts from 3/wk (i.e., 9 total bouts) to 1/wk (3 total bouts) effectively mitigated the osteogenic benefit of cyclic loading, the same reduction did not significantly reduce periosteal bone formation parameters induced by rest-inserted loading. The osteogenic response was robust to the timing of the rest-inserted loading bouts (3 bouts in the first week vs 1 bout/wk for three weeks). However, elimination of any single bout of the three 1/wk bouts mitigated the osteogenic response to rest-inserted loading. Finally, periosteal osteoblast function assessed after the 3 wk intervention was not sensitive to the timing or number of rest-inserted loading bouts. Conclusions We conclude that rest-inserted loading holds potential to retain the osteogenic benefits of mechanical loading with significantly reduced frequency of bouts of activity while also enabling greater flexibility in the timing of the activity. PMID:25207932

Solar concentrator

NASA Technical Reports Server (NTRS)

Simpson, J. G. (Inventor)

1979-01-01

An improved solar concentrator is characterized by a number of elongated supporting members arranged in substantial horizontal parallelism with the axis and intersecting a common curve. A tensioned sheet of flexible reflective material is disposed in engaging relation with the supporting members in order to impart to the tensioned sheet a catenary configuration.

Plasticity of mesenchymal stem cells under microgravity: from cytoskeletal reorganization to commitment shift

NASA Astrophysics Data System (ADS)

Buravkova, Ludmila

Mesenchymal stem cells (MSCs) can be used to examine osteogenesis of uncommitted cells maintaining the bone differentiation potential such as osteogenic gene expression, osteogenic markers, matrix maturation and mineralization. MSCs are therefore a good model for studying osteogenesis in the space environment. Recent investigations have demonstrated that MSCs change in response to microgravity and, consequently, can be involved in the development of osteopenia detected in space travelers. This is a factor that can limit human space missions due to potential risks of osteoporosis and its aftereffects during and after flight. Simulated microgravity inhibited MSC differentiation towards osteoblasts and accelerated adipocyte development due to cytoskeleton modifications, including its structure and regulation associated with signal transduction cascades. We identified transient changes in the actin cytoskeleton of non-committed human bone marrow MSCs in short-term RPM experiments. In addition, we detected transient changes in the expression of genes encoding actin cytoskeleton proteins and associated elements (ACTA1, ACTG, RHOA, CFL1, VCL). When discussing the microgravity effects on MSC osteogenic differentiation, it should be mentioned the inhibition of Runx2 and ALPL and stimulation of PPARg2 in the MSCs induced for osteogenesis. It is probable that the reciprocal regulation of the two transcription factors is a molecular mechanism underlying progenitor cell response to microgravity. It is very likely that these genes are involved in the universal circuits within which mechanical (or gravity ) signals are sensed by MSCs. Recently, the list of osteogenic markers was extended to include several new proteins as microgravity targets (proteoglycans, osteomodulin, osteoglycin). It can be believed that exposure to microgravity produces similar effects on mature bone cells (osteoblasts) and non-committed osteogenic cells (MSCs). This finds a support in the fact that terminal differentiation stages, i.e., bone matrix mineralization, are inhibited to the same extent in both osteoblasts and MSCs. When examining gravity-dependent molecular processes responsible for susceptibility and/or adaptation of progenitor cells to microgravity, it is important to concentrate not only on recognized pathways of signal transduction, such as MAPK-kinase and cytoskeleton kinase, but also on the expression pattern of genes, which are allegedly not directly involved in the MSC differentiation. Progenitor cells change their transcriptomic profile in the course of their growth, differentiation and maturation It is important to take into account the fact that MSCs can display their differentiation potential as a result of up- or down-regulation of associated or independent genes or their groups. Any interference in this process may cause significant changes in MSC metabolism and commitment. Although the number of relevant studies is much smaller than that of investigations into the typical markers of MSC differentiation in microgravity, there are publications suggesting that the pattern of MSC gene expression undergoes changes when exposed to microgravity. Our RPM experiments with human MSCs revealed significant changes in the so-called stem cell markers: up-regulation of genes associated with cell proliferation, adhesion and intracellular signaling and down-regulation of genes, most of which are involved in cell differentiation. In spite of significant progress achieved in our understanding of the cell gravitational biology, we, however, need to gain better insight into the specific molecular mechanisms underlying the susceptibility of MSCs and more committed osteogenic precursor cells to microgravity effects in vivo and in vitro. A comprehensive study of the biology of these cells is of particular importance in view of the fact that at present age- and drug-related osteoporosis has transformed into a major medical and social problem. This work was supported by grant NSc #371.2014.4

Regeneration of dental pulp/dentine complex with a three-dimensional and scaffold-free stem-cell sheet-derived pellet.

PubMed

Na, Sijia; Zhang, Hao; Huang, Fang; Wang, Weiqi; Ding, Yin; Li, Dechao; Jin, Yan

2016-03-01

Dental pulp/dentine complex regeneration is indispensable to the construction of biotissue-engineered tooth roots and represents a promising approach to therapy for irreversible pulpitis. We used a tissue-engineering method based on odontogenic stem cells to design a three-dimensional (3D) and scaffold-free stem-cell sheet-derived pellet (CSDP) with the necessary physical and biological properties. Stem cells were isolated and identified and stem cells from root apical papilla (SCAPs)-based CSDPs were then fabricated and examined. Compact cell aggregates containing a high proportion of extracellular matrix (ECM) components were observed, and the CSDP culture time was prolonged. The expression of alkaline phosphatase (ALP), dentine sialoprotein (DSPP), bone sialoprotein (BSP) and runt-related gene 2 (RUNX2) mRNA was higher in CSDPs than in cell sheets (CSs), indicating that CSDPs have greater odonto/osteogenic potential. To further investigate this hypothesis, CSDPs and CSs were inserted into human treated dentine matrix fragments (hTDMFs) and transplanted into the subcutaneous space in the backs of immunodeficient mice, where they were cultured in vivo for 6 weeks. The root space with CSDPs was filled entirely with a dental pulp-like tissue with well-established vascularity, and a continuous layer of dentine-like tissue was deposited onto the existing dentine. A layer of odontoblast-like cells was found to express DSPP, ALP and BSP, and human mitochondria lined the surface of the newly formed dentine-like tissue. These results clearly indicate that SCAP-CSDPs with a mount of endogenous ECM have a strong capacity to form a heterotopic dental pulp/dentine complex in empty root canals; this method can be used in the fabrication of bioengineered dental roots and also provides an alternative treatment approach for pulp disease. Copyright © 2013 John Wiley & Sons, Ltd.

Filter unit for use at high temperatures

DOEpatents

Ciliberti, David F.; Lippert, Thomas E.

1988-01-01

A filtering unit for filtering particulates from high temperature gases uses a spiral ceramic spring to bias a ceramic, tubular filter element into sealing contact with a flange about an aperture of a metallic tube sheet. The ceramic spiral spring may contact the upper edge of the filter element and be restrained by a stop member spaced from one end of the tube sheet, or the spring may contact the bottom of the filter element and be restrained by a support member spaced from the opposite end of the tube sheet. The stop member and support member are adjustably secured to the tube sheet. A filtering system uses the ceramic spiral spring to bias a plurality of ceramic, tubular filter elements in a respective plurality of apertures in a tube sheet which divides a vessel into upper and lower enclosed sections.

MIR146A inhibits JMJD3 expression and osteogenic differentiation in human mesenchymal stem cells

PubMed Central

Huszar, Jessica M.; Payne, Christopher J.

2014-01-01

Chromatin remodeling is important for cell differentiation. Histone methyltransferase EZH2 and histone demethylase JMJD3 (KDM6B) modulate levels of histone H3 lysine 27 trimethylation (H3K27me3). Interplay between the two modulators influence lineage specification in stem cells. Here, we identified microRNA MIR146A to be a negative regulator of JMJD3. In the osteogenic differentiation of human mesenchymal stem cells (hMSCs), we observed an upregulation of JMJD3 and a downregulation of MIR146A. Blocking JMJD3 activity in differentiating hMSCs reduced transcript levels of osteogenic gene RUNX2. H3K27me3 levels decreased at the RUNX2 promoter during cell differentiation. Modulation of MIR146A levels in hMSCs altered JMJD3 and RUNX2 expression and affected osteogenic differentiation. We conclude that JMJD3 promotes osteogenesis in differentiating hMSCs, with MIR146A regulating JMJD3. PMID:24726732

A bioactive triphasic ceramic-coated hydroxyapatite promotes proliferation and osteogenic differentiation of human bone marrow stromal cells.

PubMed

Nair, Manitha B; Bernhardt, Anne; Lode, Anja; Heinemann, Christiane; Thieme, Sebastian; Hanke, Thomas; Varma, Harikrishna; Gelinsky, Michael; John, Annie

2009-08-01

Hydroxyapatite (HA) ceramics are widely used as bone graft substitutes because of their biocompatibility and osteoconductivity. However, to enhance the success of therapeutic application, many efforts are undertaken to improve the bioactivity of HA. We have developed a triphasic, silica-containing ceramic-coated hydroxyapatite (HASi) and evaluated its performance as a scaffold for cell-based tissue engineering applications. Human bone marrow stromal cells (hBMSCs) were seeded on both HASi and HA scaffolds and cultured with and without osteogenic supplements for a period of 4 weeks. Cellular responses were determined in vitro in terms of cell adhesion, viability, proliferation, and osteogenic differentiation, where both materials exhibited excellent cytocompatibility. Nevertheless, an enhanced rate of cell proliferation and higher levels of both alkaline phosphatase expression and activity were observed for cells cultured on HASi with osteogenic supplements. These findings indicate that the bioactivity of HA endowed with a silica-containing coating has definitely influenced the cellular activity, projecting HASi as a suitable candidate material for bone regenerative therapy.

Multifunction Sr, Co and F co-doped microporous coating on titanium of antibacterial, angiogenic and osteogenic activities

PubMed Central

Zhou, Jianhong; Zhao, Lingzhou

2016-01-01

Advanced multifunction titanium (Ti) based bone implant with antibacterial, angiogenic and osteogenic activities is stringently needed in clinic, which may be accomplished via incorporation of proper inorganic bioactive elements. In this work, microporous TiO2/calcium-phosphate coating on Ti doped with strontium, cobalt and fluorine (SCF-TiCP) was developed, which had a hierarchical micro/nano-structure with a microporous structure evenly covered with nano-grains. SCF-TiCP greatly inhibited the colonization and growth of both gram-positive and gram-negative bacteria. No cytotoxicity appeared for SCF-TiCP. Furthermore, SCF-TiCP stimulated the expression of key angiogenic factors in rat bone marrow stem cells (MSCs) and dramatically enhanced MSC osteogenic differentiation. The in vivo animal test displayed that SCF-TiCP induced more new bone and tighter implant/bone bonding. In conclusion, multifunction SCF-TiCP of antibacterial, angiogenic and osteogenic activities is a promising orthopedic and dental Ti implant coating for improved clinical performance. PMID:27353337

Effects of electron pressure anisotropy on current sheet configuration

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

Artemyev, A. V., E-mail: aartemyev@igpp.ucla.edu; Angelopoulos, V.; Runov, A.

2016-09-15

Recent spacecraft observations in the Earth's magnetosphere have demonstrated that the magnetotail current sheet can be supported by currents of anisotropic electron population. Strong electron currents are responsible for the formation of very thin (intense) current sheets playing the crucial role in stability of the Earth's magnetotail. We explore the properties of such thin current sheets with hot isotropic ions and cold anisotropic electrons. Decoupling of the motions of ions and electrons results in the generation of a polarization electric field. The distribution of the corresponding scalar potential is derived from the electron pressure balance and the quasi-neutrality condition. Wemore » find that electron pressure anisotropy is partially balanced by a field-aligned component of this polarization electric field. We propose a 2D model that describes a thin current sheet supported by currents of anisotropic electrons embedded in an ion-dominated current sheet. Current density profiles in our model agree well with THEMIS observations in the Earth's magnetotail.« less

Effects of Macromolecular Crowding on Human Adipose Stem Cell Culture in Fetal Bovine Serum, Human Serum, and Defined Xeno-Free/Serum-Free Conditions

PubMed Central

Lee, Michelle Hui Ching; Mäkinen, Laura; Ang, Xiu Min; Mannerström, Bettina; Raghunath, Michael; Miettinen, Susanna

2017-01-01

Microenvironment plays an important role for stem cell proliferation and differentiation. Macromolecular crowding (MMC) was recently shown to assist stem cells in forming their own matrix microenvironment in vitro. The ability of MMC to support adipose stem cell (ASC) proliferation, metabolism, and multilineage differentiation was studied under different conditions: fetal bovine serum- (FBS-) and human serum- (HS-) based media and xeno- and serum-free (XF/SF) media. Furthermore, the immunophenotype of ASCs under MMC was evaluated. The proliferative capacity of ASCs under MMC was attenuated in each condition. However, osteogenic differentiation was enhanced under MMC, shown by increased deposition of mineralized matrix in FBS and HS cultures. Likewise, significantly greater lipid droplet accumulation and increased collagen IV deposition indicated enhanced adipogenesis under MMC in FBS and HS cultures. In contrast, chondrogenic differentiation was attenuated in ASCs expanded under MMC. The ASC immunophenotype was maintained under MMC with significantly higher expression of CD54. However, MMC impaired metabolic activity and differentiation capacity of ASCs in XF/SF conditions. Both the supportive and inhibitory effects of MMC on ASC are culture condition dependent. In the presence of serum, MMC maintains ASC immunophenotype and enhances adipogenic and osteogenic differentiation at the cost of reduced proliferation. PMID:28465691

Effects of Macromolecular Crowding on Human Adipose Stem Cell Culture in Fetal Bovine Serum, Human Serum, and Defined Xeno-Free/Serum-Free Conditions.

PubMed

Patrikoski, Mimmi; Lee, Michelle Hui Ching; Mäkinen, Laura; Ang, Xiu Min; Mannerström, Bettina; Raghunath, Michael; Miettinen, Susanna

2017-01-01

Microenvironment plays an important role for stem cell proliferation and differentiation. Macromolecular crowding (MMC) was recently shown to assist stem cells in forming their own matrix microenvironment in vitro. The ability of MMC to support adipose stem cell (ASC) proliferation, metabolism, and multilineage differentiation was studied under different conditions: fetal bovine serum- (FBS-) and human serum- (HS-) based media and xeno- and serum-free (XF/SF) media. Furthermore, the immunophenotype of ASCs under MMC was evaluated. The proliferative capacity of ASCs under MMC was attenuated in each condition. However, osteogenic differentiation was enhanced under MMC, shown by increased deposition of mineralized matrix in FBS and HS cultures. Likewise, significantly greater lipid droplet accumulation and increased collagen IV deposition indicated enhanced adipogenesis under MMC in FBS and HS cultures. In contrast, chondrogenic differentiation was attenuated in ASCs expanded under MMC. The ASC immunophenotype was maintained under MMC with significantly higher expression of CD54. However, MMC impaired metabolic activity and differentiation capacity of ASCs in XF/SF conditions. Both the supportive and inhibitory effects of MMC on ASC are culture condition dependent. In the presence of serum, MMC maintains ASC immunophenotype and enhances adipogenic and osteogenic differentiation at the cost of reduced proliferation.

Chitosan stabilizes platelet growth factors and modulates stem cell differentiation toward tissue regeneration.

PubMed

Busilacchi, Alberto; Gigante, Antonio; Mattioli-Belmonte, Monica; Manzotti, Sandra; Muzzarelli, Riccardo A A

2013-10-15

The idea of using chitosan as a functional delivery aid to support simultaneously PRP, stem cells and growth factors (GF) is associated with the intention to use morphogenic biomaterials to modulate the natural healing sequence in bone and other tissues. For example, chitosan-chondroitin sulfate loaded with platelet lysate was included in a poly(D,L-lactate) foam that was then seeded with human adipose-derived stem cells and cultured in vitro under osteogenic stimulus: the platelet lysate provided to the bone tissue the most suitable assortment of GF which induces the osteogenic differentiation of the mesenchymal stem cells. PDGF, FGF, IGF and TGF-β were protagonists in the repair of callus fractures. The release of GF from the composites of chitosan-PRP and either nano-hydroxyapatite or tricalcium phosphate was highly beneficial for enhancing MSC proliferation and differentiation, thus qualifying chitosan as an excellent vehicle. A number of biochemical characteristics of chitosan exert synergism with stem cells in the regeneration of soft tissues. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cobalt doped proangiogenic hydroxyapatite for bone tissue engineering application.

PubMed

Kulanthaivel, Senthilguru; Roy, Bibhas; Agarwal, Tarun; Giri, Supratim; Pramanik, Krishna; Pal, Kunal; Ray, Sirsendu S; Maiti, Tapas K; Banerjee, Indranil

2016-01-01

The present study delineates the synthesis and characterization of cobalt doped proangiogenic-osteogenic hydroxyapatite. Hydroxyapatite samples, doped with varying concentrations of bivalent cobalt (Co(2+)) were prepared by the ammoniacal precipitation method and the extent of doping was measured by ICP-OES. The crystalline structure of the doped hydroxyapatite samples was confirmed by XRD and FTIR studies. Analysis pertaining to the effect of doped hydroxyapatite on cell cycle progression and proliferation of MG-63 cells revealed that the doping of cobalt supported the cell viability and proliferation up to a threshold limit. Furthermore, such level of doping also induced differentiation of the bone cells, which was evident from the higher expression of differentiation markers (Runx2 and Osterix) and better nodule formation (SEM study). Western blot analysis in conjugation with ELISA study confirmed that the doped HAp samples significantly increased the expression of HIF-1α and VEGF in MG-63 cells. The analysis described here confirms the proangiogenic-osteogenic properties of the cobalt doped hydroxyapatite and indicates its potential application in bone tissue engineering. Copyright © 2015 Elsevier B.V. All rights reserved.

Clay-Enriched Silk Biomaterials for Bone Formation

PubMed Central

Mieszawska, Aneta J.; Llamas, Jabier Gallego; Vaiana, Christopher A.; Kadakia, Madhavi P.; Naik, Rajesh R.; Kaplan, David L.

2011-01-01

The formation of silk protein/clay composite biomaterials for bone tissue formation is described. Silk fibroin serves as an organic scaffolding material offering mechanical stability suitable for bone specific uses. Clay montmorillonite (Cloisite ® Na+) and sodium silicate are sources of osteoinductive silica-rich inorganic species, analogous to bioactive bioglass-like bone repair biomaterial systems. Different clay particle-silk composite biomaterial films were compared to silk films doped with sodium silicate as controls for support of human bone marrow derived mesenchymal stem cells (hMSCs) in osteogenic culture. The cells adhered and proliferated on the silk/clay composites over two weeks. Quantitative real-time RT-PCR analysis revealed increased transcript levels for alkaline phosphatase (ALP), bone sialoprotein (BSP), and collagen type 1 (Col I) osteogenic markers in the cells cultured on the silk/clay films in comparison to the controls. Early evidence for bone formation based on collagen deposition at the cell-biomaterial interface was also found, with more collagen observed for the silk films with higher contents of clay particles. The data suggest that the silk/clay composite systems may be useful for further study toward bone regenerative needs. PMID:21549864

Twin-Mirrored-Galvanometer Laser-Light-Sheet Generator

NASA Technical Reports Server (NTRS)

Rhodes, David B.; Franke, John M.; Jones, Stephen B.; Leighty, Bradley D.

1991-01-01

Multiple, rotating laser-light sheets generated to illuminate flows in wind tunnels. Designed and developed to provide flexibility and adaptability to wide range of applications. Design includes capability to control size and location of laser-light sheet in real time, to generate horizontal or vertical sheets, to sweep sheet repeatedly through volume, to generate multiple sheets with controllable separation, and to rotate single or multiple laser-light sheets. Includes electronic equipment and laser mounted on adjustable-height platform. Twin-mirrored galvanometer unit supported by tripod to reduce vibration. Other possible applications include use in construction industry to align beams of building. Artistic or display applications also possible.

Perforating Thin Metal Sheets

NASA Technical Reports Server (NTRS)

Davidson, M. E.

1985-01-01

Sheets only few mils thick bonded together, punched, then debonded. Three-step process yields perforated sheets of metal. (1): Individual sheets bonded together to form laminate. (2): laminate perforated in desired geometric pattern. (3): After baking, laminate separates into individual sheets. Developed for fabricating conductive layer on blankets that collect and remove ions; however, perforated foils have other applications - as conductive surfaces on insulating materials; stiffeners and conductors in plastic laminates; reflectors in antenna dishes; supports for thermal blankets; lightweight grille cover materials; and material for mockup of components.

Molecular and functional expression of voltage-operated calcium channels during osteogenic differentiation of human mesenchymal stem cells.

PubMed

Zahanich, Ihor; Graf, Eva M; Heubach, Jürgen F; Hempel, Ute; Boxberger, Sabine; Ravens, Ursula

2005-09-01

We used the patch-clamp technique and RT-PCR to study the molecular and functional expression of VOCCs in undifferentiated hMSCs and in cells undergoing osteogenic differentiation. L-type Ca2+ channel blocker nifedipine did not influence alkaline phosphatase activity, calcium, and phosphate accumulation of hMSCs during osteogenic differentiation. This study suggests that osteogenic differentiation of hMSCs does not require L-type Ca2+ channel function. During osteogenic differentiation, mesenchymal stem cells from human bone marrow (hMSCs) must adopt the calcium handling of terminally differentiated osteoblasts. There is evidence that voltage-operated calcium channels (VOCCs), including L-type calcium channels, are involved in regulation of osteoblast function. We therefore studied whether VOCCs play a critical role during osteogenic differentiation of hMSCs. Osteogenic differentiation was induced in hMSCs cultured in maintenance medium (MM) by addition of ascorbate, beta-glycerophosphate, and dexamethasone (ODM) and was assessed by measuring alkaline phosphatase activity, expression of osteopontin, osteoprotegerin, RANKL, and mineralization. Expression of Ca2+ channel alpha1 subunits was shown by semiquantitative or single cell RT-PCR. Voltage-activated calcium currents of hMSCs were measured with the whole cell voltage-clamp technique. mRNA for the pore-forming alpha1C and alpha1G subunits of the L-type and T-type Ca2+ channels, respectively, was found in comparable amounts in cells cultured in MM or ODM. The limitation of L-type Ca2+ currents to a subpopulation of hMSCs was confirmed by single cell RT-PCR, where mRNA for the alpha1C subunits was detectable in only 50% of the cells cultured in MM. Dihydropyridine-sensitive L-type Ca2+ currents were found in 13% of cells cultured in MM and in 12% of the cells cultured in ODM. Under MM and ODM culture conditions, the cells positive for L-type Ca2+ currents were significantly larger than cells without Ca2+ currents as deduced from membrane capacitance; thus, current densities were comparable. Addition of the L-type Ca2+ channel blocker nifedipine to the culture media did not influence alkaline phosphatase activity and the extent of mineralization. These results suggest that, in the majority of hMSCs, Ca2+ entry through the plasma membrane is mediated by some channels other than VOCCs, and blockade of the L-type Ca2+ channels does not affect early osteogenic differentiation of hMSCs.

Topography of calcium phosphate ceramics regulates primary cilia length and TGF receptor recruitment associated with osteogenesis.

PubMed

Zhang, Jingwei; Dalbay, Melis T; Luo, Xiaoman; Vrij, Erik; Barbieri, Davide; Moroni, Lorenzo; de Bruijn, Joost D; van Blitterswijk, Clemens A; Chapple, J Paul; Knight, Martin M; Yuan, Huipin

2017-07-15

The surface topography of synthetic biomaterials is known to play a role in material-driven osteogenesis. Recent studies show that TGFβ signalling also initiates osteogenic differentiation. TGFβ signalling requires the recruitment of TGFβ receptors (TGFβR) to the primary cilia. In this study, we hypothesize that the surface topography of calcium phosphate ceramics regulates stem cell morphology, primary cilia structure and TGFβR recruitment to the cilium associated with osteogenic differentiation. We developed a 2D system using two types of tricalcium phosphate (TCP) ceramic discs with identical chemistry. One sample had a surface topography at micron-scale (TCP-B, with a bigger surface structure dimension) whilst the other had a surface topography at submicron scale (TCP-S, with a smaller surface structure dimension). In the absence of osteogenic differentiation factors, human bone marrow stromal cells (hBMSCs) were more spread on TCP-S than on TCP-B with alterations in actin organization and increased primary cilia prevalence and length. The cilia elongation on TCP-S was similar to that observed on glass in the presence of osteogenic media and was followed by recruitment of transforming growth factor-β RII (p-TGFβ RII) to the cilia axoneme. This was associated with enhanced osteogenic differentiation of hBMSCs on TCP-S, as shown by alkaline phosphatase activity and gene expression for key osteogenic markers in the absence of additional osteogenic growth factors. Similarly, in vivo after a 12-week intramuscular implantation in dogs, TCP-S induced bone formation while TCP-B did not. It is most likely that the surface topography of calcium phosphate ceramics regulates primary cilia length and ciliary recruitment of p-TGFβ RII associated with osteogenesis and bone formation. This bioengineering control of osteogenesis via primary cilia modulation may represent a new type of biomaterial-based ciliotherapy for orthopedic, dental and maxillofacial surgery applications. The surface topography of synthetic biomaterials plays important roles in material-driven osteogenesis. The data presented herein have shown that the surface topography of calcium phosphate ceramics regulates mesenchymal stromal cells (e.g., human bone marrow mesenchymal stromal cells, hBMSCs) with respect to morphology, primary cilia structure and TGFβR recruitment to the cilium associated with osteogenic differentiation in vitro. Together with bone formation in vivo, our results suggested a new type of biomaterial-based ciliotherapy for orthopedic, dental and maxillofacial surgery by the bioengineering control of osteogenesis via primary cilia modulation. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Clonal precursor of bone, cartilage, and hematopoietic niche stromal cells

PubMed Central

Chan, Charles K. F.; Lindau, Paul; Jiang, Wen; Chen, James Y.; Zhang, Lillian F.; Chen, Ching-Cheng; Seita, Jun; Sahoo, Debashis; Kim, Jae-Beom; Lee, Andrew; Park, Sujin; Nag, Divya; Gong, Yongquan; Kulkarni, Subhash; Luppen, Cynthia A.; Theologis, Alexander A.; Wan, Derrick C.; DeBoer, Anthony; Seo, Eun Young; Vincent-Tompkins, Justin D.; Loh, Kyle; Walmsley, Graham G.; Kraft, Daniel L.; Wu, Joseph C.; Longaker, Michael T.; Weissman, Irving L.

2013-01-01

Organs are composites of tissue types with diverse developmental origins, and they rely on distinct stem and progenitor cells to meet physiological demands for cellular production and homeostasis. How diverse stem cell activity is coordinated within organs is not well understood. Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells. PMID:23858471

Application of Green Tea Catechin for Inducing the Osteogenic Differentiation of Human Dedifferentiated Fat Cells in Vitro

PubMed Central

Kaida, Koji; Honda, Yoshitomo; Hashimoto, Yoshiya; Tanaka, Masahiro; Baba, Shunsuke

2015-01-01

Despite advances in stem cell biology, there are few effective techniques to promote the osteogenic differentiation of human primary dedifferentiated fat (DFAT) cells. We attempted to investigate whether epigallocatechin-3-gallate (EGCG), the main component of green tea catechin, facilitates early osteogenic differentiation and mineralization on DFAT cells in vitro. DFAT cells were treated with EGCG (1.25–10 μM) in osteogenic medium (OM) with or without 100 nM dexamethasone (Dex) for 12 days (hereafter two osteogenic media were designated as OM(Dex) and OM). Supplementation of 1.25 μM EGCG to both the media effectively increased the mRNA expression of collagen 1 (COL1A1) and runt-related transcription factor 2 (RUNX2) and also increased proliferation and mineralization. Compared to OM(Dex) with EGCG, OM with EGCG induced earlier expression for COL1A1 and RUNX2 at day 1 and higher mineralization level at day 12. OM(Dex) with 10 μM EGCG remarkably hampered the proliferation of the DFAT cells. These results suggest that OM(without Dex) with EGCG might be a preferable medium to promote proliferation and to induce osteoblast differentiation of DFAT cells. Our findings provide an insight for the combinatory use of EGCG and DFAT cells for bone regeneration and stem cell-based therapy. PMID:26602917

Boron enhances odontogenic and osteogenic differentiation of human tooth germ stem cells (hTGSCs) in vitro.

PubMed

Taşlı, Pakize Neslihan; Doğan, Ayşegül; Demirci, Selami; Şahin, Fikrettin

2013-06-01

Stem cell technology has been a great hope for the treatment of many common problems such as Parkinson's disease, Alzheimer's disease, diabetes, cancer, and tissue regeneration. Therefore, the main challenge in hard tissue engineering is to make a successful combination of stem cells and efficient inductors in the concept of stem cell differentiation into odontogenic and osteogenic cell types. Although some boron derivatives have been reported to promote bone and teeth growth in vivo, the molecular mechanism of bone formation has not been elucidated yet. Different concentrations of sodium pentaborate pentahydrate (NaB) were prepared for the analysis of cell toxicity and differentiation evaluations. The odontogenic, osteogenic differentiation and biomineralization of human tooth germ stem cells (hTGSCs) were evaluated by analyzing the mRNA expression levels, odontogenic and osteogenic protein expressions, alkaline phosphatase (ALP) activity, mineralization, and calcium deposits. The NaB-treated group displayed the highest ALP activity and expression of osteo- and odontogenic-related genes and proteins compared to the other groups and baseline. In the current study, increased in vitro odontogenic and osteogenic differentiation capacity of hTGSCs by NaB application has been shown for the first time. The study offers considerable promise for the development of new scaffold systems combined with NaB in both functional bone and tooth tissue engineering.

Effects of nicotine in the presence and absence of vitamin E on morphology, viability and osteogenic gene expression in MG-63 osteoblast-like cells.

PubMed

Torshabi, Maryam; Esfahrood, Zeinab Rezaei; Gholamin, Parisan; Karami, Elahe

2016-11-01

Evidence shows that oxidative stress induced by nicotine plays an important role in bone loss. Vitamin E with its antioxidative properties may be able to reverse the effects of nicotine on bone. This study aimed to assess the effects of nicotine in the presence and absence of vitamin E on morphology, viability and osteogenic gene expression in MG-63 (osteosarcoma) human osteoblast-like cells. We treated the cells with 5 mM nicotine. The viability and morphology of cells were evaluated respectively using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) and crystal violet assays. The effect of nicotine on osteogenic gene expression in MG-63 cells was assessed by real-time reverse-transcription polymerase chain reaction of osteoblast markers, namely, alkaline phosphatase, osteocalcin and bone sialoprotein. The results revealed that survival and proliferation of MG-63 cells were suppressed following exposure to nicotine, and cytoplasm vacuolization occurred in the cells. Nicotine significantly down-regulated the expression of osteogenic marker genes. Such adverse effects on morphology, viability and osteogenic gene expression of MG-63 cells were reversed by vitamin E therapy. In conclusion, vitamin E supplementation may play a role in proliferation and differentiation of osteoblasts, and vitamin E can be considered as an anabolic agent to treat nicotine-induced bone loss.

Effect of TAK1 on osteogenic differentiation of mesenchymal stem cells by regulating BMP-2 via Wnt/β-catenin and MAPK pathway.

PubMed

Yang, Hongpeng; Guo, Yue; Wang, Dawei; Yang, Xiaofei; Ha, Chengzhi

2018-01-02

Mesenchymal stem cells (MSCs) have the ability to differentiate into osteoblasts and chondrocytes. In vitro osteogenic differentiation is critical but the molecular mechanism has yet to be further clarified. The role of TGF-β activated kinase 1 (TAK1) in MSCs osteogenesis differentiation has not been reported. By adding si-TAK1 and rhTAK1, the osteogenic differentiation of MSCs was measured. Expression levels of the osteoblastic marker genes during osteogenic differentiation of MSCs were checked. As well as molecules involved in BMP and Wnt/β-catenin signaling pathways. The phosphorylation of p38 and JNK was also checked. TAK1 is essential for mineralization of MSCs at low concentration, but excessive rhTAK1 inhibits mineralization of MSCs. It up regulates the expression levels of bone sialoprotein (BSP), osteocalcin (OSC), Alkaline phosphatase (ALP), and RUNX2 during osteogenic differentiation of MSCs. It can also promote TGF-β/BMP-2 gene expression and β-catenin expression, and down regulate GSK-3β expression. Meanwhile, TAK1 promotes the phosphorylation of p38 and JNK. Additionally, TAK1 up regulates the expression of BMP-2 at all concentration under the inhibition of p38 and JNK. Our results suggested that TAK1 is essential in MSCs osteogenesis differentiation, and functions as a double-edged sword, probably through regulation of β-catenin and p38/JNK.

A Long-Acting BMP-2 Release System Based on Poly(3-hydroxybutyrate) Nanoparticles Modified by Amphiphilic Phospholipid for Osteogenic Differentiation

PubMed Central

Peng, Xiaochun; Chen, Yunsu; Li, Yamin; Wang, Yiming

2016-01-01

We explored a novel poly(3-hydroxybutyrate) (PHB) nanoparticle loaded with hydrophilic recombinant human BMP-2 with amphiphilic phospholipid (BPC-PHB NP) for a rapid-acting and long-acting delivery system of BMP-2 for osteogenic differentiation. The BPC-PHB NPs were prepared by a solvent evaporation method and showed a spherical particle with a mean particle size of 253.4 nm, mean zeta potential of −22.42 mV, and high entrapment efficiency of 77.18%, respectively. For BPC-PHB NPs, a short initial burst release of BMP-2 from NPs in 24 h was found and it has steadily risen to reach about 80% in 20 days for in vitro test. BPC-PHB NPs significantly reduced the burst release of BMP-2, as compared to that of PHB NPs loading BMP-2 without PL (B-PHB NPs). BPC-PHB NPs maintained the content of BMP-2 for a long-term osteogenic differentiation. The OCT-1 cells with BPC-PHB NPs have high ALP activity in comparison with others. The gene markers for osteogenic differentiation were significantly upregulated for sample with BPC-PHB NPs, implying that BPC-PHB NPs can be used as a rapid-acting and long-acting BMP-2 delivery system for osteogenic differentiation. PMID:27379249

Osteoinductive-nanoscaled silk/HA composite scaffolds for bone tissue engineering application.

PubMed

Huang, Xiaowei; Bai, Shumeng; Lu, Qiang; Liu, Xi; Liu, Shanshan; Zhu, Hesun

2015-10-01

Osteoinductive silk/hydroxyapatite (HA) composite scaffolds for bone regeneration were prepared by combining silk with HA/silk core-shell nanoparticles. The HA/silk nanoparticles were directly dispersed in silk solution to form uniform silk/HA blend and then composite scaffolds after a freeze-drying process. The HA/silk nanoparticles uniformly distributed in silk scaffolds at nanometer scale at varying HA content up to 40%, and substantially improved the compressive strength of the scaffolds produced. Rat bone mesenchymal stem cells (rBMSCs) were cultured in these scaffolds and cell proliferation was analyzed by confocal microscopy and DNA assay. Gene expression and biochemical assays were employed to study the influence of increasing HA/silk nanoparticles on in vitro osteogenic differentiation of rBMSCs. Increasing HA/silk nanoparticles inside silk scaffolds improved the growth and osteogenic capability of rBMSCs in the absence of osteogenic growth factors, and also significantly increased the calcium and collagen I deposition. In addition, compared to silk/HA composite scaffolds containing HA aggregates, the scaffolds loaded with HA/silk nanoparticles showed remarkably higher stiffness and better osteogenic property at same HA content, implying a preferable microenvironment for rBMSCs. These results suggest that the osteogenic property as well as mechanical property of silk/HA scaffolds could be further improved through fabricating their structure and topography at nanometer scale, providing more suitable systems for bone regeneration. © 2014 Wiley Periodicals, Inc.

UV-activated 7-dehydrocholesterol-coated titanium implants promote differentiation of human umbilical cord mesenchymal stem cells into osteoblasts.

PubMed

Satué, María; Ramis, Joana M; Monjo, Marta

2016-01-01

Vitamin D metabolites are essential for bone regeneration and mineral homeostasis. The vitamin D precursor 7-dehydrocholesterol can be used after UV irradiation to locally produce active vitamin D by osteoblastic cells. Furthermore, UV-irradiated 7-dehydrocholesterol is a biocompatible coating for titanium implants with positive effects on osteoblast differentiation. In this study, we examined the impact of titanium implants surfaces coated with UV-irradiated 7-dehydrocholesterol on the osteogenic differentiation of human umbilical cord mesenchymal stem cells. First, the synthesis of cholecalciferol (D3) was achieved through the incubation of the UV-activated 7-dehydrocholesterol coating for 48 h at 23℃. Further, we investigated in vitro the biocompatibility of this coating in human umbilical cord mesenchymal stem cells and its potential to enhance their differentiation towards the osteogenic lineage. Human umbilical cord mesenchymal stem cells cultured onto UV-irradiated 7-dehydrocholesterol-coated titanium implants surfaces, combined with osteogenic supplements, upregulated the gene expression of several osteogenic markers and showed higher alkaline phosphatase activity and calcein blue staining, suggesting increased mineralization. Thus, our results show that the use of UV irradiation on 7-dehydrocholesterol -treated titanium implants surfaces generates a bioactive coating that promotes the osteogenic differentiation of human umbilical cord mesenchymal stem cells, with regenerative potential for improving osseointegration in titanium-based bone anchored implants. © The Author(s) 2015.

Synergetic effect of topological cue and periodic mechanical tension-stress on osteogenic differentiation of rat bone mesenchymal stem cells.

PubMed

Liu, Yao; Yang, Guang; Ji, Huanzhong; Xiang, Tao; Luo, En; Zhou, Shaobing

2017-06-01

Mesenchymal stem cells (MSCs) are able to self-renew and differentiate into tissues of mesenchymal origin, making them to be significant for cell-based therapies, such as metabolic bone diseases and bone repair. Regulating the differentiation of MSCs is significant for bone regeneration. Electrospun fibers mimicking natural extracellular matrix (ECM), is an effective artificial ECM to regulate the behaviors and fates of MSCs. The aligned electrospun fibers can modulate polar cell pattern of bone mesenchymal stem cells, which leads to more obvious osteogenic differentiation. Apart from the topographic effect of electrospun fibers, mechanical cues can also intervene the cell behaviors. In this study, the osteogenic differentiation of rat bone mesenchymal stem cells was evaluated, which were cultured on aligned/random electrospun fiber mats materials under mechanical tension intervention. Scanning electron microscope and immune-fluorescent staining were used to directly observe the polarity changing of cellular morphology and cytoskeleton. The results proved that aligned electrospun fibers could be more conducive to promote osteogenic differentiation of rat bone mesenchymal stem cells and this promotion of osteogenic differentiation was enhanced by tension intervention. These results were correlated to the quantitative real-time PCR assay. In general, culturing rat bone mesenchymal stem cells on electrospun fibers under the intervention of mechanical tension is an effective way to mimic a more real cellular microenvironment. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of extracellular calcium on viability and osteogenic differentiation of bone marrow stromal cells in vitro.

PubMed

Cheng, Shaowen; Wang, Wei; Lin, Zhongqin; Zhou, Ping; Zhang, Xiaolei; Zhang, Wei; Chen, Qingyu; Kou, Dongquan; Ying, Xiaozhou; Shen, Yue; Cheng, Xiaojie; Yu, Ziming; Peng, Lei; Lu, Chuanzhu

2013-09-01

Bone marrow stromal cells (BMSCs) have been extensively used for tissue engineering. However, the effect of Ca(2+) on the viability and osteogenic differentiation of BMSCs has yet to be evaluated. To determine the dose-dependent effect of Ca(2+) on viability and osteogenesis of BMSCs in vitro, BMSCs were cultured in calcium-free DMEM medium supplemented with various concentrations of Ca(2+) (0, 1, 2, 3, 4, and 5 mM) from calcium citrate. Cell viability was analyzed by MTT assay and osteogenic differentiation was evaluated by alkaline phosphatase (ALP) assay, Von Kossa staining, and real-time PCR. Ca(2+) stimulated BMSCs viability in a dose-dependent manner. At slightly higher concentrations (4 and 5 mM) in the culture, Ca(2+) significantly inhibited the activity of ALP on days 7 and 14 (P < 0.01 or P < 0.05), significantly suppressed collagen synthesis (P < 0.01 or P < 0.05), and significantly elevated calcium deposition (P < 0.01) and mRNA levels of osteocalcin (P < 0.01 or P < 0.05) and osteopontin (P < 0.01 or P < 0.05). Therefore, elevated concentrations of extracellular calcium may promote cell viability and late-stage osteogenic differentiation, but may suppress early-stage osteogenic differentiation in BMSCs.

[Survival of bone marrow mesenchymal stem cells and periodontal ligament stem cells in cell sheets].

PubMed

An, Kangkang; Liu, Hongwei

2014-11-01

To evaluate the survival of bone marrow mesenchymal stem cells (BMMSC) and periodontal ligament stem cells (PDLSC) in BMMSC/PDLSC cell sheets which transplanted ectopically into subcutaneous dorsum of nude mice. The canine BMMSC and PDLSC from primary culture were tranfected with lentiviral vectors carrying green fluorescent protein (GFP) gene (Lentivirus-GFP) or red fluorescent protein (RFP) gene (Lentivirus-RFP) respectively. The immunophenotypes of GFP-labeled BMMSC and RFP-labeled PDLSC were identified by flow cytometry. Adipogenic and osteogenic differentiation of them were detected by alizarin red or oil red O respectively. Then, both GFP-labeled BMMSC cell sheets and RFP-labeled PDLSC cell sheets were fabricated respectively using normal culture dish (6 cm) after stimulation of extracellular matrix formation. Each was enveloped by collagen membrane (Bio-Gide) and then transplanted into the subcutaneous dorsum of nude mice. In vivo non-invasive biofluorescence imaging(BFI) was performed at 1, 2, 4 and 8 w post-tranplantation to trace and quantify the survival and growth of RFP-labeled PDLSC and GFP-labeled BMMSC via the BFI system of the NightOWL. The fluorescence intensity change of GFP/RFP signal was monitored and compared. The mice were sacrificed 8 weeks after cell sheets transplantation and the survival of stem cells was verified by fluorescence immunohistochemistry. The flow cytometry showed that GFP-labeled BMMSC positively expressed CD29, CD44, CD34, STRO-1 were 93.07%, 92.84%, 3.23%, 67.67%, and RFP-labeled PDLSCs were 89.91%, 88.47%, 6.04%, 74.11%, respectively. Both of them had the potency of differentiating into osteoblasts and adipocytes. The stemness of both of them was almost same. After being transplanted into nude mice, the signal strength of GFP(BMMSC) was weaker and weaker in 1, 2, 4 and 8 w [(83.1±3.1)×10(6), (65.1±2.3)×10(6), (51.5 ± 2.3)×10(6), (33.8 ± 2.0)×10(6) ph/s, respectively.]. The signal strength of RFP(PDLSC) was weakenedin 1, 2 and 4 w [(53.8±3.0)×10(6), (42.2±2.6)×10(6), (34.5±2.1)×10(6) ph/s], then recovered in 8 w ([ 45.1±2.9)×10(6) ph/s]. The signal strength of RFP(PDLSC) was signifcantly stronger in 8 w than in 4 w(P < 0.01). The survival of RFP-labeled PDLSC was significant higher than that of GFP-labeled BMMSC. After 8 weeks, lots of RFP-labeled PDLSC were observed by microscope, but less GFP-labeled BMMSC were observed. Histometric analysis revealed that the survival of stem cells in the RFP-labeled PDLSC cell sheets was significantly higher than that of in the GFP-labeled BMMSCs cell sheets.

[Effect of nerve growth factor on osteogenic potential of type 2 diabetic mice bone marrow stromal cell in vitro].

PubMed

Cui, G S; Zeng, J Y; Zhang, J; Lu, R

2018-02-09

Objective: To study the effects of nerve growth factor (NGF) on the proliferation, osteogenic differentiation and mineralization of type 2 diabetic mice bone marrow stromal cell (BMSC), providing basis for clinical application of NGF. Methods: Three 8-week-old male db/db mice and two 8-week-old male C57BL/6J mice were used in the study. BMSC derived from femur were cultured though adherence method. BMSC of C57BL/6J mice and db/db mice was divided into normal group and diabetic group to conduct the osteogenic potential experiment, named experiment one. In experiment two, diabetic BMSC was divided into 3 groups: diabetic control group, NGF group, and K252a+NGF group [K252a was the inhibitor of tyrosine kinase A (TrkA), which was the high affinity receptor of NGF], to investigate effect of NGF on osteogenic potential of diabetic mice BMSC. After seeding BMSC, K252a was added into K252a+NGF group, then NGF was added 30 min later. NGF was added into NGF group and K252a+NGF group, but not diabetic control group. The proliferation of BMSC at 1, 3, 5 and 7 d in experiment one and the proliferation of BMSC at 1, 2 and 3 d in experiment two were evaluated through methyl thiazolyl tetrazolium, and the level of alkaline phosphatase (ALP) at 3, 5 and 7 d in both experiments were measured. After being osteogenic induced for 14 d, mineralized nodules in both experiments were quantitated by alizarin red calcium stain. Five holes were set in every group, and all experiments were repeated 3 times. Results: The BMSC proliferation of diabetic group was significantly higher than that of the normal group at 3, 5 and 7 d ( P< 0.05). After being osteogenic inducted for 3, 5 and 7 d, ALP level of diabetic group were significantly lower than that of normal group ( P< 0.05). After being osteogenic inducted for 14 d, calcium nodule count of diabetic group [(23.1±6.4) nodule/field] were significantly lower than that of normal group [(36.9±7.9) nodule/field]( P< 0.05). At 1, 2 and 3 d, BMSC proliferations of diabetic control group, NGF group and K252a+NGF group were not statistically different ( P> 0.05). After being osteogenic inducted for 3 and 5 d, ALP level of NGF group was significantly higher than that of diabetic control group ( P< 0.05). After being osteogenic inducted for 3, 5, and 7 d, ALP level of K252a+NGF group was significantly lower than that of NGF group ( P< 0.05) and diabetic control group ( P< 0.05). After being osteogenic induced for 14 d, calcium nodule count of NGF group [(45.2±6.8) nodule/field] was significantly more than that of diabetic control group [(23.1±6.4) nodule/field]( P< 0.05); while calcium nodule count of K252a+NGF group [(18.0±4.5) nodule/field] was significantly less than that of NGF group ( P< 0.05) and diabetic control group ( P< 0.05). Conclusions: The differentiation and mineralization of type 2 diabetic mice BMSC was significantly reduced. NGF promoted the osteoblastic differentiation and mineralization of diabetic mice BMSC in viro though combining with TrkA.

Gold nanoparticle size and shape influence on osteogenesis of mesenchymal stem cells

NASA Astrophysics Data System (ADS)

Li, Jingchao; Li, Jia'en Jasmine; Zhang, Jing; Wang, Xinlong; Kawazoe, Naoki; Chen, Guoping

2016-04-01

Gold nanoparticles (AuNPs) have been extensively explored for biomedical applications due to their advantages of facile synthesis and surface functionalization. Previous studies have suggested that AuNPs can induce differentiation of stem cells into osteoblasts. However, how the size and shape of AuNPs affect the differentiation response of stem cells has not been elucidated. In this work, a series of bovine serum albumin (BSA)-coated Au nanospheres, Au nanostars and Au nanorods with different diameters of 40, 70 and 110 nm were synthesized and their effects on osteogenic differentiation of human mesenchymal stem cells (hMSCs) were investigated. All the AuNPs showed good cytocompatibility and did not influence proliferation of hMSCs at the studied concentrations. Osteogenic differentiation of hMSCs was dependent on the size and shape of AuNPs. Sphere-40, sphere-70 and rod-70 significantly increased the alkaline phosphatase (ALP) activity and calcium deposition of cells while rod-40 reduced the ALP activity and calcium deposition. Gene profiling revealed that the expression of osteogenic marker genes was down-regulated after incubation with rod-40. However, up-regulation of these genes was found in the sphere-40, sphere-70 and rod-70 treatment. Moreover, it was found that the size and shape of AuNPs affected the osteogenic differentiation of hMSCs through regulating the activation of Yes-associated protein (YAP). These results indicate that the size and shape of AuNPs had an influence on the osteogenic differentiation of hMSCs, which should provide useful guidance for the preparation of AuNPs with defined size and shape for their biomedical applications.Gold nanoparticles (AuNPs) have been extensively explored for biomedical applications due to their advantages of facile synthesis and surface functionalization. Previous studies have suggested that AuNPs can induce differentiation of stem cells into osteoblasts. However, how the size and shape of AuNPs affect the differentiation response of stem cells has not been elucidated. In this work, a series of bovine serum albumin (BSA)-coated Au nanospheres, Au nanostars and Au nanorods with different diameters of 40, 70 and 110 nm were synthesized and their effects on osteogenic differentiation of human mesenchymal stem cells (hMSCs) were investigated. All the AuNPs showed good cytocompatibility and did not influence proliferation of hMSCs at the studied concentrations. Osteogenic differentiation of hMSCs was dependent on the size and shape of AuNPs. Sphere-40, sphere-70 and rod-70 significantly increased the alkaline phosphatase (ALP) activity and calcium deposition of cells while rod-40 reduced the ALP activity and calcium deposition. Gene profiling revealed that the expression of osteogenic marker genes was down-regulated after incubation with rod-40. However, up-regulation of these genes was found in the sphere-40, sphere-70 and rod-70 treatment. Moreover, it was found that the size and shape of AuNPs affected the osteogenic differentiation of hMSCs through regulating the activation of Yes-associated protein (YAP). These results indicate that the size and shape of AuNPs had an influence on the osteogenic differentiation of hMSCs, which should provide useful guidance for the preparation of AuNPs with defined size and shape for their biomedical applications. Electronic supplementary information (ESI) available: Additional experimental results. See DOI: 10.1039/c5nr08808a

Heat exchanger and water tank arrangement for passive cooling system

DOEpatents

Gillett, J.E.; Johnson, F.T.; Orr, R.S.; Schulz, T.L.

1993-11-30

A water storage tank in the coolant water loop of a nuclear reactor contains a tubular heat exchanger. The heat exchanger has tube sheets mounted to the tank connections so that the tube sheets and tubes may be readily inspected and repaired. Preferably, the tubes extend from the tube sheets on a square pitch and then on a rectangular pitch there between. Also, the heat exchanger is supported by a frame so that the tank wall is not required to support all of its weight. 6 figures.

A stable planar bilayer membrane of phospholipid supported by cellulose sheets.

PubMed

Setaka, M; Yamamoto, T; Sato, N; Yano, M; Kwan, T

1982-01-01

A new method is reported for preparing a thin planar membrane of 1,2-distearoylsn-glycero-3-phosphocholine and egg yolk lecithin-cholesterol (molar ratio of 1:1) between a pair of cellulose sheets. This technique, developed from the method of the multilayer planar membrane preparation (Setaka, M., et al. (1979) J. Biochem. 86, 355-362; 1619-1622; (1980) J. Biochem. 88, 1819-1829), consisted of three experimental processes. First, a phospholipid monolayer was prepared at an air-water interface, then taken up on a stretched cellulose sheet. A thin lipid membrane, supported from both sides by cellulose sheets, was constructed by combining two of these lipid monolayer-cellulose sheets. The permeability coefficient of the thin lipid membrane was estimated by removing the effect of two outer cellulose sheets, and this permeability was found to be larger than those of other model membranes of a lipid bilayer, indicating that the present lipid membrane is not a perfect single lipid bilayer. However, certain experimental evidence suggests that the bulk of the phospholipids formed a bilayer between the two cellulose sheets. Since this lipid membrane is particularily stable, larger membranes can be prepared by the present method than other planar bilayer membranes of lipid, which are usually constructed inside a pin hole in a thin teflon sheet.

Scaffold preferences of mesenchymal stromal cells and adipose-derived stem cells from green fluorescent protein transgenic mice influence the tissue engineering of bone.

PubMed

Wittenburg, Gretel; Flade, Viktoria; Garbe, Annette I; Lauer, Günter; Labudde, Dirk

2014-05-01

We have analysed the growth and differentiation of mesenchymal stromal cells (MSC) from bone marrow, and of adipose derived stem cells (ASC) from murine abdominal fat tissue, of green fluorescent protein (GFP) transgenic animals grown directly on two types of hydroxyapatite ceramic bone substitutes. BONITmatrix® and NanoBone® have specific mechanical and physiochemical properties such as porosity and an inner surface that influence cellular growth. Both MSC and ASC were separately seeded on 200mg of each biomaterial and cultured for 3 weeks under osteogenic differentiation conditions. The degree of mineralisation was assessed by alizarin red dye and the specific alkaline phosphatase activity of the differentiated cells. The morphology of the cells was examined by scanning electron microscopy and confocal microscopy. The osteoblastic phenotype of the cells was confirmed by analysing the expression of bone-specific genes (Runx2, osteocalcin, osteopontin, and osteonectin) by semiquantitative reverse transcriptase polymerase chain reaction (PCR). Comparison of BONITmatrix® and NanoBone® showed cell type-specific preferences in terms of osteogenic differentiation. MSC-derived osteoblast-like cells spread optimally on the surface of NanoBone® but not BONITmatrix® granules. In contrast BONITmatrix® granules conditioned the growth of osteoblast-like cells derived from ASC. The osteoblastic phenotype of the cultured cells on all matrices was confirmed by specific gene expression. Our results show that the in vitro growth and osteogenic differentiation of murine MSC or ASC of GFP transgenic mice are distinctly influenced by the ceramic substratum. While NanoBone® granules support the proliferation and differentiation of murine MSC isolated from bone marrow, the growth of murine ASC is supported by BONITmatrix® granules. NanoBone® is therefore recommended for use as scaffold in tissue engineering that requires MSC, whereas ASC can be combined with BONITmatrix® for in vitro bone engineering. Copyright © 2014 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

High surface area graphene-supported metal chalcogenide assembly

DOEpatents

Worsley, Marcus A.; Kuntz, Joshua D.; Orme, Christine A.

2017-04-25

Disclosed here is a method for hydrocarbon conversion, comprising contacting at least one graphene-supported assembly with at least one hydrocarbon feedstock, wherein the graphene-supported assembly comprises (i) a three-dimensional network of graphene sheets crosslinked by covalent carbon bonds and (ii) at least one metal chalcogenide compound disposed on the graphene sheets, wherein the chalcogen of the metal chalcogenide compound is selected from S, Se and Te, and wherein the metal chalcogenide compound accounts for at least 20 wt. % of the graphene-supported assembly.

Buckling Behavior of Substrate Supported Graphene Sheets

PubMed Central

Yang, Kuijian; Chen, Yuli; Pan, Fei; Wang, Shengtao; Ma, Yong; Liu, Qijun

2016-01-01

The buckling of graphene sheets on substrates can significantly degrade their performance in materials and devices. Therefore, a systematic investigation on the buckling behavior of monolayer graphene sheet/substrate systems is carried out in this paper by both molecular mechanics simulations and theoretical analysis. From 70 simulation cases of simple-supported graphene sheets with different sizes under uniaxial compression, two different buckling modes are investigated and revealed to be dominated by the graphene size. Especially, for graphene sheets with length larger than 3 nm and width larger than 1.1 nm, the buckling mode depends only on the length/width ratio. Besides, it is revealed that the existence of graphene substrate can increase the critical buckling stress and strain to 4.39 N/m and 1.58%, respectively, which are about 10 times those for free-standing graphene sheets. Moreover, for graphene sheets with common size (longer than 20 nm), both theoretical and simulation results show that the critical buckling stress and strain are dominated only by the adhesive interactions with substrate and independent of the graphene size. Results in this work provide valuable insight and guidelines for the design and application of graphene-derived materials and nano-electromechanical systems. PMID:28787831

A web-based fact sheet series for grandparents raising grandchildren and the professionals who serve them.

PubMed

Brintnall-Peterson, Mary; Poehlmann, Julie; Morgan, Kari; Shlafer, Rebecca

2009-04-01

To develop and evaluate a series of web-based fact sheets for grandparents raising grandchildren. The fact sheets focus on child development issues that grandparents may face when raising their grandchildren. The fact sheets were developed using research on attachment theory, child development, and the needs of grandparents raising grandchildren. The fact sheets can be viewed online or downloaded for free. Evaluation data for the fact sheets were gathered using an online survey. Results of the survey revealed that the fact sheets are used by grandparents and professionals. Respondents reported sharing the fact sheets with others and using them for personal use, in support groups, and as a general agency resource. The fact sheet series is a useful way to reach both grandparents and professionals working with this audience in a variety of settings. Modifications to the fact sheet series are suggested to address additional needs of grandparents raising grandchildren.

Rapid prototyped porous nickel–titanium scaffolds as bone substitutes

PubMed Central

Hoffmann, Waldemar; Bormann, Therese; Rossi, Antonella; Müller, Bert; Schumacher, Ralf; Martin, Ivan; Wendt, David

2014-01-01

While calcium phosphate–based ceramics are currently the most widely used materials in bone repair, they generally lack tensile strength for initial load bearing. Bulk titanium is the gold standard of metallic implant materials, but does not match the mechanical properties of the surrounding bone, potentially leading to problems of fixation and bone resorption. As an alternative, nickel–titanium alloys possess a unique combination of mechanical properties including a relatively low elastic modulus, pseudoelasticity, and high damping capacity, matching the properties of bone better than any other metallic material. With the ultimate goal of fabricating porous implants for spinal, orthopedic and dental applications, nickel–titanium substrates were fabricated by means of selective laser melting. The response of human mesenchymal stromal cells to the nickel–titanium substrates was compared to mesenchymal stromal cells cultured on clinically used titanium. Selective laser melted titanium as well as surface-treated nickel–titanium and titanium served as controls. Mesenchymal stromal cells had similar proliferation rates when cultured on selective laser melted nickel–titanium, clinically used titanium, or controls. Osteogenic differentiation was similar for mesenchymal stromal cells cultured on the selected materials, as indicated by similar gene expression levels of bone sialoprotein and osteocalcin. Mesenchymal stromal cells seeded and cultured on porous three-dimensional selective laser melted nickel–titanium scaffolds homogeneously colonized the scaffold, and following osteogenic induction, filled the scaffold’s pore volume with extracellular matrix. The combination of bone-related mechanical properties of selective laser melted nickel–titanium with its cytocompatibility and support of osteogenic differentiation of mesenchymal stromal cells highlights its potential as a superior bone substitute as compared to clinically used titanium. PMID:25383165

Experimental variation of the level and the ratio of angiogenic and osteogenic signaling affects the spatiotemporal expression of bone-specific markers and organization of bone formation in ectopic sites.

PubMed

Moser, Norman; Goldstein, Jan; Kauffmann, Phillip; Epple, Matthias; Schliephake, Henning

2018-04-01

The aim of the present study was to test the hypothesis that the ratio of angiogenic and osteogenic signaling affects ectopic bone formation when delivered in different amounts. Porous composite PDLLA/CaCO 3 scaffolds were loaded with rhBMP2 and rhVEGF in different dosage combinations and implanted into the gluteal muscles of 120 adult male Wistar rats. Bone formation and expression of alkaline phosphatase and Runx2 were quantified by histomorphometry. Spatial distribution across the scaffolds was assessed by using a grid that discriminated between the periphery and center of the scaffolds. The evaluation showed that the combined delivery of bone morphogenetic protein BMP2 and VEGF in different dosage combinations did not enhance the overall quantity of ectopic bone formation compared to the delivery of BMP2 alone. The addition of VEGF generally upregulated Runx2 after 4 weeks, which may have retarded terminal osteogenic differentiation. However, slow combined delivery of 1.5-2.0 μg BMP2 combined with 50 ng VEGF165 over a period of 5 weeks supported a more even distribution of bone formation across the implanted scaffolds whereas higher amounts of VEGF did not elicit this effect. The findings suggest that structural organization rather than the quantity of ectopic bone formation is affected by the dosage and the ratio of BMP2 and VEGF levels at the observed intervals. The development of carriers for dual growth factor delivery has to take into account the necessity to carefully balance the ratio of growth release.

Osteoblastic phenotype of rat marrow stromal cells cultured in the presence of dexamethasone, beta-glycerolphosphate, and L-ascorbic acid

NASA Technical Reports Server (NTRS)

Peter, S. J.; Liang, C. R.; Kim, D. J.; Widmer, M. S.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

1998-01-01

We investigated the effects of the time course of addition of osteogenic supplements dexamethasone, beta-glycerolphosphate, and L-ascorbic acid to rat marrow stromal cells, and the exposure time on the proliferation and differentiation of the cells. It was the goal of these experiments to determine the time point for supplement addition to optimize marrow stromal cell proliferation and osteoblastic differentiation. To determine this, two studies were performed; one study was based on the age of the cells from harvest, and the other study was based on the duration of exposure to supplemented medium. Cells were seen to proliferate rapidly at early time points in the presence and absence of osteogenic supplements as determined by 3H-thymidine incorporation into the DNA of replicating cells. These results were supported by cell counts ascertained through total DNA analysis. Alkaline phosphatase (ALP) activity and osteocalcin production at 21 days were highest for both experimental designs when the cells were exposed to supplemented medium immediately upon harvest. The ALP levels at 21 days were six times greater for cells maintained in supplements throughout than for control cells cultured in the absence of supplements for both studies, reaching an absolute value of 75 x 10(-7) micromole/min/cell. Osteocalcin production reached 20 x 10(-6) ng/cell at 21 days in both studies for cells maintained in supplemented medium throughout the study, whereas the control cells produced an insignificant amount of osteocalcin. These results suggest that the addition of osteogenic supplements to marrow-derived cells early in the culture period did not inhibit proliferation and greatly enhanced the osteoblastic phenotype of cells in a rat model.

Pooled thrombin-activated platelet-rich plasma: a substitute for fetal bovine serum in the engineering of osteogenic/vasculogenic grafts.

PubMed

Tchang, Laurent A; Pippenger, Benjamin E; Todorov, Atanas; Wolf, Francine; Burger, Maximilian G; Jaquiery, Claude; Bieback, Karen; Martin, Ivan; Schaefer, Dirk J; Scherberich, Arnaud

2017-05-01

The use of fetal bovine serum (FBS) as a culture medium supplement in cell therapy and clinical tissue engineering is challenged by immunological concerns and the risk of disease transmission. Here we tested whether human, thrombin-activated, pooled, platelet-rich plasma (tPRP) can be substituted for FBS in the engineering of osteogenic and vasculogenic grafts, using cells from the stromal vascular fraction (SVF) of human adipose tissue. SVF cells were cultured under perfusion flow into porous hydroxyapatite scaffolds for 5 days, with the medium supplemented with either 10% tPRP or 10% FBS and implanted in an ectopic mouse model. Following in vitro culture, as compared to FBS, the use of tPRP did not modify the fraction of clonogenic cells or the different cell phenotypes, but increased by 1.9-fold the total number of cells. After 8 weeks in vivo, bone tissue was formed more reproducibly and in higher amounts (3.7-fold increase) in constructs cultured with tPRP. Staining for human-specific ALU sequences and for the human isoforms of CD31/CD34 revealed the human origin of the bone, the formation of blood vessels by human vascular progenitors and a higher density of human cells in implants cultured with tPRP. In summary, tPRP supports higher efficiency of bone formation by SVF cells than FBS, likely by enhancing cell expansion in vitro while maintaining vasculogenic properties. The use of tPRP may facilitate the clinical translation of osteogenic grafts with intrinsic capacity for vascularization, based on the use of adipose-derived cells. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Osteoimmunomodulatory properties of magnesium scaffolds coated with β-tricalcium phosphate.

PubMed

Chen, Zetao; Mao, Xueli; Tan, Lili; Friis, Thor; Wu, Chengtie; Crawford, Ross; Xiao, Yin

2014-10-01

The osteoimmunomodulatory property of bone biomaterials is a vital property determining the in vivo fate of the implants. Endowing bone biomaterials with favorable osteoimmunomodulatory properties is of great importance in triggering desired immune response and thus supports the bone healing process. Magnesium (Mg) has been recognized as a revolutionary metal for applications in orthopedics due to it being biodegradable, biocompatible, and having osteoconductive properties. However, Mg's high rate of degradation leads to an excessive inflammatory response and this has restricted its application in bone tissue engineering. In this study, β-tricalcium phosphate (β-TCP) was used to coat Mg scaffolds in an effort to modulate the detrimental osteoimmunomodulatory properties of Mg scaffolds, due to the reported favorable osteoimmunomodulatory properties of β-TCP. It was noted that macrophages switched to the M2 extreme phenotype in response to the Mg-β-TCP scaffolds, which could be due to the inhibition of the toll like receptor (TLR) signaling pathway. VEGF and BMP2 were significantly upregulated in the macrophages exposed to Mg-β-TCP scaffolds, indicating pro-osteogenic properties of macrophages in β-TCP modified Mg scaffolds. This was further demonstrated by the macrophage-mediated osteogenic differentiation of bone marrow stromal cells (BMSCs). When BMSCs were stimulated by conditioned medium from macrophages cultured on Mg-β-TCP scaffolds, osteogenic differentiation of BMSCs was significantly enhanced; whereas osteoclastogenesis was inhibited, as indicated by the downregualtion of MCSF, TRAP and inhibition of the RANKL/RANK system. These findings suggest that β-TCP coating of Mg scaffolds can modulate the scaffold's osteoimmunomodulatory properties, shift the immune microenvironment towards one that favors osteogenesis over osteoclastogenesis. Endowing bone biomaterials with favorable osteoimmunomodulatory properties can be a highly valuable strategy for the development or modification of advanced bone biomaterials. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparative evaluation of different calcium phosphate-based bone graft granules - an in vitro study with osteoblast-like cells.

PubMed

Bernhardt, Anne; Lode, Anja; Peters, Fabian; Gelinsky, Michael

2013-04-01

Granule-shaped calcium phosphate-based bone graft materials are often required for bone regeneration especially in implant dentistry. Two newly developed bone graft materials are Ceracell(®) , an open-celled highly porous bioceramic from β-tricalcium phosphate (β-TCP) under addition of bioglass and Osseolive(®) , an open porous glass ceramic with the general formula Ca2 KNa(PO4 )2 . The goal of this study was to characterize different modifications of the two bone graft materials in vitro in comparison to already established ceramic bone grafts Cerasorb M(®) , NanoBone(®) and BONIT Matrix(®) . Adhesion and proliferation of SaOS-2 osteoblast-like cells were evaluated quantitatively by determining DNA content and lactate dehydrogenase (LDH) activity and qualitatively by scanning electron microscopy (SEM). In addition, MTT cell-vitality staining was applied to confirm the attachment of viable cells to the different materials. Osteogenic differentiation was evaluated by measurement of alkaline phosphatase (ALP) activity as well as gene expression analysis of osteogenic markers using reverse transcriptase PCR. DNA content and LDH activity revealed good cell attachment and proliferation for Ceracell and Cerasorb M. When pre-incubated with cell-culture medium, also Osseolive showed good cell attachment and proliferation. Attachment and proliferation of osteoblast-like cells on NanoBone and BONIT Matrix was very low, even after pre-incubation with cell-culture medium. Specific ALP activity on Ceracell(®) , Osseolive (®) and Cerasorb M(®) increased with time and expression of bone-related genes ALP, osteonectin, osteopontin and bone sialoprotein II was demonstrated. Ceracell as well as Osseolive granules support proliferation and osteogenic differentiation in vitro and may be promising candidates for in vivo applications. © 2011 John Wiley & Sons A/S.

To Find a Safe Dose and Show Early Clinical Activity of Weekly Nab-paclitaxel in Pediatric Patients With Recurrent/ Refractory Solid Tumors

ClinicalTrials.gov

2018-04-23

Neuroblastoma; Rhabdomyosarcoma; Ewing's Sarcoma; Ewing's Tumor; Sarcoma, Ewing's; Sarcomas, Epitheliod; Sarcoma, Soft Tissue; Sarcoma, Spindle Cell; Melanoma; Malignant Melanoma; Clinical Oncology; Oncology, Medical; Pediatrics, Osteosarcoma; Osteogenic Sarcoma; Osteosarcoma Tumor; Sarcoma, Osteogenic; Tumors; Cancer; Neoplasia; Neoplasm; Histiocytoma; Fibrosarcoma; Dermatofibrosarcoma

Effects of TGF-β1 and VEGF-A transgenes on the osteogenic potential of bone marrow stromal cells in vitro and in vivo

PubMed Central

Sumner, Dale R; Virdi, Amarjit S

2012-01-01

An exogenous supply of growth factors and bioreplaceable scaffolds may help bone regeneration. The aim of this study was to examine the effects of TGF-β1 and VEGF-A transgenes on the osteogenic potential of bone marrow stromal cells. Rat bone marrow stromal cells were transfected with plasmids encoding mouse TGF-β1 and/or VEGF-A complementary DNAs and cultured for up to 28 days. Furthermore, collagen scaffolds carrying combinations of the plasmids-transfected cells were implanted subcutaneously in rats. The transgenes increased alkaline phosphatase activity, enhanced mineralized nodule formation, and elevated osteogenic gene expressions in vitro. In vivo, messenger RNA expression of osteogenic genes such as BMPs and Runx2 elevated higher by the transgenes. The data indicate that exogenous TGF-β1 and VEGF-A acted synergistically and could induce osteoblastic differentiation of bone marrow stromal cells in both cell culture and an animal model. The results may provide valuable information to optimize protocols for transgene-and-cell-based tissue engineering. PMID:22962632

Synergetic topography and chemistry cues guiding osteogenic differentiation in bone marrow stromal cells through ERK1/2 and p38 MAPK signaling pathway.

PubMed

Zhang, Xinran; Li, Haotian; Lin, Chucheng; Ning, Congqin; Lin, Kaili

2018-01-30

Both the topographic surface and chemical composition modification can enhance rapid osteogenic differentiation and bone formation. Till now, the synergetic effects of topography and chemistry cues guiding biological responses have been rarely reported. Herein, the ordered micro-patterned topography and classically essential trace element of strontium (Sr) ion doping were selected to imitate topography and chemistry cues, respectively. The ordered micro-patterned topography on Sr ion-doped bioceramics was successfully duplicated using the nylon sieve as the template. Biological response results revealed that the micro-patterned topography design or Sr doping could promote cell attachment, ALP activity, and osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Most importantly, the samples both with micro-patterned topography and Sr doping showed the highest promotion effects, and could synergistically activate the ERK1/2 and p38 MAPK signaling pathways. The results suggested that the grafts with both specific topography and chemistry cues have synergetic effects on osteogenic activity of BMSCs and provide an effective approach to design functional bone grafts and cell culture substrates.

Osteogenic differentiation of mesenchymal progenitor cells in computer designed fibrin-polymer-ceramic scaffolds manufactured by fused deposition modeling.

PubMed

Schantz, Jan-Thorsten; Brandwood, Arthur; Hutmacher, Dietmar Werner; Khor, Hwei Ling; Bittner, Katharina

2005-09-01

Biomimetic scaffolds offer great potentials in the development of bone analogs for tissue engineering. The studies presented in this paper focus specifically on the osteogenic potential of the novel PCL/CaP matrices and its degradation behavior. Biodegradable Polymer-ceramic Scaffolds were fabricated using the solid free form fabrication technology: Fused Deposition Modeling (FDM). The scaffold architecture was characterized by a honeycomb-like design and a complete interconnectivity of the pores. Human mesenchymal stem cells (MSCs) were seeded together with fibrin glue into PCL/CaP scaffolds and cultured in vitro for periods of up to eight weeks. Cellular adhesion, proliferation and osteogenic differentiation were assessed in these constructs using a range of histological and microscopic techniques. In additional experiments, degradation was assessed by measuring mass loss, diameter change, molecular weight change and by scanning electron micrographs. MSCs were able to adhere, migrate, and differentiate along the osteogenic lineage with in these scaffolds. The PCL/CaP scaffolds showed up to 27 fold increased degradation of compared to PCL scaffolds.

The use of bone marrow stromal cells (bone marrow-derived multipotent mesenchymal stromal cells) for alveolar bone tissue engineering: basic science to clinical translation.

PubMed

Kagami, Hideaki; Agata, Hideki; Inoue, Minoru; Asahina, Izumi; Tojo, Arinobu; Yamashita, Naohide; Imai, Kohzoh

2014-06-01

Bone tissue engineering is a promising field of regenerative medicine in which cultured cells, scaffolds, and osteogenic inductive signals are used to regenerate bone. Human bone marrow stromal cells (BMSCs) are the most commonly used cell source for bone tissue engineering. Although it is known that cell culture and induction protocols significantly affect the in vivo bone forming ability of BMSCs, the responsible factors of clinical outcome are poorly understood. The results from recent studies using human BMSCs have shown that factors such as passage number and length of osteogenic induction significantly affect ectopic bone formation, although such differences hardly affected the alkaline phosphatase activity or gene expression of osteogenic markers. Application of basic fibroblast growth factor helped to maintain the in vivo osteogenic ability of BMSCs. Importantly, responsiveness of those factors should be tested under clinical circumstances to improve the bone tissue engineering further. In this review, clinical application of bone tissue engineering was reviewed with putative underlying mechanisms.

Indentation of a floating elastic sheet: geometry versus applied tension

NASA Astrophysics Data System (ADS)

Box, Finn; Vella, Dominic; Style, Robert W.; Neufeld, Jerome A.

2017-10-01

The localized loading of an elastic sheet floating on a liquid bath occurs at scales from a frog sitting on a lily pad to a volcano supported by the Earth's tectonic plates. The load is supported by a combination of the stresses within the sheet (which may include applied tensions from, for example, surface tension) and the hydrostatic pressure in the liquid. At the same time, the sheet deforms, and may wrinkle, because of the load. We study this problem in terms of the (relatively weak) applied tension and the indentation depth. For small indentation depths, we find that the force-indentation curve is linear with a stiffness that we characterize in terms of the applied tension and bending stiffness of the sheet. At larger indentations, the force-indentation curve becomes nonlinear and the sheet is subject to a wrinkling instability. We study this wrinkling instability close to the buckling threshold and calculate both the number of wrinkles at onset and the indentation depth at onset, comparing our theoretical results with experiments. Finally, we contrast our results with those previously reported for very thin, highly bendable membranes.

Indentation of a floating elastic sheet: geometry versus applied tension.

PubMed

Box, Finn; Vella, Dominic; Style, Robert W; Neufeld, Jerome A

2017-10-01

The localized loading of an elastic sheet floating on a liquid bath occurs at scales from a frog sitting on a lily pad to a volcano supported by the Earth's tectonic plates. The load is supported by a combination of the stresses within the sheet (which may include applied tensions from, for example, surface tension) and the hydrostatic pressure in the liquid. At the same time, the sheet deforms, and may wrinkle, because of the load. We study this problem in terms of the (relatively weak) applied tension and the indentation depth. For small indentation depths, we find that the force-indentation curve is linear with a stiffness that we characterize in terms of the applied tension and bending stiffness of the sheet. At larger indentations, the force-indentation curve becomes nonlinear and the sheet is subject to a wrinkling instability. We study this wrinkling instability close to the buckling threshold and calculate both the number of wrinkles at onset and the indentation depth at onset, comparing our theoretical results with experiments. Finally, we contrast our results with those previously reported for very thin, highly bendable membranes.

Graphene-supported metal oxide monolith

DOEpatents

Worsley, Marcus A.; Baumann, Theodore F.; Biener, Juergen; Biener, Monika A.; Wang, Yinmin; Ye, Jianchao; Tylski, Elijah

2017-01-10

A composition comprising at least one graphene-supported metal oxide monolith, said monolith comprising a three-dimensional structure of graphene sheets crosslinked by covalent carbon bonds, wherein the graphene sheets are coated by at least one metal oxide such as iron oxide or titanium oxide. Also provided is an electrode comprising the aforementioned graphene-supported metal oxide monolith, wherein the electrode can be substantially free of any carbon-black and substantially free of any binder.

Effect of micro-nano-hybrid structured hydroxyapatite bioceramics on osteogenic and cementogenic differentiation of human periodontal ligament stem cell via Wnt signaling pathway.

PubMed

Mao, Lixia; Liu, Jiaqiang; Zhao, Jinglei; Chang, Jiang; Xia, Lunguo; Jiang, Lingyong; Wang, Xiuhui; Lin, Kaili; Fang, Bing

2015-01-01

The surface structure of bioceramic scaffolds is crucial for its bioactivity and osteoinductive ability, and in recent years, human periodontal ligament stem cells have been certified to possess high osteogenic and cementogenic differential ability. In the present study, hydroxyapatite (HA) bioceramics with micro-nano-hybrid surface (mnHA [the hybrid of nanorods and microrods]) were fabricated via hydrothermal reaction of the α-tricalcium phosphate granules as precursors in aqueous solution, and the effects of mnHA on the attachment, proliferation, osteogenic and cementogenic differentiations of human periodontal ligament stem cells as well as the related mechanisms were systematically investigated. The results showed that mnHA bioceramics could promote cell adhesion, proliferation, alkaline phosphatase (ALP) activity, and expression of osteogenic/cementogenic-related markers including runt-related transcription factor 2 (Runx2), ALP, osteocalcin (OCN), cementum attachment protein (CAP), and cementum protein (CEMP) as compared to the HA bioceramics with flat and dense surface. Moreover, mnHA bioceramics stimulated gene expression of low-density lipoprotein receptor-related protein 5 (LRP5) and β-catenin, which are the key genes of canonical Wnt signaling. Moreover, the stimulatory effect on ALP activity and osteogenic and cementogenic gene expression, including that of ALP, OCN, CAP, CEMP, and Runx2 of mnHA bioceramics could be repressed by canonical Wnt signaling inhibitor dickkopf1 (Dkk1). The results suggested that the HA bioceramics with mnHA could act as promising grafts for periodontal tissue regeneration.

A comparative study of proliferation and osteogenic differentiation of adipose-derived stem cells on akermanite and beta-TCP ceramics.

PubMed

Liu, Qihai; Cen, Lian; Yin, Shuo; Chen, Lei; Liu, Guangpeng; Chang, Jiang; Cui, Lei

2008-12-01

This study investigated the in vitro effects of akermanite, a new kind of Ca-, Mg-, Si-containing bioceramic, on the attachment, proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs). Parallel comparison of the cellular behaviors of hASCs on the akermanite was made with those on beta-tricalcium phosphate (beta-TCP). Scanning electron microscope (SEM) observation and fluorescent DiO labeling were carried out to reveal the attachment and growth of hASCs on the two ceramic surfaces, while the quantitative assay of cell proliferation with time was detected by DNA assay. Osteogenic differentiation of hASCs cultured on the akermanite and beta-TCP was assayed by ALP expression and osteocalcin (OCN) deposition, which was further confirmed by Real-time PCR analysis for markers of osteogenic differentiation. It was shown that hASCs attached and spread well on the akermanite as those on beta-TCP, and similar proliferation behaviors of hASCs were observed on the two ceramics. Both of them exhibited good compatibility to hASCs with only minor cytotoxicity as compared with the tissue culture plates. Interestingly, the osteogenic differentiation of hASCs could be enhanced on the akermanite compared with that on the beta-TCP when the culture time was extended to approximately 10 days. Thus, it can be ascertained that akermanite ceramics may serve as a potential scaffold for bone tissue engineering.

Chip-based comparison of the osteogenesis of human bone marrow- and adipose tissue-derived mesenchymal stem cells under mechanical stimulation.

PubMed

Park, Sang-Hyug; Sim, Woo Young; Min, Byoung-Hyun; Yang, Sang Sik; Khademhosseini, Ali; Kaplan, David L

2012-01-01

Adipose tissue-derived stem cells (ASCs) are considered as an attractive stem cell source for tissue engineering and regenerative medicine. We compared human bone marrow-derived mesenchymal stem cells (hMSCs) and hASCs under dynamic hydraulic compression to evaluate and compare osteogenic abilities. A novel micro cell chip integrated with microvalves and microscale cell culture chambers separated from an air-pressure chamber was developed using microfabrication technology. The microscale chip enables the culture of two types of stem cells concurrently, where each is loaded into cell culture chambers and dynamic compressive stimulation is applied to the cells uniformly. Dynamic hydraulic compression (1 Hz, 1 psi) increased the production of osteogenic matrix components (bone sialoprotein, oateopontin, type I collagen) and integrin (CD11b and CD31) expression from both stem cell sources. Alkaline phosphatase and Alrizarin red staining were evident in the stimulated hMSCs, while the stimulated hASCs did not show significant increases in staining under the same stimulation conditions. Upon application of mechanical stimulus to the two types of stem cells, integrin (β1) and osteogenic gene markers were upregulated from both cell types. In conclusion, stimulated hMSCs and hASCs showed increased osteogenic gene expression compared to non-stimulated groups. The hMSCs were more sensitive to mechanical stimulation and more effective towards osteogenic differentiation than the hASCs under these modes of mechanical stimulation.

Effect of dynamic three-dimensional culture on osteogenic potential of human periodontal ligament-derived mesenchymal stem cells entrapped in alginate microbeads.

PubMed

Vecchiatini, R; Penolazzi, L; Lambertini, E; Angelozzi, M; Morganti, C; Mazzitelli, S; Trombelli, L; Nastruzzi, C; Piva, R

2015-08-01

Bioreactors are devices that efficiently create an environment that enables cell cultures to grow in a three-dimensional (3D) context mimicking in vivo conditions. In this study, we investigate the effect of dynamic fluid flow on the osteogenic potential of human mesenchymal stem cells obtained from periodontal ligament and entrapped in alginate microbeads. After proper immunophenotyping, cells were encapsulated in barium alginate, cultured in 3D static or 3D dynamic conditions represented by a bioreactor system. Calcein-AM/propidium iodide staining was used to assess cellular viability. Quantitative real-time polymerase chain reaction was used to analyze the expression of osteogenic markers (Runx2 and COL1). Alizarin Red S staining and the Fourier transform infrared spectroscopy were used to assess mineral matrix deposition. Optimal encapsulation procedure, in terms of polymer pumping rate, distance from droplet generator to the gelling bath and atomizing airflow was assessed. Cell viability was not affected by encapsulation in alginate microbeads. Bioreactor cell exposure was effective in anticipating osteogenic differentiation and improving mineral matrix deposition. For the first time human mesenchymal stem cells obtained from periodontal ligaments encapsulated in alginate microbeads were cultured in a bioreactor system. This combination could represent a promising strategy to create a cell-based smart system with enhanced osteogenic potential useful for many different dental applications. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Transient changes in oxygen tension inhibit osteogenic differentiation and Runx2 expression in osteoblasts.

PubMed

Salim, Ali; Nacamuli, Randall P; Morgan, Elise F; Giaccia, Amato J; Longaker, Michael T

2004-09-17

Vascular disruption following bony injury results in a hypoxic gradient within the wound microenvironment. Nevertheless, the effects of low oxygen tension on osteogenic precursors remain to be fully elucidated. In the present study, we investigated in vitro osteoblast and mesenchymal stem cell differentiation following exposure to 21% O(2) (ambient oxygen), 2% O(2) (hypoxia), and <0.02% O(2) (anoxia). Hypoxia had little effect on osteogenic differentiation. In contrast, short-term anoxic treatment of primary osteoblasts and mesenchymal precursors inhibited in vitro bone nodule formation and extracellular calcium deposition. Cell viability assays revealed that this effect was not caused by immediate or delayed cell death. Microarray profiling implicated down-regulation of the key osteogenic transcription factor Runx2 as a potential mechanism for the anoxic inhibition of differentiation. Subsequent analysis revealed not only a short-term differential regulation of Runx2 and its targets by anoxia and hypoxia, but a long-term inhibition of Runx2 transcriptional and protein levels after only 12-24 h of anoxic insult. Furthermore, we present evidence that Runx2 inhibition may, at least in part, be because of anoxic repression of BMP2, and that restoring Runx2 levels during anoxia by pretreatment with recombinant BMP2 rescued the anoxic inhibition of differentiation. Taken together, our findings indicate that brief exposure to anoxia (but not 2% hypoxia) down-regulated BMP2 and Runx2 expression, thus inhibiting critical steps in the osteogenic differentiation of pluripotent mesenchymal precursors and committed osteoblasts.

Nanotopographic Substrates of Poly (Methyl Methacrylate) Do Not Strongly Influence the Osteogenic Phenotype of Mesenchymal Stem Cells In Vitro

PubMed Central

Janson, Isaac A.; Kong, Yen P.; Putnam, Andrew J.

2014-01-01

The chemical, mechanical, and topographical features of the extracellular matrix (ECM) have all been documented to influence cell adhesion, gene expression, migration, proliferation, and differentiation. Topography plays a key role in the architecture and functionality of various tissues in vivo, thus raising the possibility that topographic cues can be instructive when incorporated into biomaterials for regenerative applications. In the literature, there are discrepancies regarding the potential roles of nanotopography to enhance the osteogenic phenotype of mesenchymal stem cells (MSC). In this study, we used thin film substrates of poly(methyl methacrylate) (PMMA) with nanoscale gratings to investigate the influence of nanotopography on the osteogenic phenotype of MSCs, focusing in particular on their ability to produce mineral similar to native bone. Topography influenced focal adhesion size and MSC alignment, and enhanced MSC proliferation after 14 days of culture. However, the osteogenic phenotype was minimally influenced by surface topography. Specifically, alkaline phosphatase (ALP) expression was not increased on nanotopographic films, nor was calcium deposition improved after 21 days in culture. Ca: P ratios were similar to native mouse bone on films with gratings of 415 nm width and 200 nm depth (G415) and 303 nm width and 190 nm depth (G303). Notably, all surfaces had Ca∶P ratios significantly lower than G415 films. Collectively, these data suggest that, PMMA films with nanogratings are poor drivers of an osteogenic phenotype. PMID:24594848

Physical characterization and osteogenic activity of the quaternized chitosan-loaded PMMA bone cement.

PubMed

Tan, Honglue; Guo, Shengrong; Yang, Shengbing; Xu, Xiaofen; Tang, Tingting

2012-07-01

Gentamicin-loaded polymethylmethacrylate (PMMA), widely used for primary cemented arthroplasty and revision surgery for preventing or treating infections, may lead to the evolution of antibiotic-resistant bacteria and dysfunction of osteogenic cells, which further influence the osteointegration of bone cement. In a previous study, we reported that a new quaternized chitosan derivative (hydroxypropyltrimethyl ammonium chloride chitosan, HACC) that was loaded into PMMA significantly inhibited the formation of biofilms caused by methicillin-resistant Staphylococcus strains. In the present study, we further investigated the surface morphology, hydrophilicity, apatite formation ability and osteogenic activity of HACC-loaded PMMA. Chitosan-loaded PMMA, gentamicin-loaded PMMA and PMMA without antibiotic were also investigated and compared. The results showed that, compared to other PMMA-based cements, HACC-loaded PMMA had improved properties such as a lower polymerization temperature, prolonged setting time, porous structures after immersion in phosphate-buffered saline, higher hydrophilicity, more apatite formation on the surface after immersion in simulated body fluid, and better attachment and spreading of the human-marrow-derived mesenchymal stem cells. We also found better stem cell proliferation, osteogenic differentiation, and osteogenesis-associated genes expression on the surface of the HACC-loaded PMMA compared to the gentamicin-loaded PMMA. Therefore, this new anti-infective bone cement had improved physical properties and osteogenic activity, which may lead to better osteointegration of the bone cement in cemented arthroplasty. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Ultrasound-responsive gene-activated matrices for osteogenic gene therapy using matrix-assisted sonoporation.

PubMed

Nomikou, N; Feichtinger, G A; Saha, S; Nuernberger, S; Heimel, P; Redl, H; McHale, A P

2018-01-01

Gene-activated matrix (GAM)-based therapeutics for tissue regeneration are limited by efficacy, the lack of spatiotemporal control and availability of target cells, all of which impact negatively on their translation to the clinic. Here, an advanced ultrasound-responsive GAM is described containing target cells that facilitates matrix-assisted sonoporation (MAS) to induce osteogenic differentiation. Ultrasound-responsive GAMs consisting of fibrin/collagen hybrid-matrices containing microbubbles, bone morphogenetic protein BMP2/7 coexpression plasmids together with C2C12 cells were treated with ultrasound either in vitro or following parenteral intramuscular implantation in vivo. Using direct measurement for alkaline phosphatase activity, von Kossa staining and immunohistochemical analysis for osteocalcin expression, MAS-stimulated osteogenic differentiation was confirmed in the GAMs in vitro 7 days after treatment with ultrasound. At day 30 post-treatment with ultrasound, ectopic osteogenic differentiation was confirmed in vivo using X-ray microcomputed tomography and histological analysis. Osteogenic differentiation was indicated by the presence of ectopic bone structures in all animals treated with MAS. In addition, bone volumes in this group were statistically greater than those in the control groups. This novel approach of incorporating a MAS capability into GAMs could be exploited to facilitate ex vivo gene transfer with subsequent surgical implantation or alternatively provide a minimally invasive means of stimulating in situ transgene delivery for osteoinductive gene-based therapies. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Mechanical signals promote osteogenic fate through a primary cilia-mediated mechanism

PubMed Central

Chen, Julia C.; Hoey, David A.; Chua, Mardonn; Bellon, Raymond; Jacobs, Christopher R.

2016-01-01

It has long been suspected, but never directly shown, that bone formed to accommodate an increase in mechanical loading is related to the creation of osteoblasts from skeletal stem cells. Indeed, biophysical stimuli potently regulate osteogenic lineage commitment in vitro. In this study, we transplanted bone marrow cells expressing green fluorescent protein, to enable lineage tracing, and subjected mice to a biophysical stimulus, to elicit a bone-forming response. We detected cells derived from transplanted progenitors embedded within the bone matrix near active bone-forming surfaces in response to loading, demonstrating for the first time, that mechanical signals enhance the homing and attachment of bone marrow cells to bone surfaces and the commitment to an osteogenic lineage of these cells in vivo. Furthermore, we used an inducible Cre/Lox recombination system to delete kinesin family member 3A (Kif3a), a gene that is essential for primary cilia formation, at will in transplanted cells and their progeny, regardless of which tissue may have incorporated them. Disruption of the mechanosensing organelle, the primary cilium in a progenitor population, significantly decreased the amount of bone formed in response to mechanical stimulation. The collective results of our study directly demonstrate that, in a novel experimental stem cell mechanobiology model, mechanical signals enhance osteogenic lineage commitment in vivo and that the primary cilium contributes to this process.—Chen, J. C., Hoey, D. A., Chua, M., Bellon, R., Jacobs, C. R. Mechanical signals promote osteogenic fate through a primary cilia-mediated mechanism. PMID:26675708

The use of SHP-2 gene transduced bone marrow mesenchymal stem cells to promote osteogenic differentiation and bone defect repair in rat.

PubMed

Fan, Dapeng; Liu, Shen; Jiang, Shichao; Li, Zhiwei; Mo, Xiumei; Ruan, Hongjiang; Zou, Gang-Ming; Fan, Cunyi

2016-08-01

Bone tissue engineering is a promising approach for bone regeneration, in which growth factors play an important role. The tyrosine phosphatase Src-homology region 2-containing protein tyrosine phosphatase 2 (SHP2), encoded by the PTPN11 gene, is essential for the differentiation, proliferation and metabolism of osteoblasts. However, SHP-2 has never been systematically studied for its effect in osteogenesis. We predicted that overexpression of SHP-2 could promote bone marrow-derived mesenchymal stem cell (BMSC)osteogenic differentiation and SHP-2 transduced BMSCs could enhance new bone formation, determined using the following study groups: (1) BMSCs transduced with SHP-2 and induced with osteoblast-inducing liquid (BMSCs/SHP-2/OL); (2) BMSCs transduced with SHP-2 (BMSCs/-SHP-2); (3) BMSCs induced with osteoblast-inducing liquid (BMSCs/OL) and (4) pure BMSCs. Cells were assessed for osteogenic differentiation by quantitative real-time polymerase chain reaction analysis, western blot analysis, alkaline phosphatase activity and alizarin red S staining. For in vivo assessment, cells were combined with beta-tricalcium phosphate scaffolds and transplanted into rat calvarial defects for 8 weeks. Following euthanasia, skull samples were explanted for osteogenic evaluation, including micro-computed tomography measurement, histology and immunohistochemistry staining. SHP-2 and upregulation of its gene promoted BMSC osteogenic differentiation and therefore represents a potential new therapeutic approach to bone repair. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1871-1881, 2016. © 2016 Wiley Periodicals, Inc.

[Effect of KH2PO4 on the odonto- and osteogenic differentiation potential of human stem cells from apical papillae].

PubMed

Wang, Yan-ping; Wu, Jin-tao; Wang, Zi-lu; Zheng, Yang-yu; Zhang, Guang-dong; Yu, Jin-hua

2013-01-01

To determine the effects of KH2PO4 on the odonto- and osteogenic differentiation potential of human stem cells from apical papillae (SCAP) in vitro. SCAP were isolated and cultured respectively in alpha minimum essential medium (α-MEM) or α-MEM containing 1.8 mmol/L KH2PO4. Alkaline phosphatase (ALP) activity, alizarin red staining, real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to examine the odonto and osteogenic potential of SCAP in the two media. SCAP cultured in α-MEM containing 1.8 mmol/L KH2PO4 exhibited a higher ALP activity [(0.370 ± 0.013) Sigma unit×min(-1)×mg(-1)] at day 3 than control group [(0.285 ± 0.008) Sigma unit×min(-1)×mg(-1)] and KH2PO4-treated SCAP formed more calcified nodules at day 5 [(0.539 ± 0.007) µg/g] and day 7 [(1.617 ± 0.042) µg/g] than those in normal medium [(0.138 ± 0.037) µg/g, P < 0.01]. The expression of odonto- and osteogenic markers were significantly up-regulated after the stimulation of KH2PO4 at day 3 and 7 respectively, as compared with control group. 1.8 mmol/L KH2PO4 can promote the odonto and osteogenic differentiation potential of human SCAP.

Chip-Based Comparison of the Osteogenesis of Human Bone Marrow- and Adipose Tissue-Derived Mesenchymal Stem Cells under Mechanical Stimulation

PubMed Central

Min, Byoung-Hyun; Yang, Sang Sik; Khademhosseini, Ali; Kaplan, David L.

2012-01-01

Adipose tissue-derived stem cells (ASCs) are considered as an attractive stem cell source for tissue engineering and regenerative medicine. We compared human bone marrow-derived mesenchymal stem cells (hMSCs) and hASCs under dynamic hydraulic compression to evaluate and compare osteogenic abilities. A novel micro cell chip integrated with microvalves and microscale cell culture chambers separated from an air-pressure chamber was developed using microfabrication technology. The microscale chip enables the culture of two types of stem cells concurrently, where each is loaded into cell culture chambers and dynamic compressive stimulation is applied to the cells uniformly. Dynamic hydraulic compression (1 Hz, 1 psi) increased the production of osteogenic matrix components (bone sialoprotein, oateopontin, type I collagen) and integrin (CD11b and CD31) expression from both stem cell sources. Alkaline phosphatase and Alrizarin red staining were evident in the stimulated hMSCs, while the stimulated hASCs did not show significant increases in staining under the same stimulation conditions. Upon application of mechanical stimulus to the two types of stem cells, integrin (β1) and osteogenic gene markers were upregulated from both cell types. In conclusion, stimulated hMSCs and hASCs showed increased osteogenic gene expression compared to non-stimulated groups. The hMSCs were more sensitive to mechanical stimulation and more effective towards osteogenic differentiation than the hASCs under these modes of mechanical stimulation. PMID:23029565

Deformation strain is the main physical driver for skeletal precursors to undergo osteogenesis in earlier stages of osteogenic cell maturation.

PubMed

Ramani-Mohan, Ram-Kumar; Schwedhelm, Ivo; Finne-Wistrand, Anna; Krug, Melanie; Schwarz, Thomas; Jakob, Franz; Walles, Heike; Hansmann, Jan

2018-03-01

Mesenchymal stem cells play a major role during bone remodelling and are thus of high interest for tissue engineering and regenerative medicine applications. Mechanical stimuli, that is, deformation strain and interstitial fluid-flow-induced shear stress, promote osteogenic lineage commitment. However, the predominant physical stimulus that drives early osteogenic cell maturation is not clearly identified. The evaluation of each stimulus is challenging, as deformation and fluid-flow-induced shear stress interdepend. In this study, we developed a bioreactor that was used to culture mesenchymal stem cells harbouring a strain-responsive AP-1 luciferase reporter construct, on porous scaffolds. In addition to the reporter, mineralization and vitality of the cells was investigated by alizarin red staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Quantification of the expression of genes associated to bone regeneration and bone remodelling was used to confirm alizarin red measurements. Controlled perfusion and deformation of the 3-dimensional scaffold facilitated the alteration of the expression of osteogenic markers, luciferase activity, and calcification. To isolate the specific impact of scaffold deformation, a computational model was developed to derive a perfusion flow profile that results in dynamic shear stress conditions present in periodically loaded scaffolds. In comparison to actually deformed scaffolds, a lower expression of all measured readout parameters indicated that deformation strain is the predominant stimulus for skeletal precursors to undergo osteogenesis in earlier stages of osteogenic cell maturation. Copyright © 2017 John Wiley & Sons, Ltd.

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

Application of low-frequency alternating current electric fields via interdigitated electrodes: effects on cellular viability, cytoplasmic calcium, and osteogenic differentiation of human adipose-derived stem cells.

PubMed

McCullen, Seth D; McQuilling, John P; Grossfeld, Robert M; Lubischer, Jane L; Clarke, Laura I; Loboa, Elizabeth G

2010-12-01

Electric stimulation is known to initiate signaling pathways and provides a technique to enhance osteogenic differentiation of stem and/or progenitor cells. There are a variety of in vitro stimulation devices to apply electric fields to such cells. Herein, we describe and highlight the use of interdigitated electrodes to characterize signaling pathways and the effect of electric fields on the proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs). The advantage of the interdigitated electrode configuration is that cells can be easily imaged during short-term (acute) stimulation, and this identical configuration can be utilized for long-term (chronic) studies. Acute exposure of hASCs to alternating current (AC) sinusoidal electric fields of 1 Hz induced a dose-dependent increase in cytoplasmic calcium in response to electric field magnitude, as observed by fluorescence microscopy. hASCs that were chronically exposed to AC electric field treatment of 1 V/cm (4 h/day for 14 days, cultured in the osteogenic differentiation medium containing dexamethasone, ascorbic acid, and β-glycerol phosphate) displayed a significant increase in mineral deposition relative to unstimulated controls. This is the first study to evaluate the effects of sinusoidal AC electric fields on hASCs and to demonstrate that acute and chronic electric field exposure can significantly increase intracellular calcium signaling and the deposition of accreted calcium under osteogenic stimulation, respectively.

The Training and Support Programme for Parents of Children with Ataxia: Parents' Perspectives

ERIC Educational Resources Information Center

Powell, L. A.; Barlow, J. H.

2007-01-01

The aim of the study was to assess the Training and Support Programme (TSP) among parents of children with ataxia. Twenty-seven parents and their children completed the TSP. Data were collected by home record sheets and observation sheets completed by parents and therapists, respectively, and telephone interviews with 10 parents. Benefits reported…

Summary of Internet Terms and Resources. NRC Fact Sheet

ERIC Educational Resources Information Center

Zubal, Rachael; Hall, Mair

2010-01-01

What is the Internet? The Internet is a worldwide network of computers communicating with each other. This paper offers some basic, easy-to-understand meanings of words about the Internet that individuals may have questions about.[The preparation of this fact sheet was supported in part by the National Resource Center on Supported Living and…

ToF-SIMS study of differentiation of human bone-derived stromal cells: new insights into osteoporosis.

PubMed

Schaepe, Kaija; Werner, Janina; Glenske, Kristina; Bartges, Tessa; Henss, Anja; Rohnke, Marcus; Wenisch, Sabine; Janek, Jürgen

2017-07-01

Lipids have numerous important functions in the human body, as they form the cells' plasma membranes and play a key role in many disease states, presumably also in osteoporosis. Here, the fatty acid composition of the outer plasma membranes of cells differentiated into the osteogenic and adipogenic direction is studied with surface-sensitive time-of-flight secondary ion mass spectrometry (ToF-SIMS). For data evaluation, principal component analysis (PCA) is applied. Human (bone-derived) mesenchymal stromal cells (hMSCs) from an osteoporotic donor and a control donor are compared to reveal differences in the fatty acid composition of the membranes. The chemical information is correlated to staining and real-time quantitative polymerase chain reaction (rt-qPCR) results to provide insight into the gene expression of several differentiation markers on the RNA level. Adipogenic differentiation of hMSCs from a non-osteoporotic donor correlates with increased relative intensities of all fatty acids under investigation. After osteogenic differentiation of non-osteoporotic cells, the relative mass signal intensities of unsaturated fatty acids such as oleic and linoleic acids are increased. However, the osteoporotic cells show increased levels of palmitic acid in the plasma membrane after exposure to osteogenic differentiation conditions, which correlates to an immature differentiation state relative to non-osteoporotic osteogenic cells. This immature differentiation state is confirmed by increased early osteogenic differentiation factor Runx2 on RNA level and by less calcium mineralization spots seen in von Kossa staining and ToF-SIMS images. Graphical abstract Time-of-flight secondary ion mass spectrometry is applied to analyze the fatty acid composition of the outer plasma membranes of cells differentiated into the adipogenic and osteogenic direction. Cells from an osteoporotic and a control donor are compared to reveal differences due to differentiation and disease stage of the cells.

Influence of exercise mode and osteogenic index on bone biomarker responses during short-term physical training.

PubMed

Lester, Mark E; Urso, Maria L; Evans, Rachel K; Pierce, Joseph R; Spiering, Barry A; Maresh, Carl M; Hatfield, Disa L; Kraemer, William J; Nindl, Bradley C

2009-10-01

Prescribing exercise based on intensity, frequency, and duration of loading may maximize osteogenic responses in bone, but a model of the osteogenic potential of exercise has not been established in humans. In rodents, an osteogenic index (OI) has been used to predict the osteogenic potential of exercise. The current study sought to determine whether aerobic, resistance, or combined aerobic and resistance exercise programs conducted over eight weeks and compared to a control group could produce changes in biochemical markers of bone turnover indicative of bone formation. We further sought to determine whether an OI could be calculated for each of these programs that would reflect observed biochemical changes. We collected serum biomarkers [bone-specific alkaline phosphatase (BAP), osteocalcin, tartrate-resistant acid phosphatase (TRAP), C-terminal telopeptide fragment of type I collagen (CTx), deoxypyridinoline (DPD), 25-hydroxy vitamin D (25(OH)D), and parathyroid hormone (PTH)] in 56 women (20.3+/-1.8 years) before, during and after eight weeks of training. We also measured bone mineral density (BMD) at regional areas of interest using DXA and pQCT. Biomarkers of bone formation (BAP and osteocalcin) increased in the Resistance and Combined groups (p<0.05), while biomarkers of bone resorption (TRAP and DPD) decreased and increased, respectively, after training (p<0.05) in all groups. Small changes in volumetric and areal BMD (p<0.05) were observed in the distal tibia in the Aerobic and Combined groups, respectively. Mean weekly OIs were 16.0+/-1.9, 20.6+/-2.2, and 36.9+/-5.2 for the Resistance, Aerobic, and Combined groups, respectively. The calculated osteogenic potential of our programs did not correlate with the observed changes in biomarkers of bone turnover. The results of the present study demonstrate that participation in an eight week physical training program that incorporates a resistance component by previously inactive young women results in alterations in biomarkers of bone remodeling indicative of increased formation without substantial alterations in markers of resorption.

On the role of subtype selective adenosine receptor agonists during proliferation and osteogenic differentiation of human primary bone marrow stromal cells.

PubMed

Costa, M Adelina; Barbosa, A; Neto, E; Sá-e-Sousa, A; Freitas, R; Neves, J M; Magalhães-Cardoso, T; Ferreirinha, F; Correia-de-Sá, P

2011-05-01

Purines are important modulators of bone cell biology. ATP is metabolized into adenosine by human primary osteoblast cells (HPOC); due to very low activity of adenosine deaminase, the nucleoside is the end product of the ecto-nucleotidase cascade. We, therefore, investigated the expression and function of adenosine receptor subtypes (A(1) , A(2A) , A(2B) , and A(3) ) during proliferation and osteogenic differentiation of HPOC. Adenosine A(1) (CPA), A(2A) (CGS21680C), A(2B) (NECA), and A(3) (2-Cl-IB-MECA) receptor agonists concentration-dependently increased HPOC proliferation. Agonist-induced HPOC proliferation was prevented by their selective antagonists, DPCPX, SCH442416, PSB603, and MRS1191. CPA and NECA facilitated osteogenic differentiation measured by increases in alkaline phosphatase (ALP) activity. This contrasts with the effect of CGS21680C which delayed HPOC differentiation; 2-Cl-IB-MECA was devoid of effect. Blockade of the A(2B) receptor with PSB603 prevented osteogenic differentiation by NECA. In the presence of the A(1) antagonist, DPCPX, CPA reduced ALP activity at 21 and 28 days in culture. At the same time points, blockade of A(2A) receptors with SCH442416 transformed the inhibitory effect of CGS21680C into facilitation. Inhibition of adenosine uptake with dipyridamole caused a net increase in osteogenic differentiation. The presence of all subtypes of adenosine receptors on HPOC was confirmed by immunocytochemistry. Data show that adenosine is an important regulator of osteogenic cell differentiation through the activation of subtype-specific receptors. The most abundant A(2B) receptor seems to have a consistent role in cell differentiation, which may be balanced through the relative strengths of A(1) or A(2A) receptors determining whether osteoblasts are driven into proliferation or differentiation. Copyright © 2010 Wiley-Liss, Inc.

Insulin-like growth factor-1 (IGF-1) enhances the osteogenic activity of bone morphogenetic protein-6 (BMP-6) in vitro and in vivo, and together have a stronger osteogenic effect than when IGF-1 is combined with BMP-2.

PubMed

Rico-Llanos, Gustavo A; Becerra, Jose; Visser, Rick

2017-07-01

Bone morphogenetic protein-2 (BMP-2) is widely used in orthopedic surgery and bone tissue engineering because of its strong osteogenic activity. However, BMP-2 treatments have several drawbacks and many groups are actively exploring alternatives. Since BMP-6 has been demonstrated to be more osteoinductive, its use, either alone or together with other growth factors, might be an interesting option. In this work, we have compared the effect of BMP-2, BMP-6, or insulin-like growth factor-1 (IGF-1), either alone or in combination. Murine preosteoblasts were treated with 15 nM IGF-1 and/or 6 nM BMP-2 or -6 and the expression of osteogenic marker genes, proliferation, and alkaline phosphatase (ALP) activity in vitro were analyzed. The results showed that IGF-1 greatly enhanced the BMP-induced osteogenic differentiation of these cells in general and that the ALP activity in the cultures was higher when the combination was made with BMP-6 than with BMP-2. Furthermore, we tested the osteogenic potential of these treatments in vivo by loading 25 pmoles of IGF-1 and/or 10 pmoles of BMP-2 or -6 onto absorbable collagen sponges and implanting them into an ectopic bone formation model in rats. This study revealed that only BMP-6 was able to induce bone formation at the used dose and that the addition of IGF-1 contributed to an increase of the mineralization in the implants. Hence, the combination of BMP-6 with IGF-1 might be a better alternative than BMP-2 for orthopedic surgery or bone tissue engineering approaches. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1867-1875, 2017. © 2017 Wiley Periodicals, Inc.

Vitamin D3 contributes to enhanced osteogenic differentiation of MSCs under oxidative stress condition via activating the endogenous antioxidant system.

PubMed

Zhou, J; Wang, F; Ma, Y; Wei, F

2018-06-02

The anti-oxidative effects of vitamin D3 (Vd3) on mesenchymal stem cells (MSCs) have not been studied before. The present study suggested that Vd3 could not only promote the osteogenic differentiation of MSCs under normal condition but also partly protect it from oxidative stress damage by activating the endogenous antioxidant system. Evolving evidence proved that oxidative stress caused by reactive oxygen species (ROS) overproduction might lead to bone loss. Vd3, a commonly used osteogenic induction drug, was proved to exhibit potent anti-oxidative effects on other cell types. The present study aims to investigate the protective effects of Vd3 on oxidative stress-induced dysfunctions of MSCs, as well as its underlying mechanisms. The H 2 O 2 was used as exogenous reactive oxygen species (ROS). The influence of ROS and anti-oxidative protection of Vd3 on MSCs were analyzed too. Multi-techniques were used to assess the beneficial effects of Vd3 on MSCs under oxidative stress condition. The results demonstrated that Vd3 could significantly attenuate the H 2 O 2 -induced cell injury of MSCs via Sirt1/FoxO1 signaling pathway, and reduced the H 2 O 2 exposure-induced intracellular oxidative stress status of MSCs. What's more, the H 2 O 2 exposure resulted in the decreased osteogenic differentiation of MSCs, as evidenced by decreased alkaline phosphatase activity, calcium deposition level, and osteogenic differentiation gene mRNA levels, but the injury was restored via Vd3 administration. The results suggested that Vd3 could not only promote the osteogenic differentiation of osteoblastic cells under normal condition but also partly protect the cell from oxidative stress damage by activating endogenous antioxidant system. The study shed light on the new roles of Vd3 in bone modeling and remodeling regulation.

Osteogenic Differentiation of Mesenchymal Stromal Cells: A Comparative Analysis Between Human Subcutaneous Adipose Tissue and Dental Pulp.

PubMed

D'Alimonte, Iolanda; Mastrangelo, Filiberto; Giuliani, Patricia; Pierdomenico, Laura; Marchisio, Marco; Zuccarini, Mariachiara; Di Iorio, Patrizia; Quaresima, Raimondo; Caciagli, Francesco; Ciccarelli, Renata

2017-06-01

White adipose tissue is a source of mesenchymal stromal/stem cells (MSCs) that are actively studied for their possible therapeutic use in bone tissue repair/remodeling. To better appreciate the osteogenic potential of these cells, we compared some properties of MSCs from human subcutaneous adipose tissue [subcutaneous-adipose stromal cells (S-ASCs)] and dental pulp stem cell (DPSCs) of third-impacted molars, the latter representing a well-established MSC source. Both undifferentiated cell types showed similar fibroblast-like morphology and mesenchymal marker expression. However, undifferentiated S-ASCs displayed a faster doubling time coupled to greater proliferation and colony-forming ability than DPSCs. Also, the osteogenic differentiation of S-ASCs was greater than that of DPSCs, as evaluated by the higher levels of expression of early osteogenic markers Runt-related transcription factor-2 (RUNX2) and alkaline phosphatase at days 3-14 and of extracellular matrix mineralization at days 14-21. Moreover, S-ASCs showed a better colonization of the titanium scaffold. In addition, we investigated whether S-ASC osteogenic commitment was enhanced by adenosine A1 receptor (A1R) stimulation, as previously shown for DPSCs. Although A1R expression was constant during DPSC differentiation, it increased in S-ASC at day 21 from osteogenesis induction. Accordingly, A1R stimulation by the agonist 2-chloro-N 6 -cyclopentyl-adenosine, added to the cultures at each medium change, stimulated proliferation only in differentiating DPSC and enhanced the osteogenic differentiation earlier in DPSCs than in S-ASCs. These effects were counteracted by cell pretreatment with a selective A1R antagonist. Thus, our findings suggest that S-ASCs could be advantageously used in regenerative orthopedics/dentistry, and locally released or exogenously added purines may play a role in bone repair/remodeling, even though this aspect should be more thoroughly evaluated.

Preparation of poly(ethylene glycol)/polylactide hybrid fibrous scaffolds for bone tissue engineering.

PubMed

Ni, PeiYan; Fu, ShaoZhi; Fan, Min; Guo, Gang; Shi, Shuai; Peng, JinRong; Luo, Feng; Qian, ZhiYong

2011-01-01

Polylactide (PLA) electrospun fibers have been reported as a scaffold for bone tissue engineering application, however, the great hydrophobicity limits its broad application. In this study, the hybrid amphiphilic poly(ethylene glycol) (PEG)/hydrophobic PLA fibrous scaffolds exhibited improved morphology with regular and continuous fibers compared to corresponding blank PLA fiber mats. The prepared PEG/PLA fibrous scaffolds favored mesenchymal stem cell (MSC) attachment and proliferation by providing an interconnected porous extracellular environment. Meanwhile, MSCs can penetrate into the fibrous scaffold through the interstitial pores and integrate well with the surrounding fibers, which is very important for favorable application in tissue engineering. More importantly, the electrospun hybrid PEG/PLA fibrous scaffolds can enhance MSCs to differentiate into bone-associated cells by comprehensively evaluating the representative markers of the osteogenic procedure with messenger ribonucleic acid quantitation and protein analysis. MSCs on the PEG/PLA fibrous scaffolds presented better differentiation potential with higher messenger ribonucleic acid expression of the earliest osteogenic marker Cbfa-1 and mid-stage osteogenic marker Col I. The significantly higher alkaline phosphatase activity of the PEG/PLA fibrous scaffolds indicated that these can enhance the differentiation of MSCs into osteoblast-like cells. Furthermore, the higher messenger ribonucleic acid level of the late osteogenic differentiation markers OCN (osteocalcin) and OPN (osteopontin), accompanied by the positive Alizarin red S staining, showed better maturation of osteogenic induction on the PEG/PLA fibrous scaffolds at the mineralization stage of differentiation. After transplantation into the thigh muscle pouches of rats, and evaluating the inflammatory cells surrounding the scaffolds and the physiological characteristics of the surrounding tissues, the PEG/PLA scaffolds presented good biocompatibility. Based on the good cellular response and excellent osteogenic potential in vitro, as well as the biocompatibility with the surrounding tissues in vivo, the electrospun PEG/PLA fibrous scaffolds could be one of the most promising candidates in bone tissue engineering.

MicroRNA hsa-let-7b suppresses the odonto/osteogenic differentiation capacity of stem cells from apical papilla by targeting MMP1.

PubMed

Wang, Yanqiu; Pang, Xiyao; Wu, Jintao; Jin, Lin; Yu, Yan; Gobin, Romila; Yu, Jinhua

2018-01-31

MicroRNA let-7 family acts as the key regulator of the differentiation of mesenchymal stem cells (MSCs). However, the influence of let-7b on biological characteristics of stem cells from apical papilla (SCAPs) is still controversial. In this study, the expression of hsa-let-7b was obviously downregulated during the osteogenic differentiation of SCAPs. SCAPs were then infected with hsa-let-7b or hsa-let-7b inhibitor lentiviruses. The proliferation ability was determined by CCK-8 and flow cytometry. The odonto/osteogenic differentiation capacity was analyzed by alkaline phosphatase (ALP) activity, alizarin red staining, Western blot assay, and real-time RT-PCR. Bioinformatics analysis was used to screen out the target of hsa-let-7b and the target relationship was confirmed by dual luciferase reporter assay. Hsa-let-7b was of no influence on the proliferation of SCAPs. Interferential expression of hsa-let-7b increased the ALP activity as well as the formation of calcified nodules of SCAPs. Moreover, the mRNA levels of osteoblastic markers (ALP, RUNX2, OSX, OPN, and OCN) were upregulated while the protein levels of DSPP, ALP, RUNX2, OSX, OPN, and OCN also increased considerably. Conversely, overexpression of hsa-let-7b inhibited the odonto/osteogenic differentiation capacity of SCAPs. Bioinformatics analysis revealed a putative binding site of hsa-let-7b in the matrix metalloproteinase 1 (MMP1) 3'-untranslated region (3'-UTR). Dual luciferase reporter assay confirmed that hsa-let-7b targets MMP1. The odonto/osteogenic differentiation ability of SCAPs ascended after repression of hsa-let-7b, which was then reversed after co-transfection with siMMP1. Together, hsa-let-7b can suppress the odonto/osteogenic differentiation capacity of SCAPs by targeting MMP1. © 2018 Wiley Periodicals, Inc.

Adiponectin enhances osteogenic differentiation in human adipose-derived stem cells by activating the APPL1-AMPK signaling pathway

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

Chen, Tong; Wu, Yu-wei; Lu, Hui

Human adipose-derived stem cells (hASCs) are multipotent progenitor cells with multi-lineage differentiation potential including osteogenesis and adipogenesis. While significant progress has been made in understanding the transcriptional control of hASC fate, little is known about how hASC differentiation is regulated by the autocrine loop. The most abundant adipocytokine secreted by adipocytes, adiponectin (APN) plays a pivotal role in glucose metabolism and energy homeostasis. Growing evidence suggests a positive association between APN and bone formation yet little is known regarding the direct effects of APN on hASC osteogenesis. Therefore, this study was designed to investigate the varied osteogenic effects and regulatorymore » mechanisms of APN in the osteogenic commitment of hASCs. We found that APN enhanced the expression of osteoblast-related genes in hASCs, such as osteocalcin, alkaline phosphatase, and runt-related transcription factor-2 (Runx2, also known as CBFa1), in a dose- and time-dependent manner. This was further confirmed by the higher expression levels of alkaline phosphatase and increased formation of mineralization nodules, along with the absence of inhibition of cell proliferation. Importantly, APN at 1 μg/ml was the optimal concentration, resulting in maximum deposition of calcium nodules, and was significant superior to bone morphogenetic protein 2. Mechanistically, we found for the first time that APN increased nuclear translocation of the leucine zipper motif (APPL)-1 as well as AMP-activated protein kinase (AMPK) phosphorylation, which were reversed by pretreatment with APPL1 siRNA. Our results indicate that APN promotes the osteogenic differentiation of hASCs by activating APPL1-AMPK signaling, suggesting that manipulation of APN is a novel therapeutic target for controlling hASC fate. - Highlights: • Adiponectin enhances osteogenic differentiation in human adipose-derived stem cells. • The knock-down of APPL1 block the enhancement of osteogenic differentiation in hASC. • AMPK signaling cascade is activated by adiponectin through APPL1.« less

Enhancement of osteogenic differentiation of rat adipose tissue-derived mesenchymal stem cells by zinc sulphate under electromagnetic field via the PKA, ERK1/2 and Wnt/β-catenin signaling pathways

PubMed Central

Fathi, Ezzatollah; Farahzadi, Raheleh

2017-01-01

Zinc ion as an essential trace element and electromagnetic fields (EMFs) has been reported to be involved in the regulation of bone metabolism. The aim of this study was to elucidate the effects of zinc sulphate (ZnSO4) on the osteogenic differentiation of adipose tissue-derived mesenchymal stem cells (ADSCs) in the presence of EMF as a strategy in osteoporosis therapy. Alkaline phophatase (ALP) activity measurement, calcium assay and expression of several osteoblastic marker genes were examined to assess the effect of ZnSO4 on the osteogenic differentiation of ADSCs under EMF. The expression of cAMP and PKA was evaluated by ELISA. The expression of β-catenin, Wnt1, Wnt3a, low-density lipoprotein receptor-related protein 5 (LRP5) and reduced dickkopf1 (DKK1) genes were used to detect the Wnt/β-catenin pathway. It was found that ZnSO4, in the presence of EMF, resulted in an increase in the expression of osteogenic genes, ALP activity and calcium levels. EMF, in the presence of ZnSO4, increased the cAMP level and protein kinase A (PKA) activity. Treatment of ADSCs with (MAPK)/ERK kinase 1/2 inhibitor, or PKA inhibitor, significantly inhibited the promotion of osteogenic markers, indicating that the induction of osteogenesis was dependent on the ERK and PKA signaling pathways. Real-time PCR analysis showed that ZnSO4, in the presence of EMF, increased the mRNA expressions of β-catenin, Wnt1, Wnt3a, LRP5 and DKK1. In this study, it was shown that 0.432 μg/ml ZnSO4, in the presence of 50 Hz, 20 mT EMF, induced the osteogenic differentiation of ADSCs via PKA, ERK1/2 and Wnt/β-catenin signaling pathways. PMID:28339498

Inflammation and Toll-like receptor ligation differentially affect the osteogenic potential of human mesenchymal stromal cells depending on their tissue origin.

PubMed

Raicevic, Gordana; Najar, Mehdi; Pieters, Karlien; De Bruyn, Cecile; Meuleman, Nathalie; Bron, Dominique; Toungouz, Michel; Lagneaux, Laurence

2012-07-01

Mesenchymal stromal cells (MSCs) can be isolated not only from bone marrow (BM) but also from other tissues, including adipose tissue (AT) and umbilical cord Wharton's Jelly (WJ). Thanks to their ability to differentiate into various cell types, MSC are considered attractive candidates for cell-based regenerative therapy. In degenerative clinical settings, inflammation or infection is often involved. In the present work, we hypothesized that an inflammatory environment and/or Toll-like receptor (TLR) ligation could affect the MSC differentiation potential. MSC were isolated from BM, AT, and WJ. Inflammation was mimicked by a cytokine cocktail, and TLR activation was induced through TLR3 and TLR4 ligation. Osteogenesis was chosen as a model for differentiation. Osteogenic parameters were evaluated by measuring Ca2+ deposits and alkaline phosphatase (ALP) activity at day 7, 14, and 21 of the culture in an osteogenic medium. Our results show that WJ-MSC exhibit a much lower osteogenic potential than the other two MSC types. However, inflammation was able to strongly increase the osteogenic differentiation of WJ-MSC as calcification, and ALP activity appeared as early as day 7. However, this latter enzymatic activity remained much lower than that disclosed by BM-MSC. TLR3 or TLR4 triggering increased the osteogenesis in AT- and, to lesser extent, in BM-MSC. In conclusion, WJ-MSC constitutively disclose a lower osteogenic potential as compared with BM and AT-MSC, which is not affected by TLR triggering but is strongly increased by inflammation, then reaching the level of BM-MSC. These observations suggest that WJ-MSC could constitute an alternative of BM-MSC for bone regenerative applications, as WJ is an easy access source of large amounts of MSC that can effectively differentiate into osteoblasts in an inflammatory setting.

Interleukin-18 Enhances Vascular Calcification and Osteogenic Differentiation of Vascular Smooth Muscle Cells Through TRPM7 Activation.

PubMed

Zhang, Kun; Zhang, Yinyin; Feng, Weijing; Chen, Renhua; Chen, Jie; Touyz, Rhian M; Wang, Jingfeng; Huang, Hui

2017-10-01

Vascular calcification (VC) is an important predictor of cardiovascular morbidity and mortality. Osteogenic differentiation of vascular smooth muscle cells (VSMCs) is a key mechanism of VC. Recent studies show that IL-18 (interleukin-18) favors VC while TRPM7 (transient receptor potential melastatin 7) channel upregulation inhibits VC. However, the relationship between IL-18 and TRPM7 is unclear. We questioned whether IL-18 enhances VC and osteogenic differentiation of VSMCs through TRPM7 channel activation. Coronary artery calcification and serum IL-18 were measured in patients by computed tomographic scanning and enzyme-linked immunosorbent assay, respectively. Primary rat VSMCs calcification were induced by high inorganic phosphate and exposed to IL-18. VSMCs were also treated with TRPM7 antagonist 2-aminoethoxy-diphenylborate or TRPM7 small interfering RNA to block TRPM7 channel activity and expression. TRPM7 currents were recorded by patch-clamp. Human studies showed that serum IL-18 levels were positively associated with coronary artery calcium scores ( r =0.91; P <0.001). In VSMCs, IL-18 significantly decreased expression of contractile markers α-smooth muscle actin, smooth muscle 22 α, and increased calcium deposition, alkaline phosphatase activity, and expression of osteogenic differentiation markers bone morphogenetic protein-2, Runx2 (runt-related transcription factor 2), and osteocalcin ( P <0.05). IL-18 increased TRPM7 expression through ERK1/2 (extracellular signal-regulated kinase 1/2) signaling activation, and TRPM7 currents were augmented by IL-18 treatment. Inhibition of TRPM7 channel by 2-aminoethoxy-diphenylborate or TRPM7 small interfering RNA prevented IL-18-enhanced osteogenic differentiation and VSMCs calcification. These findings suggest that coronary artery calcification is associated with increased IL-18 levels. IL-18 enhances VSMCs osteogenic differentiation and subsequent VC induced by β-glycerophosphate via TRPM7 channel activation. Accordingly, IL-18 may contribute to VC in proinflammatory conditions. © 2017 American Heart Association, Inc.

Identification and integrated analysis of differentially expressed lncRNAs and circRNAs reveal the potential ceRNA networks during PDLSC osteogenic differentiation.

PubMed

Gu, Xiuge; Li, Mengying; Jin, Ye; Liu, Dongxu; Wei, Fulan

2017-12-02

Researchers have been exploring the molecular mechanisms underlying the control of periodontal ligament stem cell (PDLSC) osteogenic differentiation. Recently, long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) were shown to function as competitive endogenous RNAs (ceRNAs) to regulate the effect of microRNAs (miRNAs) on their target genes during cell differentiation. However, comprehensive identification and integrated analysis of lncRNAs and circRNAs acting as ceRNAs during PDLSC osteogenic differentiation have not been performed. PDLSCs were derived from healthy human periodontal ligament and cultured separately with osteogenic induction and normal media for 7 days. Cultured PDLSCs were positive for STRO-1 and CD146 and negative for CD31 and CD45. Osteo-induced PDLSCs showed increased ALP (alkaline phosphatase) activity and up-regulated expression levels of the osteogenesis-related markers ALP, Runt-related transcription factor 2 and osteocalcin. Then, a total of 960 lncRNAs and 1456 circRNAs were found to be differentially expressed by RNA sequencing. The expression profiles of eight lncRNAs and eight circRNAs were measured with quantitative real-time polymerase chain reaction and were shown to agree with the RNA-seq results. Furthermore, the potential functions of lncRNAs and circRNAs as ceRNAs were predicted based on miRanda and were investigated using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. In total, 147 lncRNAs and 1382 circRNAs were predicted to combine with 148 common miRNAs and compete for miRNA binding sites with 744 messenger RNAs. These mRNAs were predicted to significantly participate in osteoblast differentiation, the MAPK pathway, the Wnt pathway and the signaling pathways regulating pluripotency of stem cells. Among them, lncRNAs coded as TCONS_00212979 and TCONS_00212984, as well as circRNA BANP and circRNA ITCH, might interact with miRNA34a and miRNA146a to regulate PDLSC osteogenic differentiation via the MAPK pathway. This study comprehensively identified lncRNAs/circRNAs and first integrated their potential ceRNA function during PDLSC osteogenic differentiation. These findings suggest that specific lncRNAs and circRNAs might function as ceRNAs to promote PDLSC osteogenic differentiation and periodontal regeneration.

Magnetically actuated mechanical stimuli on Fe3O4/mineralized collagen coatings to enhance osteogenic differentiation of the MC3T3-E1 cells.

PubMed

Zhuang, Junjun; Lin, Suya; Dong, Lingqing; Cheng, Kui; Weng, Wenjian

2018-04-15

Mechanical stimuli at the bone-implant interface are considered to activate the mechanotransduction pathway of the cell to improve the initial osseointegration establishment and to guarantee clinical success of the implant. However, control of the mechanical stimuli at the bone-implant interface still remains a challenge. In this study, we have designed a strategy of a magnetically responsive coating on which the mechanical stimuli is controlled because of coating deformation under static magnetic field (SMF). The iron oxide nanoparticle/mineralized collagen (IOP-MC) coatings were electrochemically codeposited on titanium substrates in different quantities of IOPs and distributions; the resulting coatings were verified to possess swelling behavior with flexibility same as that of hydrogel. The relative quantity of IOP to collagen and the IOP distribution in the coatings were demonstrated to play a critical role in mediating cell behavior. The cells present on the outer layer of the distributed IOP-MC (O-IOP-MC) coating with a mass ratio of 0.67 revealed the most distinct osteogenic differentiation activity being promoted, which could be attributed to the maximized mechanical stimuli with exposure to SMF. Furthermore, the enhanced osteogenic differentiation of the stimulated MC3T3-E1 cells originated from magnetically actuated mechanotransduction signaling pathway, embodying the upregulated expression of osteogenic-related and mechanotransduction-related genes. This work therefore provides a promising strategy for implementing mechanical stimuli to activate mechanotransduction on the bone-implant interface and thus to promote osseointegration. The magnetically actuated coating is designed to produce mechanical stimuli to cells for promoting osteogenic differentiation based on the coating deformation. Iron oxide nanoparticles (IOPs) were incorporated into the mineralized collagen coatings (MC) forming the composite coatings (IOP-MC) with spatially distributed IOPs, and the IOP-MC coatings with outer distributed IOPs (O-IOPs-MC) shows the maximized mechanical stimuli to cells with enhanced osteogenic differentiation under static magnetic field. The upregulated expression of the associated genes reveals that the enabled mechanotransduction signaling pathway is responsible for the promoted cellular osteogenic differentiation. This work therefore provides a promising strategy for implementing mechanical stimuli to activate mechanotransduction on the bone-implant interface to promote osseointegration. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Resveratrol counteracts bone loss via mitofilin-mediated osteogenic improvement of mesenchymal stem cells in senescence-accelerated mice

PubMed Central

Lv, Ya-Jie; Yang, Yi; Sui, Bing-Dong; Hu, Cheng-Hu; Zhao, Pan; Liao, Li; Chen, Ji; Zhang, Li-Qiang; Yang, Tong-Tao; Zhang, Shao-Feng; Jin, Yan

2018-01-01

Rational: Senescence of mesenchymal stem cells (MSCs) and the related functional decline of osteogenesis have emerged as the critical pathogenesis of osteoporosis in aging. Resveratrol (RESV), a small molecular compound that safely mimics the effects of dietary restriction, has been well documented to extend lifespan in lower organisms and improve health in aging rodents. However, whether RESV promotes function of senescent stem cells in alleviating age-related phenotypes remains largely unknown. Here, we intend to investigate whether RESV counteracts senescence-associated bone loss via osteogenic improvement of MSCs and the underlying mechanism. Methods: MSCs derived from bone marrow (BMMSCs) and the bone-specific, senescence-accelerated, osteoblastogenesis/osteogenesis-defective mice (the SAMP6 strain) were used as experimental models. In vivo application of RESV was performed at 100 mg/kg intraperitoneally once every other day for 2 months, and in vitro application of RESV was performed at 10 μM. Bone mass, bone formation rates and osteogenic differentiation of BMMSCs were primarily evaluated. Metabolic statuses of BMMSCs and the mitochondrial activity, transcription and morphology were also examined. Mitofilin expression was assessed at both mRNA and protein levels, and short hairpin RNA (shRNA)-based gene knockdown was applied for mechanistic experiments. Results: Chronic intermittent application of RESV enhances bone formation and counteracts accelerated bone loss, with RESV improving osteogenic differentiation of senescent BMMSCs. Furthermore, in rescuing osteogenic decline under BMMSC senescence, RESV restores cellular metabolism through mitochondrial functional recovery via facilitating mitochondrial autonomous gene transcription. Molecularly, in alleviating senescence-associated mitochondrial disorders of BMMSCs, particularly the mitochondrial morphological alterations, RESV upregulates Mitofilin, also known as inner membrane protein of mitochondria (Immt) or Mic60, which is the core component of the mitochondrial contact site and cristae organizing system (MICOS). Moreover, Mitofilin is revealed to be indispensable for mitochondrial homeostasis and osteogenesis of BMMSCs, and that insufficiency of Mitofilin leads to BMMSC senescence and bone loss. More importantly, Mitofilin mediates resveratrol-induced mitochondrial and osteogenic improvements of BMMSCs in senescence. Conclusion: Our findings uncover osteogenic functional improvements of senescent MSCs as critical impacts in anti-osteoporotic practice of RESV, and unravel Mitofilin as a novel mechanism mediating RESV promotion on mitochondrial function in stem cell senescence. PMID:29721087

Resveratrol counteracts bone loss via mitofilin-mediated osteogenic improvement of mesenchymal stem cells in senescence-accelerated mice.

PubMed

Lv, Ya-Jie; Yang, Yi; Sui, Bing-Dong; Hu, Cheng-Hu; Zhao, Pan; Liao, Li; Chen, Ji; Zhang, Li-Qiang; Yang, Tong-Tao; Zhang, Shao-Feng; Jin, Yan

2018-01-01

Rational: Senescence of mesenchymal stem cells (MSCs) and the related functional decline of osteogenesis have emerged as the critical pathogenesis of osteoporosis in aging. Resveratrol (RESV), a small molecular compound that safely mimics the effects of dietary restriction, has been well documented to extend lifespan in lower organisms and improve health in aging rodents. However, whether RESV promotes function of senescent stem cells in alleviating age-related phenotypes remains largely unknown. Here, we intend to investigate whether RESV counteracts senescence-associated bone loss via osteogenic improvement of MSCs and the underlying mechanism. Methods: MSCs derived from bone marrow (BMMSCs) and the bone-specific, senescence-accelerated, osteoblastogenesis/osteogenesis-defective mice (the SAMP6 strain) were used as experimental models. In vivo application of RESV was performed at 100 mg/kg intraperitoneally once every other day for 2 months, and in vitro application of RESV was performed at 10 μM. Bone mass, bone formation rates and osteogenic differentiation of BMMSCs were primarily evaluated. Metabolic statuses of BMMSCs and the mitochondrial activity, transcription and morphology were also examined. Mitofilin expression was assessed at both mRNA and protein levels, and short hairpin RNA (shRNA)-based gene knockdown was applied for mechanistic experiments. Results: Chronic intermittent application of RESV enhances bone formation and counteracts accelerated bone loss, with RESV improving osteogenic differentiation of senescent BMMSCs. Furthermore, in rescuing osteogenic decline under BMMSC senescence, RESV restores cellular metabolism through mitochondrial functional recovery via facilitating mitochondrial autonomous gene transcription. Molecularly, in alleviating senescence-associated mitochondrial disorders of BMMSCs, particularly the mitochondrial morphological alterations, RESV upregulates Mitofilin, also known as inner membrane protein of mitochondria (Immt) or Mic60, which is the core component of the mitochondrial contact site and cristae organizing system (MICOS). Moreover, Mitofilin is revealed to be indispensable for mitochondrial homeostasis and osteogenesis of BMMSCs, and that insufficiency of Mitofilin leads to BMMSC senescence and bone loss. More importantly, Mitofilin mediates resveratrol-induced mitochondrial and osteogenic improvements of BMMSCs in senescence. Conclusion: Our findings uncover osteogenic functional improvements of senescent MSCs as critical impacts in anti-osteoporotic practice of RESV, and unravel Mitofilin as a novel mechanism mediating RESV promotion on mitochondrial function in stem cell senescence.

Patient-Derived Human Induced Pluripotent Stem Cells From Gingival Fibroblasts Composited With Defined Nanohydroxyapatite/Chitosan/Gelatin Porous Scaffolds as Potential Bone Graft Substitutes.

PubMed

Ji, Jun; Tong, Xin; Huang, Xiaofeng; Zhang, Junfeng; Qin, Haiyan; Hu, Qingang

2016-01-01

Human embryonic stem cells and adult stem cells have always been the cell source for bone tissue engineering. However, their limitations are obvious, including ethical concerns and/or a short lifespan. The use of human induced pluripotent stem cells (hiPSCs) could avoid these problems. Nanohydroxyapatite (nHA) is an important component of natural bone and bone tissue engineering scaffolds. However, its regulation on osteogenic differentiation with hiPSCs from human gingival fibroblasts (hGFs) is unknown. The purpose of the present study was to investigate the osteogenic differentiation of hiPSCs from patient-derived hGFs regulated by nHA/chitosan/gelatin (HCG) scaffolds with different nHA ratios, such as HCG-111 (1 wt/vol% nHA) and HCG-311 (3 wt/vol% nHA). First, hGFs were reprogrammed into hiPSCs, which have enhanced osteogenic differentiation capability. Second, HCG-111 and HCG-311 scaffolds were successfully synthesized. Finally, hiPSC/HCG complexes were cultured in vitro or subcutaneously transplanted into immunocompromised mice in vivo. The osteogenic differentiation effects of two types of HCG scaffolds on hiPSCs were assessed for up to 12 weeks. The results showed that HCG-311 increased osteogenic-related gene expression of hiPSCs in vitro proved by quantitative real-time polymerase chain reaction, and hiPSC/HCG-311 complexes formed much bone-like tissue in vivo, indicated by cone-beam computed tomography imaging, H&E staining, Masson staining, and RUNX-2, OCN immunohistochemistry staining. In conclusion, our study has shown that osteogenic differentiation of hiPSCs from hGFs was improved by HCG-311. The mechanism might be that the nHA addition stimulates osteogenic marker expression of hiPSCs from hGFs. Our work has provided an innovative autologous cell-based bone tissue engineering approach with soft tissues such as clinically abundant gingiva. The present study focused on patient-personalized bone tissue engineering. Human induced pluripotent stem cells (hiPSCs) were established from clinically easily derived human gingival fibroblasts (hGFs) and defined nanohydroxyapatite/chitosan/gelatin (HCG) scaffolds. hiPSCs derived from hGFs had better osteogenesis capability than that of hGFs. More interestingly, osteogenic differentiation of hiPSCs from hGFs was elevated significantly when composited with HCG-311 scaffolds in vitro and in vivo. The present study has uncovered the important role of different nHA ratios in HCG scaffolds in osteogenesis induction of hiPSCs derived from hGFs. This technique could serve as a potential innovative approach for bone tissue engineering, especially large bone regeneration clinically. ©AlphaMed Press.

Knockdown of SLC41A1 magnesium transporter promotes mineralization and attenuates magnesium inhibition during osteogenesis of mesenchymal stromal cells.

PubMed

Tsao, Yu-Tzu; Shih, Ya-Yi; Liu, Yu-An; Liu, Yi-Shiuan; Lee, Oscar K

2017-02-21

Magnesium is essential for numerous physiological functions. Magnesium exists mostly in bone and the amount is dynamically regulated by skeletal remodeling. Accelerating bone mass loss occurs when magnesium intake is insufficient; whereas high magnesium could lead to mineralization defects. However, the underlying magnesium regulatory mechanisms remain elusive. In the present study, we investigated the effects of high extracellular magnesium concentration on osteogenic differentiation of mesenchymal stromal/stem cells (MSCs) and the role of magnesium transporter SLC41A1 in the mineralization process. Murine MSCs derived from the bone marrow of BALB/c mouse or commercially purchased human MSCs were treated with osteogenic induction medium containing 5.8 mM magnesium chloride and the osteogenic differentiation efficiency was compared with that of MSCs in normal differentiation medium containing 0.8 mM magnesium chloride by cell morphology, gene expression profile of osteogenic markers, and Alizarin Red staining. Slc41a1 gene knockdown in MSCs was performed by siRNA transfection using Lipofectamine RNAiMAX, and the differentiation efficiency of siRNA-treated MSCs was also assessed. High concentration of extracellular magnesium ion inhibited mineralization during osteogenic differentiation of MSCs. Early osteogenic marker genes including osterix, alkaline phosphatase, and type I collagen were significantly downregulated in MSCs under high concentration of magnesium, whereas late marker genes such as osteopontin, osteocalcin, and bone morphogenetic protein 2 were upregulated with statistical significance compared with those in normal differentiation medium containing 0.8 mM magnesium. siRNA treatment targeting SLC41A1 magnesium transporter, a member of the solute carrier family with a predominant Mg 2+ efflux system, accelerated the mineralization process and ameliorated the inhibition of mineralization caused by high concentration of magnesium. High concentration of magnesium significantly upregulated Dkk1 gene expression and the upregulation was attenuated after the Slc41a1 gene was knocked down. Immunofluorescent staining showed that Slc41a1 gene knockdown promoted the translocation of phosphorylated β-catenin into nuclei. In addition, secreted MGP protein was elevated after Slc41a1 was knocked down. High concentration of extracellular magnesium modulates gene expression of MSCs during osteogenic differentiation and inhibits the mineralization process. Additionally, we identified magnesium transporter SLC41A1 that regulates the interaction of magnesium and MSCs during osteogenic differentiation. Wnt signaling is suggested to be involved in SLC41A1-mediated regulation. Tissue-specific SLC41A1 could be a potential treatment for bone mass loss; in addition, caution should be taken regarding the role of magnesium in osteoporosis and the design of magnesium alloys for implantation.

Patient-Derived Human Induced Pluripotent Stem Cells From Gingival Fibroblasts Composited With Defined Nanohydroxyapatite/Chitosan/Gelatin Porous Scaffolds as Potential Bone Graft Substitutes

PubMed Central

Ji, Jun; Tong, Xin; Huang, Xiaofeng; Zhang, Junfeng

2016-01-01

Human embryonic stem cells and adult stem cells have always been the cell source for bone tissue engineering. However, their limitations are obvious, including ethical concerns and/or a short lifespan. The use of human induced pluripotent stem cells (hiPSCs) could avoid these problems. Nanohydroxyapatite (nHA) is an important component of natural bone and bone tissue engineering scaffolds. However, its regulation on osteogenic differentiation with hiPSCs from human gingival fibroblasts (hGFs) is unknown. The purpose of the present study was to investigate the osteogenic differentiation of hiPSCs from patient-derived hGFs regulated by nHA/chitosan/gelatin (HCG) scaffolds with different nHA ratios, such as HCG-111 (1 wt/vol% nHA) and HCG-311 (3 wt/vol% nHA). First, hGFs were reprogrammed into hiPSCs, which have enhanced osteogenic differentiation capability. Second, HCG-111 and HCG-311 scaffolds were successfully synthesized. Finally, hiPSC/HCG complexes were cultured in vitro or subcutaneously transplanted into immunocompromised mice in vivo. The osteogenic differentiation effects of two types of HCG scaffolds on hiPSCs were assessed for up to 12 weeks. The results showed that HCG-311 increased osteogenic-related gene expression of hiPSCs in vitro proved by quantitative real-time polymerase chain reaction, and hiPSC/HCG-311 complexes formed much bone-like tissue in vivo, indicated by cone-beam computed tomography imaging, H&E staining, Masson staining, and RUNX-2, OCN immunohistochemistry staining. In conclusion, our study has shown that osteogenic differentiation of hiPSCs from hGFs was improved by HCG-311. The mechanism might be that the nHA addition stimulates osteogenic marker expression of hiPSCs from hGFs. Our work has provided an innovative autologous cell-based bone tissue engineering approach with soft tissues such as clinically abundant gingiva. Significance The present study focused on patient-personalized bone tissue engineering. Human induced pluripotent stem cells (hiPSCs) were established from clinically easily derived human gingival fibroblasts (hGFs) and defined nanohydroxyapatite/chitosan/gelatin (HCG) scaffolds. hiPSCs derived from hGFs had better osteogenesis capability than that of hGFs. More interestingly, osteogenic differentiation of hiPSCs from hGFs was elevated significantly when composited with HCG-311 scaffolds in vitro and in vivo. The present study has uncovered the important role of different nHA ratios in HCG scaffolds in osteogenesis induction of hiPSCs derived from hGFs. This technique could serve as a potential innovative approach for bone tissue engineering, especially large bone regeneration clinically. PMID:26586776

The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder

NASA Astrophysics Data System (ADS)

Dalby, Matthew J.; Gadegaard, Nikolaj; Tare, Rahul; Andar, Abhay; Riehle, Mathis O.; Herzyk, Pawel; Wilkinson, Chris D. W.; Oreffo, Richard O. C.

2007-12-01

A key tenet of bone tissue engineering is the development of scaffold materials that can stimulate stem cell differentiation in the absence of chemical treatment to become osteoblasts without compromising material properties. At present, conventional implant materials fail owing to encapsulation by soft tissue, rather than direct bone bonding. Here, we demonstrate the use of nanoscale disorder to stimulate human mesenchymal stem cells (MSCs) to produce bone mineral in vitro, in the absence of osteogenic supplements. This approach has similar efficiency to that of cells cultured with osteogenic media. In addition, the current studies show that topographically treated MSCs have a distinct differentiation profile compared with those treated with osteogenic media, which has implications for cell therapies.

Fact Sheet: Summary of Self-Determination. NRC Fact Sheet

ERIC Educational Resources Information Center

Kennedy, Michael; Lewin, Lori

2010-01-01

This fact sheet provides an explanation of what self determination is, provides the four principles of self determination, describes the values supported by self determination. The authors contend that if self-determination is going to be successful, it requires that those who supply services and fund them make certain changes in both the way they…

High surface area graphene-supported metal chalcogenide assembly

DOEpatents

Worsley, Marcus A.; Kuntz, Joshua; Orme, Christine A.

2016-04-19

A composition comprising at least one graphene-supported assembly, which comprises a three-dimensional network of graphene sheets crosslinked by covalent carbon bonds, and at least one metal chalcogenide compound disposed on said graphene sheets, wherein the chalcogen of said metal chalcogenide compound is selected from S, Se and Te. Also disclosed are methods for making and using the graphene-supported assembly, including graphene-supported MoS.sub.2. Monoliths with high surface area and conductivity can be achieved. Lower operating temperatures in some applications can be achieved. Pore size and volume can be tuned.

A space network structure constructed by tetraneedlelike ZnO whiskers supporting boron nitride nanosheets to enhance comprehensive properties of poly(L-lacti acid) scaffolds

PubMed Central

Feng, Pei; Peng, Shuping; Wu, Ping; Gao, Chengde; Huang, Wei; Deng, Youwen; Shuai, Cijun

2016-01-01

In this study, the mechanical strength and modulus of poly(L-lacti acid) (PLLA) scaffolds were enhanced with the mechanical properties of boron nitride nanosheets (BNNSs) and tetraneedlelike ZnO whiskers (T-ZnOw). The adhesion and proliferation of cells were improved as well as osteogenic differentiation of stem cells was increased. Their dispersion statues in PLLA matrix were improved through a space network structure constructed by three-dimensional T-ZnOw supporting two-dimensional BNNSs. The results showed that the compressive strength, modulus and Vickers hardness of the scaffolds with incorporation of 1 wt% BNNSs and 7 wt% T-ZnOw together were about 96.15%, 32.86% and 357.19% higher than that of the PLLA scaffolds, respectively. This might be due to the effect of the pull out and bridging of BNNSs and T-ZnOw as well as the crack deflection, facilitating the formation of effective stress transfer between the reinforcement phases and the matrix. Furthermore, incorporation of BNNSs and T-ZnOw together into PLLA scaffolds was beneficial for attachment and viability of MG-63 cells. More importantly, the scaffolds significantly increased proliferation and promoted osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). The enhanced mechanical and biological properties provide the potentials of PLLA/BNNSs/T-ZnOw scaffolds for the application into bone tissue engineering. PMID:27629058

Particle and field characteristics of the high-latitude plasma sheet boundary layer

NASA Technical Reports Server (NTRS)

Parks, G. K.; Mccarthy, M.; Fitzenreiter, R. J.; Ogilvie, K. W.; Etcheto, J.; Anderson, K. A.; Lin, R. P.; Anderson, R. R.; Eastman, T. E.; Frank, L. A.

1984-01-01

Particle and field data obtained by eight ISEE spacecraft experiments are used to define more precisely the characteristics of the high-latitude boundary region of the plasma sheet. A region immediately adjacent to the high-latitude plasma sheet boundary has particle and field characteristics distinctly different from those observed in the lobe and deeper in the central plasma sheet. Electrons over a broad energy interval are 'field-aligned' and bidirectional, whereas in the plasma sheet the distributions are more isotropic. The region supports intense ion flows, large-amplitude electric fields, and enhanced broad-band electrostatic noise.

Science support for the Earth radiation budget sensor on the Nimbus-7 spacecraft

NASA Technical Reports Server (NTRS)

Ingersoll, A. P.

1982-01-01

Experimental data supporting the Earth radiation budget sensor on the Nimbus 7 Satellite is given. The data deals with the empirical relations between radiative flux, cloudiness, and other meteorological parameters; response of a zonal climate ice sheet model to the orbital perturbations during the quaternary ice ages; and a simple parameterization for ice sheet ablation rate.

Interhemispheric ice-sheet synchronicity during the last glacial maximum

USGS Publications Warehouse

Weber, Michael E.; Clark, Peter U.; Ricken, Werner; Mitrovica, Jerry X.; Hostetler, Steven W.; Kuhn, Gerhard

2011-01-01

The timing of the last maximum extent of the Antarctic ice sheets relative to those in the Northern Hemisphere remains poorly understood. We develop a chronology for the Weddell Sea sector of the East Antarctic Ice Sheet that, combined with ages from other Antarctic ice-sheet sectors, indicates that the advance to and retreat from their maximum extent was within dating uncertainties synchronous with most sectors of Northern Hemisphere ice sheets. Surface climate forcing of Antarctic mass balance would probably cause an opposite response, whereby a warming climate would increase accumulation but not surface melting. Our new data support teleconnections involving sea-level forcing from Northern Hemisphere ice sheets and changes in North Atlantic deep-water formation and attendant heat flux to Antarctic grounding lines to synchronize the hemispheric ice sheets.

Interhemispheric ice-sheet synchronicity during the Last Glacial Maximum.

PubMed

Weber, Michael E; Clark, Peter U; Ricken, Werner; Mitrovica, Jerry X; Hostetler, Steven W; Kuhn, Gerhard

2011-12-02

The timing of the last maximum extent of the Antarctic ice sheets relative to those in the Northern Hemisphere remains poorly understood. We develop a chronology for the Weddell Sea sector of the East Antarctic Ice Sheet that, combined with ages from other Antarctic ice-sheet sectors, indicates that the advance to and retreat from their maximum extent was within dating uncertainties synchronous with most sectors of Northern Hemisphere ice sheets. Surface climate forcing of Antarctic mass balance would probably cause an opposite response, whereby a warming climate would increase accumulation but not surface melting. Our new data support teleconnections involving sea-level forcing from Northern Hemisphere ice sheets and changes in North Atlantic deep-water formation and attendant heat flux to Antarctic grounding lines to synchronize the hemispheric ice sheets.

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

Enhanced osteoprogenitor elongated collagen fiber matrix formation by bioactive glass ionic silicon dependent on Sp7 (osterix) transcription.

PubMed

Varanasi, Venu G; Odatsu, Tetsurou; Bishop, Timothy; Chang, Joyce; Owyoung, Jeremy; Loomer, Peter M

2016-10-01

Bioactive glasses release ions, those enhance osteoblast collagen matrix synthesis and osteogenic marker expression during bone healing. Collagen matrix density and osteogenic marker expression depend on osteogenic transcription factors, (e.g., Osterix (OSX)). We hypothesize that enhanced expression and formation of collagen by Si(4+) depends on enhanced expression of OSX transcription. Experimental bioactive glass (6P53-b) and commercial Bioglass(TM) (45S5) were dissolved in basal medium to make glass conditioned medium (GCM). ICP-MS analysis was used to measure bioactive glass ion release rates. MC3T3-E1 cells were cultured for 20 days, and gene expression and extracellular matrix collagen formation was analyzed. In a separate study, siRNA was used to determine the effect of OSX knockdown on impacting the effect of Si(4+) on osteogenic markers and matrix collagen formation. Each bioactive glass exhibited similar ion release rates for all ions, except Mg(2+) released by 6P53-b. Gene expression results showed that GCM markedly enhanced many osteogenic markers, and 45S5 GCM showed higher levels of expression and collagen matrix fiber bundle density than 6P53-b GCM. Upon knockdown of OSX transcription, collagen type 5, alkaline phosphatase, and matrix density were not enhanced as compared to wild type cells. This study illustrates that the enhancement of elongated collagen fiber matrix formation by Si(±) depends on OSX transcription. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2604-2615, 2016. © 2016 Wiley Periodicals, Inc.

Effect of micro-nano-hybrid structured hydroxyapatite bioceramics on osteogenic and cementogenic differentiation of human periodontal ligament stem cell via Wnt signaling pathway

PubMed Central

Mao, Lixia; Liu, Jiaqiang; Zhao, Jinglei; Chang, Jiang; Xia, Lunguo; Jiang, Lingyong; Wang, Xiuhui; Lin, Kaili; Fang, Bing

2015-01-01

The surface structure of bioceramic scaffolds is crucial for its bioactivity and osteoinductive ability, and in recent years, human periodontal ligament stem cells have been certified to possess high osteogenic and cementogenic differential ability. In the present study, hydroxyapatite (HA) bioceramics with micro-nano-hybrid surface (mnHA [the hybrid of nanorods and microrods]) were fabricated via hydrothermal reaction of the α-tricalcium phosphate granules as precursors in aqueous solution, and the effects of mnHA on the attachment, proliferation, osteogenic and cementogenic differentiations of human periodontal ligament stem cells as well as the related mechanisms were systematically investigated. The results showed that mnHA bioceramics could promote cell adhesion, proliferation, alkaline phosphatase (ALP) activity, and expression of osteogenic/cementogenic-related markers including runt-related transcription factor 2 (Runx2), ALP, osteocalcin (OCN), cementum attachment protein (CAP), and cementum protein (CEMP) as compared to the HA bioceramics with flat and dense surface. Moreover, mnHA bioceramics stimulated gene expression of low-density lipoprotein receptor-related protein 5 (LRP5) and β-catenin, which are the key genes of canonical Wnt signaling. Moreover, the stimulatory effect on ALP activity and osteogenic and cementogenic gene expression, including that of ALP, OCN, CAP, CEMP, and Runx2 of mnHA bioceramics could be repressed by canonical Wnt signaling inhibitor dickkopf1 (Dkk1). The results suggested that the HA bioceramics with mnHA could act as promising grafts for periodontal tissue regeneration. PMID:26648716

Nuclear factor I-C reciprocally regulates adipocyte and osteoblast differentiation via control of canonical Wnt signaling.

PubMed

Zhou, Jie; Wang, Shan; Qi, Qi; Yang, Xiaoyue; Zhu, Endong; Yuan, Hairui; Li, Xuemei; Liu, Ying; Li, Xiaoxia; Wang, Baoli

2017-05-01

Nuclear factor I-C (NFIC) has recently been identified as an important player in osteogenesis and bone homeostasis in vivo However, the molecular mechanisms involved have yet to be defined. In the current study, Nfic expression was altered in primary marrow stromal cells and established progenitor lines after adipogenic and osteogenic treatment. Overexpression of Nfic in stromal cells ST2, mesenchymal cells C3H10T1/2, and primary marrow stromal cells inhibited adipogenic differentiation, whereas it promoted osteogenic differentiation. Conversely, silencing of endogenous Nfic in the cell lines enhanced adipogenic differentiation, whereas it blocked osteogenic differentiation. Mechanism investigations revealed that Nfic overexpression promoted nuclear translocation of β-catenin and increased nuclear protein levels of β-catenin and transcription factor 7-like 2 (TCF7L2). Promoter studies and the chromatin immunoprecipitation (ChIP) assay revealed that NFIC directly binds to the promoter of low-density lipoprotein receptor-related protein 5 (Lrp5) and thereafter transactivates the promoter. Finally, inactivation of canonical Wnt signaling in ST2 attenuated the inhibition of adipogenic differentiation and stimulation of osteogenic differentiation by NFIC. Our study suggests that NFIC balances adipogenic and osteogenic differentiation from progenitor cells through controlling canonical Wnt signaling and highlights the potential of NFIC as a target for new therapies to control metabolic disorders like osteoporosis and obesity.-Zhou, J., Wang, S., Qi, Q., Yang, X., Zhu, E., Yuan, H., Li, X., Liu, Y., Li, X., Wang, B. Nuclear factor I-C reciprocally regulates adipocyte and osteoblast differentiation via control of canonical Wnt signaling. © FASEB.

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

Morinda citrifolia Leaf Extract Enhances Osteogenic Differentiation Through Activation of Wnt/β-Catenin Signaling.

PubMed

Gu, Hanna; Boonanantanasarn, Kanitsak; Kang, Moonkyu; Kim, Ikhwi; Woo, Kyung Mi; Ryoo, Hyun-Mo; Baek, Jeong-Hwa

2018-01-01

Morinda citrifolia (Noni) leaf is an herbal medicine with application in the domestic treatment of a broad range of conditions, including bone fracture and luxation. However, the basic mechanism underlying the stimulation of osteogenic differentiation by Noni leaf extract remains poorly understood. This study aimed to examine the effect of this extract on osteogenic differentiation and the mechanism by which Noni leaf extract enhances osteogenic differentiation. Aqueous extract of Noni leaves was prepared, and rutin and kaempferol-3-O-rutinoside were identified to be two of its major components. C2C12 and human periodontal ligament (hPDL) cells were used to study the effect of Noni. Noni did not show cytotoxicity at a concentration range of 0.015%-1.0% (w/v%) and significantly enhanced the activity of alkaline phosphatase (ALP) and expression levels of osteoblast differentiation markers, including Runx2, ALP, osterix, and osteocalcin, bone morphogenetic protein 2, Wnt3a, and β-catenin. In addition, Noni enhanced the matrix mineralization of hPDL cells. In the signaling pathways, Noni increased the phosphorylation levels of Akt and GSK3β and nuclear translocation and transcriptional activity of β-catenin, which were attenuated by the addition of Dkk-1, a Wnt inhibitor, or LY294002, a PI3K inhibitor. These results suggest that Noni leaf extract enhances osteogenic differentiation through the PI3K/Akt-dependent activation of Wnt/β-catenin signaling. Noni leaf extract might be a novel alternative medicine for bone and periodontal regeneration in patients with periodontal diseases.

FOXO1-suppressed miR-424 regulates the proliferation and osteogenic differentiation of MSCs by targeting FGF2 under oxidative stress

NASA Astrophysics Data System (ADS)

Li, Liangping; Qi, Qihua; Luo, Jiaquan; Huang, Sheng; Ling, Zemin; Gao, Manman; Zhou, Zhiyu; Stiehler, Maik; Zou, Xuenong

2017-02-01

Recently, microRNAs (miRNAs) have been identified as key regulators of the proliferation and differentiation of mesenchymal stem cells (MSCs). Our previous in vivo study and other in vitro studies using miRNA microarrays suggest that miR-424 is involved in the regulation of bone formation. However, the role and mechanism of miR-424 in bone formation still remain unknown. Here, we identified that the downregulation of miR-424 mediates bone formation under oxidative stress, and we explored its underlying mechanism. Our results showed that miR-424 was significantly downregulated in an anterior lumbar interbody fusion model of pigs and in a cell model of oxidative stress induced by H2O2. The overexpression of miR-424 inhibited proliferation and osteogenic differentiation shown by a decrease in alkaline phosphatase (ALP) activity, mineralization and osteogenic markers, including RUNX2 and ALP, whereas the knockdown of miR-424 led to the opposite results. Moreover, miR-424 exerts its effects by targeting FGF2. Furthermore, we found that FOXO1 suppressed miR-424 expression and bound to its promoter region. FOXO1 enhanced proliferation and osteogenic differentiation in part through the miR-424/FGF2 pathway. These results indicated that FOXO1-suppressed miR-424 regulates both the proliferation and osteogenic differentiation of MSCs via targeting FGF2, suggesting that miR-424 might be a potential novel therapeutic strategy for promoting bone formation.

[The effect of Foxc2 overexpression on the osteogenic properties of C3H10T1/2 cells].

PubMed

Wang, Min-Jiao; Si, Jia-Wen; Li, Hong-Liang; Ouyang, Ning-Juan; Shen, Guo-Fang

2016-08-01

To investigate the effect of Foxc2 overexpression on osteogenic and adipogenic differentiation of C3H10T1/2 cells. C3H10T1/2 cells were transfected with plenti-Foxc2 and selected with puromycin for stable clones. The expression of Foxc2 was determined by real-time PCR and Western blot. Cell proliferation was detected by CCK-8 kit. Cell cycle and apoptosis were detected by flow cytometry. The level of osteogenic biomarkers Runx2, OPN, OCN and adipogenic biomarker PPARγ were quantified by real-time PCR and Western blot. Alkaline phosphatase (ALP) staining and oil red staining were conducted to evaluate the effect of Foxc2 overexpression on osteogenic and adipogenic differentiation. Statistical analysis was performed using SPSS 17.0 software package. C3H10T1/2-Foxc2 cell line was successfully constructed and verified by direct sequencing and Foxc2 overexpression in vitro. Cell proliferation was reduced and cell cycle was blocked in G1/G0 phase. Enhanced ALP staining and reduced oil red staining were observed in C3H10T1/2-Foxc2 cells as compared with the control. Foxc2 overexpression up-regulated Runx2, OPN, OCN during osteogenic differentiation and down-regulated PPARγduring adipogenic differentiation. C3H10T1/2 cell line stably expressing Foxc2 gene was successfully established, cell proliferation was reduced, osteogenesis biomarkers were up-regulated during the osteogenesis by overexpression Foxc2, PPARγwas down-regulated during adipogenesis.

CoCl2 , a mimic of hypoxia, enhances bone marrow mesenchymal stem cells migration and osteogenic differentiation via STAT3 signaling pathway.

PubMed

Yu, Xin; Wan, Qilong; Cheng, Gu; Cheng, Xin; Zhang, Jing; Pathak, Janak L; Li, Zubing

2018-06-16

Mesenchymal stem cells homing and migration is a crucial step during bone fracture healing. Hypoxic environment in fracture site induces bone marrow mesenchymal stem cells (BMSCs) migration, but its mechanism remains unclear. Our previous study and studies by other groups have reported the involvement of signal transducer and activator of transcription 3 (STAT3) pathway in cell migration. However, the role of STAT3 pathway in hypoxia-induced cell migration is still unknown. In this study, we investigated the role of STAT3 signaling in hypoxia-induced BMSCs migration and osteogenic differentiation. BMSCs isolated from C57BL/6 male mice were cultured in the presence of cobalt chloride (CoCl 2 ) to simulate intracellular hypoxia. Hypoxia enhanced BMSCs migration, and upregulated cell migration related gene expression i.e., metal-loproteinase (MMP) 7, MMP9 and C-X-C motif chemokine receptor 4. Hypoxia enhanced the phosphorylation of STAT3, and cell migration related proteins: c-jun n-terminal kinase (JNK), focal of adhesion kinase (FAK), extracellular regulated protein kinases and protein kinase B 1/2 (ERK1/2). Moreover, hypoxia enhanced expression of osteogenic differentiation marker. Inhibition of STAT3 suppressed the hy-poxia-induced BMSCs migration, cell migration related signaling molecules phos-phorylation, and osteogenic differentiation related gene expression. In conclusion, our result indicates that hypoxia-induced BMSCs migration and osteogenic differentiation is via STAT3 phosphorylation and involves the cooperative activity of the JNK, FAK and MMP9 signaling pathways. This article is protected by copyright. All rights reserved.

Silibinin promotes osteoblast differentiation of human bone marrow stromal cells via bone morphogenetic protein signaling.

PubMed

Ying, Xiaozhou; Sun, Liaojun; Chen, Xiaowei; Xu, Huazi; Guo, Xiaoshan; Chen, Hua; Hong, Jianjun; Cheng, Shaowen; Peng, Lei

2013-12-05

Silibinin is the major active constituent of the natural compound silymarin; several studies suggest that silibinin possesses antihepatotoxic properties and anticancer effects against carcinoma cells. However, no study has yet investigated the effect of silibinin on osteogenic differentiation of human bone marrow stem cells (hBMSCs). The aim of this study was to evaluate the effect of silibinin on osteogenic differentiation of hBMSCs. In this study, the hBMSCs were cultured in an osteogenic medium with 0, 1, 10 or 20 μmol/l silibinin respectively. hBMSCs viability was analyzed by cell number quantification assay and cells osteogenic differentiation was evaluated by alkaline phosphatas (ALP) activity assay, Von Kossa staining and real time-polymerase chain reaction (RT-PCR). We found that silibinin promoted ALP activity in hBMSCs without affecting their proliferation. The mineralization of hBMSCs was enhanced by treatment with silibinin. Silibinin also increased the mRNA expressions of Collagen type I (COL-I), ALP, Osteocalcin (OCN), Osterix, bone morphogenetic protein-2 (BMP-2) and Runt-related transcription factor 2 (RUNX2). The BMP antagonist noggin and its receptor kinase inhibitors dorsomorphin and LDN-193189 attenuated silibinin-promoted ALP activity. Furthermore, BMP-responsive and Runx2-responsive reporters were activated by silibinin treatment. These results indicate that silibinin enhances osteoblast differentiation probably by inducing the expressions of BMPs and activating BMP and RUNX2 pathways. Thus, silibinin may play an important therapeutic role in osteoporosis patients by improving osteogenic differentiation of BMSCs. © 2013 Elsevier B.V. All rights reserved.

Synergistic effect between bioactive glass foam and a perfusion bioreactor on osteogenic differentiation of human adipose stem cells.

PubMed

Silva, A R P; Paula, A C C; Martins, T M M; Goes, A M; Pereria, M M

2014-03-01

Tissue engineering is a multidisciplinary science that combines a structural scaffold and cells to form a construct able to promote regeneration of injured tissue. Bioactive glass foam produced by sol-gel is an osteoinductive material with a network of interconnected macropores necessary for cell colonization. The use of human adipose-derived stem cell (hASC) presents advantages as the potential for a large number of cells, rapid expansion in vitro and the capability of differentiating into osteoblasts. The use of a bioreactor in three-dimensional cell culture enables greater efficiency for cell nutrition and application of mechanical forces, important modulators of bone physiology. The hASC seeded in a bioactive glass scaffold and cultured in osteogenic Leibovitz L-15 medium in a bioreactor with a flow rate of 0.1 mL min(-1) demonstrated a significant increase in cell proliferation and viability and alkaline phosphatase (ALP) activity peak after 14 days. The immunofluorescence assay revealed an expression of osteopontin, osteocalcin and type I collagen from 7 to 21 days after culture. The cells changed from a spindle shape to a cuboidal morphology characteristic of osteoblasts. The polymerase chain reaction assay confirmed that osteopontin, osteocalcin, and ALP genes were expressed. These results indicate that hASCs differentiated into an osteogenic phenotype when cultured in bioactive glass scaffold, osteogenic Leibovitz L-15 medium and a perfusion bioreactor. Therefore, these results highlight the synergism between a bioactive glass scaffold and the effect of perfusion on cells and indicate the differentiation into an osteogenic phenotype. Copyright © 2013 Wiley Periodicals, Inc.

Stem cell regulatory gene expression in human adult dental pulp and periodontal ligament cells undergoing odontogenic/osteogenic differentiation.

PubMed

Liu, Lu; Ling, Junqi; Wei, Xi; Wu, Liping; Xiao, Yin

2009-10-01

During development and regeneration, odontogenesis and osteogenesis are initiated by a cascade of signals driven by several master regulatory genes. In this study, we investigated the differential expression of 84 stem cell-related genes in dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) undergoing odontogenic/osteogenic differentiation. Our results showed that, although there was considerable overlap, certain genes had more differential expression in PDLCs than in DPCs. CCND2, DLL1, and MME were the major upregulated genes in both PDLCs and DPCs, whereas KRT15 was the only gene significantly downregulated in PDLCs and DPCs in both odontogenic and osteogenic differentiation. Interestingly, a large number of regulatory genes in odontogenic and osteogenic differentiation interact or crosstalk via Notch, Wnt, transforming growth factor beta (TGF-beta)/bone morphogenic protein (BMP), and cadherin signaling pathways, such as the regulation of APC, DLL1, CCND2, BMP2, and CDH1. Using a rat dental pulp and periodontal defect model, the expression and distribution of both BMP2 and CDH1 have been verified for their spatial localization in dental pulp and periodontal tissue regeneration. This study has generated an overview of stem cell-related gene expression in DPCs and PDLCs during odontogenic/osteogenic differentiation and revealed that these genes may interact through the Notch, Wnt, TGF-beta/BMP, and cadherin signaling pathways to play a crucial role in determining the fate of dental derived cell and dental tissue regeneration. These findings provided a new insight into the molecular mechanisms of the dental tissue mineralization and regeneration.

Runx2 Expression in Smooth Muscle Cells Is Required for Arterial Medial Calcification in Mice

PubMed Central

Lin, Mu-En; Chen, Theodore; Leaf, Elizabeth M.; Speer, Mei Y.; Giachelli, Cecilia M.

2016-01-01

Arterial medial calcification (AMC) is a hallmark of aging, diabetes, and chronic kidney disease. Smooth muscle cell (SMC) transition to an osteogenic phenotype is a common feature of AMC, and is preceded by expression of runt-related transcription factor 2 (Runx2), a master regulator of bone development. Whether SMC-specific Runx2 expression is required for osteogenic phenotype change and AMC remains unknown. We therefore created an improved targeting construct to generate mice with floxed Runx2 alleles (Runx2f/f) that do not produce truncated Runx2 proteins after Cre recombination, thereby preventing potential off-target effects. SMC-specific deletion using SM22–recombinase transgenic allele mice (Runx2ΔSM) led to viable mice with normal bone and arterial morphology. After vitamin D overload, arterial SMCs in Runx2f/f mice expressed Runx2, underwent osteogenic phenotype change, and developed severe AMC. In contrast, vitamin D–treated Runx2ΔSM mice had no Runx2 in blood vessels, maintained SMC phenotype, and did not develop AMC. Runx2 deletion did not affect serum calcium, phosphate, fibroblast growth factor-23, or alkaline phosphatase levels. In vitro, Runx2f/f SMCs calcified to a much greater extent than those derived from Runx2ΔSM mice. These data indicate a critical role of Runx2 in SMC osteogenic phenotype change and mineral deposition in a mouse model of AMC, suggesting that Runx2 and downstream osteogenic pathways in SMCs may be useful therapeutic targets for treating or preventing AMC in high-risk patients. PMID:25987250

Novel daidzein analogs enhance osteogenic activity of bone marrow-derived mesenchymal stem cells and adipose-derived stromal/stem cells through estrogen receptor dependent and independent mechanisms

USDA-ARS?s Scientific Manuscript database

Osteoporosis is a disease characterized by low bone mineral density (BMD) and increased risk of fractures. Studies have demonstrated the use of phytoestrogens, or plant-derived estrogens, such as genistein anddaidzein, to effectively increase osteogenic activity of bone marrow-derived mesenchymal s...

Role of geometrical cues in bone marrow-derived mesenchymal stem cell survival, growth and osteogenic differentiation.

PubMed

Gupta, Dhanak; Grant, David M; Zakir Hossain, Kazi M; Ahmed, Ifty; Sottile, Virginie

2018-02-01

Mesenchymal stem cells play a vital role in bone formation process by differentiating into osteoblasts, in a tissue that offers not a flat but a discontinuous three-dimensional (3D) topography in vivo. In order to understand how geometry may be affecting mesenchymal stem cells, this study explored the influence of 3D geometry on mesenchymal stem cell-fate by comparing cell growth, viability and osteogenic potential using monolayer (two-dimensional, 2D) with microsphere (3D) culture systems normalised to surface area. The results suggested lower cell viability and reduced cell growth in 3D. Alkaline phosphatase activity was higher in 3D; however, both collagen and mineral deposition appeared significantly lower in 3D, even after osteogenic supplementation. Also, there were signs of patchy mineralisation in 3D with or without osteogenic supplementation as early as day 7. These results suggest that the convex surfaces on microspheres and inter-particulate porosity may have led to variable cell morphology and fate within the 3D culture. This study provides deeper insights into geometrical regulation of mesenchymal stem cell responses applicable for bone tissue engineering.

Effect of low-magnitude, high-frequency vibration on osteogenic differentiation of rat mesenchymal stromal cells

PubMed Central

Lau, Esther; Lee, Whitaik David; Li, Jason; Xiao, Andrew; Davies, John E.; Wu, Qianhong; Wang, Liyun; You, Lidan

2011-01-01

Whole body vibration (WBV), consisting of a low-magnitude, high-frequency (LMHF) signal, has been found to be anabolic to bone in vivo, which may act through alteration of the lineage commitment of mesenchymal stromal cells (MSC). Here, we investigated the effect of LMHF vibration on rat bone marrow-derived MSCs (rMSCs) in an in vitro system. We subjected rMSCs to repeated (six) bouts of 1-hour vibration at 0.3g and 60 Hz in the presence of osteogenic induction medium. The osteogenic differentiation of rMSCs under the loaded and non-loaded conditions was assessed by examining cell proliferation, alkaline phosphatase (ALP) activity, mRNA expression of various osteoblast-associated markers (ALP, Runx2, osterix, collagen type I alpha 1, bone sialoprotein, osteopontin, and osteocalcin), as well as matrix mineralization. We observed that LMHF vibration did not enhance the osteogenic differentiation of rMSCs. Surprisingly, we found that the mRNA level of osterix, a transcription factor necessary for osteoblast formation, was decreased, and matrix mineralization was inhibited. Our findings suggest that LMHF vibration may exert its anabolic effects in vivo via mechanosensing of a cell type different from MSCs. PMID:21344497

Osteogenic Responses to Zirconia with Hydroxyapatite Coating by Aerosol Deposition

PubMed Central

Cho, Y.; Hong, J.; Ryoo, H.; Kim, D.; Park, J.

2015-01-01

Previously, we found that osteogenic responses to zirconia co-doped with niobium oxide (Nb2O5) or tantalum oxide (Ta2O5) are comparable with responses to titanium, which is widely used as a dental implant material. The present study aimed to evaluate the in vitro osteogenic potential of hydroxyapatite (HA)-coated zirconia by an aerosol deposition method for improved osseointegration. Surface analysis by scanning electron microscopy and x-ray diffraction proved that a thin as-deposited HA film on zirconia showed a shallow, regular, crater-like surface. Deposition of dense and uniform HA films was measured by SEM, and the contact angle test demonstrated improved wettability of the HA-coated surface. Confocal laser scanning microscopy indicated that MC3T3-E1 pre-osteoblast attachment did not differ notably between the titanium and zirconia surfaces; however, cells on the HA-coated zirconia exhibited a lower proliferation than those on the uncoated zirconia late in the culture. Nevertheless, ALP, alizarin red S staining, and bone marker gene expression analysis indicated good osteogenic responses on HA-coated zirconia. Our results suggest that HA-coating by aerosol deposition improves the quality of surface modification and is favorable to osteogenesis. PMID:25586588

The Nell-1 Growth Factor Stimulates Bone Formation by Purified Human Perivascular Cells

PubMed Central

Zhang, Xinli; Péault, Bruno; Chen, Weiwei; Li, Weiming; Corselli, Mirko; James, Aaron W.; Lee, Min; Siu, Ronald K.; Shen, Pang; Zheng, Zhong; Shen, Jia; Kwak, Jinny; Zara, Janette N.; Chen, Feng; Zhang, Hong; Yin, Zack; Wu, Ben; Ting, Kang

2011-01-01

The search for novel sources of stem cells other than bone marrow mesenchymal stem cells (MSCs) for bone regeneration and repair has been a critical endeavor. We previously established an effective protocol to homogeneously purify human pericytes from multiple fetal and adult tissues, including adipose, bone marrow, skeletal muscle, and pancreas, and identified pericytes as a primitive origin of human MSCs. In the present study, we further characterized the osteogenic potential of purified human pericytes combined with a novel osteoinductive growth factor, Nell-1. Purified pericytes grown on either standard culture ware or human cancellous bone chip (hCBC) scaffolds exhibited robust osteogenic differentiation in vitro. Using a nude mouse muscle pouch model, pericytes formed significant new bone in vivo as compared to scaffold alone (hCBC). Moreover, Nell-1 significantly increased pericyte osteogenic differentiation, both in vitro and in vivo. Interestingly, Nell-1 significantly induced pericyte proliferation and was observed to have pro-angiogenic effects, both in vitro and in vivo. These studies suggest that pericytes are a potential new cell source for future efforts in skeletal regenerative medicine, and that Nell-1 is a candidate growth factor able to induce pericyte osteogenic differentiation. PMID:21615216

Understanding the influence of phosphorylation and polysialylation of gelatin on mineralization and osteogenic differentiation.

PubMed

Arora, Aditya; Katti, Dhirendra S

2016-08-01

Post-translational modifications such as phosphorylation and sialylation impart crucial functions such as mineral deposition and osteogenic differentiation to non-collagenous bone matrix proteins. In this work, the influence of phosphorylation and polysialylation of gelatin on mineralization in simulated body fluid (SBF) and on osteogenic differentiation of mesenchymal stem cells (MSC) was studied. It was observed that increase in phosphorylation could be directly correlated with the mineralization ability of phosphorylated gelatin in SBF. The total calcium and phosphate deposited increased with increase in degree of phosphorylation and was >3 fold higher on the highest degree of phosphorylation. Whereas, polysialylation did not have any significant influence on mineral deposition in SBF. On the other hand, when MSCs were cultured on polysialylated surfaces they showed relatively higher cell elongation with 1.5 fold higher cell aspect ratio, higher alkaline phosphatase activity and 3 fold higher mineral deposition when compared to control and phosphorylated gelatin surfaces. In conclusion, phosphorylation and polysialylation of gelatin show a significant influence on mineralization and osteogenic differentiation respectively which can be advantageously used for bone tissue engineering. Copyright © 2016 Elsevier B.V. All rights reserved.

Sonochemical synthesis of fructose 1,6-bisphosphate dicalcium porous microspheres and their application in promotion of osteogenic differentiation.

PubMed

Qi, Chao; Zhou, Ding; Zhu, Ying-Jie; Sun, Tuan-Wei; Chen, Feng; Zhang, Chang-Qing

2017-08-01

Human bone mesenchymal stem cells (hBMSCs) have the ability to differentiate into bone and cartilage for clinical bone regeneration. Biomaterials with an innate ability to stimulate osteogenic differentiation of hBMSCs into bone and cartilage are considered attractive candidates for the applications in bone tissue engineering and regeneration. In this paper, we synthesized fructose 1,6-bisphosphate dicalcium (Ca 2 FBP) porous microspheres by the sonochemical method, and investigated the ability of Ca 2 FBP for the promotion of the osteogenic differentiation of hBMSCs. After the hBMSCs were co-cultured with the sterilized powder of Ca 2 FBP porous microspheres for different times, the cell proliferation assay, alkaline phosphatase activity assay, quantitative real-time polymerase chain reaction and western blotting were performed to investigate the bioactivity and osteogenic differentiation performance of the as-prepared product. Compared with hydroxyapatite nanorods, Ca 2 FBP porous microspheres show a superior bioactivity and osteoinductive potential, and can promote the cell differentiation of hBMSCs in vitro, thus, they are promising for applications in the tissue engineering field such as dental and bone defect repair. Copyright © 2017 Elsevier B.V. All rights reserved.

Bioactive borate glass promotes the repair of radius segmental bone defects by enhancing the osteogenic differentiation of BMSCs.

PubMed

Zhang, Jieyuan; Guan, Junjie; Zhang, Changqing; Wang, Hui; Huang, Wenhai; Guo, Shangchun; Niu, Xin; Xie, Zongping; Wang, Yang

2015-11-20

Bioactive borate glass (BG) has emerged as a promising alternative for bone regeneration due to its high osteoinductivity, osteoconductivity, compressive strength, and biocompatibility. However, the role of BG in large segmental bone repair is unclear and little is known about the underlying mechanism of BG's osteoinductivity. In this study, we demonstrated that BG possessed pro-osteogenic effects in an experimental model of critical-sized radius defects. Transplanting BG to radius defects resulted in better repair of bone defects as compared to widely used β-TCP. Histological and morphological analysis indicated that BG significantly enhanced new bone formation. Furthermore, the degradation rate of the BG was faster than that of β-TCP, which matched the higher bone regeneration rate. In addition, ions from BG enhanced cell viability, ALP activity, and osteogenic-related genes expression. Mechanistically, the critical genes Smad1/5 and Dlx5 in the BMP pathway and p-Smad1/5 proteins were significantly elevated after BG transplantation, and these effects could be blocked by the BMP/Smad specific inhibitor. Taken together, our findings suggest that BG could repair large segmental bone defects through activating the BMP/Smad pathway and osteogenic differentiation in BMSCs.

In vitro effects of ascorbic acid and β-glycerophosphate on human gingival fibroblast cells.

PubMed

Martinez, Elizabeth F; Donato, Tatiani A G; Arana-Chavez, Victor E

2012-10-01

Ascorbic acid (AA) and β-glycerophosphate (βG) are considered in vitro osteogenic factors important to the differentiation of osteoblastic progenitor and dental pulp cells into mineralized tissue-forming cells. So, the present study investigated in vitro if these mineralizing inducible factors (AA and βG) could influence differentiation of human gingival fibroblasts when compared with human pulp cells and osteogenic cells derived from rat calvaria cultured. The expression of osteopontin (OPN) and osteoadherin (OSAD) was analyzed by indirect immunofluorescence, immunocytochemistry as well as Western-blotting. In addition, the main ultrastructural aspects were also investigated. No mineralized matrix formation occurred on gingival fibroblasts induced with AA+βG. On these cells, no expression of OPN and OSAD was observed when compared with pulp cells, pulp cells induced with AA+βG as well as osteogenic cells. Ultrastructure analysis additionally showed that gingival fibroblasts exhibited typical fibroblast morphology with no nodule formation. The present findings showed that AA and βG could not promote a mineralized cell differentiation of human gingival fibroblasts and confirm that human dental pulp cells, as the osteogenic cells, are capable to form a mineralized extracellular. Copyright © 2012 Elsevier Ltd. All rights reserved.

Indentation of a floating elastic sheet: geometry versus applied tension

PubMed Central

Box, Finn; Style, Robert W.; Neufeld, Jerome A.

2017-01-01

The localized loading of an elastic sheet floating on a liquid bath occurs at scales from a frog sitting on a lily pad to a volcano supported by the Earth’s tectonic plates. The load is supported by a combination of the stresses within the sheet (which may include applied tensions from, for example, surface tension) and the hydrostatic pressure in the liquid. At the same time, the sheet deforms, and may wrinkle, because of the load. We study this problem in terms of the (relatively weak) applied tension and the indentation depth. For small indentation depths, we find that the force–indentation curve is linear with a stiffness that we characterize in terms of the applied tension and bending stiffness of the sheet. At larger indentations, the force–indentation curve becomes nonlinear and the sheet is subject to a wrinkling instability. We study this wrinkling instability close to the buckling threshold and calculate both the number of wrinkles at onset and the indentation depth at onset, comparing our theoretical results with experiments. Finally, we contrast our results with those previously reported for very thin, highly bendable membranes. PMID:29118662

Analysis and design of composite slab by varying different parameters

NASA Astrophysics Data System (ADS)

Lambe, Kedar; Siddh, Sharda

2018-03-01

Composite deck slabs are in demand because of its faster, lighter and economical construction work. Composite slab consists of cold formed deck profiled sheet and concrete either lightweight or normal. Investigation of shear behaviour of the composite slab is very complex. Shear bond strength depends on the various parameter such as a shape of sheeting, a thickness of the sheet, type of embossment and its frequency of use, shear stiffener or intermediate stiffener, type of load, an arrangement of load, length of shear span, the thickness of concrete and support friction etc. In present study finite element analysis is carried out with ABAQUS 6.13, a simply supported composite slab is considered for the investigation of the shear bond behaviour of the composite slab by considering variation in three different parameters, the shape of a sheet, thickness of sheet and shear span. Different shear spans of two different shape of cold formed deck profiled sheet i.e. with intermediate stiffeners and without intermediate stiffeners are considered with two different thicknesses (0.8 mm and 1.2 mm) for simulation. In present work, simulation of models has done for static loading with 20 mm mesh size is considered.

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

Wang, Lifeng, E-mail: walfe@nuaa.edu.cn; Hu, Haiyan

The thermal vibration of a rectangular single-layered graphene sheet is investigated by using a rectangular nonlocal elastic plate model with quantum effects taken into account when the law of energy equipartition is unreliable. The relation between the temperature and the Root of Mean Squared (RMS) amplitude of vibration at any point of the rectangular single-layered graphene sheet in simply supported case is derived first from the rectangular nonlocal elastic plate model with the strain gradient of the second order taken into consideration so as to characterize the effect of microstructure of the graphene sheet. Then, the RMS amplitude of thermalmore » vibration of a rectangular single-layered graphene sheet simply supported on an elastic foundation is derived. The study shows that the RMS amplitude of the rectangular single-layered graphene sheet predicted from the quantum theory is lower than that predicted from the law of energy equipartition. The maximal relative difference of RMS amplitude of thermal vibration appears at the sheet corners. The microstructure of the graphene sheet has a little effect on the thermal vibrations of lower modes, but exhibits an obvious effect on the thermal vibrations of higher modes. The quantum effect is more important for the thermal vibration of higher modes in the case of smaller sides and lower temperature. The relative difference of maximal RMS amplitude of thermal vibration of a rectangular single-layered graphene sheet decreases monotonically with an increase of temperature. The absolute difference of maximal RMS amplitude of thermal vibration of a rectangular single-layered graphene sheet increases slowly with the rising of Winkler foundation modulus.« less

Noninvasive in situ evaluation of osteogenic differentiation by time-resolved laser-induced fluorescence spectroscopy.

PubMed

Ashjian, Peter; Elbarbary, Amir; Zuk, Patricia; DeUgarte, Daniel A; Benhaim, Prosper; Marcu, Laura; Hedrick, Marc H

2004-01-01

The clinical implantation of bioengineered tissues requires an in situ nondestructive evaluation of the quality of tissue constructs developed in vitro before transplantation. Time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) is demonstrated here to noninvasively monitor the formation of osteogenic extracellular matrix (ECM) produced by putative stem cells (PLA cells) derived from human adipose tissue. We show that this optical spectroscopy technique can assess the relative expression of collagens (types I, III, IV, and V) within newly forming osteogenic ECM. The results are consistent with those obtained by conventional histochemical techniques (immunofluorescence and Western blot) and demonstrate that TR-LIFS is a potential tool for monitoring the expression of distinct collagen types and the formation of collagen cross-links in intact tissue constructs.

Geometric Design Laboratory Fact Sheet

DOT National Transportation Integrated Search

2006-08-02

This fact sheet provides concise information about the Geometric Design Laboratory (GDL) at the Turner-Fairbank Highway Research Center. The mission of the GDL is to provide technical support to the Federal Highway Administration's Office of Safety R...

CD90 (Thy-1)-positive selection enhances osteogenic capacity of human adipose-derived stromal cells.

PubMed

Chung, Michael T; Liu, Chunjun; Hyun, Jeong S; Lo, David D; Montoro, Daniel T; Hasegawa, Masakazu; Li, Shuli; Sorkin, Michael; Rennert, Robert; Keeney, Michael; Yang, Fan; Quarto, Natalina; Longaker, Michael T; Wan, Derrick C

2013-04-01

Stem cell-based bone tissue engineering with adipose-derived stromal cells (ASCs) has shown great promise for revolutionizing treatment of large bone deficits. However, there is still a lack of consensus on cell surface markers identifying osteoprogenitors. Fluorescence-activated cell sorting has identified a subpopulation of CD105(low) cells with enhanced osteogenic differentiation. The purpose of the present study was to compare the ability of CD90 (Thy-1) to identify osteoprogenitors relative to CD(105). Unsorted cells, CD90(+), CD90(-), CD105(high), and CD105(low) cells were treated with an osteogenic differentiation medium. For evaluation of in vitro osteogenesis, alkaline phosphatase (ALP) staining and alizarin red staining were performed at 7 days and 14 days, respectively. RNA was harvested after 7 and 14 days of differentiation, and osteogenic gene expression was examined by quantitative real-time polymerase chain reaction. For evaluation of in vivo osteogenesis, critical-sized (4-mm) calvarial defects in nude mice were treated with the hydroxyapatite-poly(lactic-co-glycolic acid) scaffold seeded with the above-mentioned subpopulations. Healing was followed using micro-CT scans for 8 weeks. Calvaria were harvested at 8 weeks postoperatively, and sections were stained with Movat's Pentachrome. Transcriptional analysis revealed that the CD90(+) subpopulation was enriched for a more osteogenic subtype relative to the CD105(low) subpopulation. Staining at day 7 for ALP was greatest in the CD90(+) cells, followed by the CD105(low) cells. Staining at day 14 for alizarin red demonstrated the greatest amount of mineralized extracellular matrix in the CD90(+) cells, again followed by the CD105(low) cells. Quantification of in vivo healing at 2, 4, 6, and 8weeks postoperatively demonstrated increased bone formation in defects treated with CD90(+) ASCs relative to all other groups. On Movat's Pentachrome-stained sections, defects treated with CD90(+) cells showed the most robust bony regeneration. Defects treated with CD90(-) cells, CD105(high) cells, and CD105(low) cells demonstrated some bone formation, but to a lesser degree when compared with the CD90(+) group. While CD105(low) cells have previously been shown to possess an enhanced osteogenic potential, we found that CD90(+) cells are more capable of forming bone both in vitro and in vivo. These data therefore suggest that CD90 may be a more effective marker than CD105 to isolate a highly osteogenic subpopulation for bone tissue engineering.

CD90 (Thy-1)-Positive Selection Enhances Osteogenic Capacity of Human Adipose-Derived Stromal Cells

PubMed Central

Chung, Michael T.; Liu, Chunjun; Hyun, Jeong S.; Lo, David D.; Montoro, Daniel T.; Hasegawa, Masakazu; Li, Shuli; Sorkin, Michael; Rennert, Robert; Keeney, Michael; Yang, Fan; Quarto, Natalina; Longaker, Michael T.

2013-01-01

Background Stem cell-based bone tissue engineering with adipose-derived stromal cells (ASCs) has shown great promise for revolutionizing treatment of large bone deficits. However, there is still a lack of consensus on cell surface markers identifying osteoprogenitors. Fluorescence-activated cell sorting has identified a subpopulation of CD105low cells with enhanced osteogenic differentiation. The purpose of the present study was to compare the ability of CD90 (Thy-1) to identify osteoprogenitors relative to CD105. Methods Unsorted cells, CD90+, CD90−, CD105high, and CD105low cells were treated with an osteogenic differentiation medium. For evaluation of in vitro osteogenesis, alkaline phosphatase (ALP) staining and alizarin red staining were performed at 7 days and 14 days, respectively. RNA was harvested after 7 and 14 days of differentiation, and osteogenic gene expression was examined by quantitative real-time polymerase chain reaction. For evaluation of in vivo osteogenesis, critical-sized (4-mm) calvarial defects in nude mice were treated with the hydroxyapatite-poly(lactic-co-glycolic acid) scaffold seeded with the above-mentioned subpopulations. Healing was followed using micro-CT scans for 8 weeks. Calvaria were harvested at 8 weeks postoperatively, and sections were stained with Movat's Pentachrome. Results Transcriptional analysis revealed that the CD90+ subpopulation was enriched for a more osteogenic subtype relative to the CD105low subpopulation. Staining at day 7 for ALP was greatest in the CD90+ cells, followed by the CD105low cells. Staining at day 14 for alizarin red demonstrated the greatest amount of mineralized extracellular matrix in the CD90+ cells, again followed by the CD105low cells. Quantification of in vivo healing at 2, 4, 6, and 8weeks postoperatively demonstrated increased bone formation in defects treated with CD90+ ASCs relative to all other groups. On Movat's Pentachrome-stained sections, defects treated with CD90+ cells showed the most robust bony regeneration. Defects treated with CD90− cells, CD105high cells, and CD105low cells demonstrated some bone formation, but to a lesser degree when compared with the CD90+ group. Conclusions While CD105low cells have previously been shown to possess an enhanced osteogenic potential, we found that CD90+ cells are more capable of forming bone both in vitro and in vivo. These data therefore suggest that CD90 may be a more effective marker than CD105 to isolate a highly osteogenic subpopulation for bone tissue engineering. PMID:23216074

Mechanisms of Cdc42-mediated rat MSC differentiation on micro/nano-textured topography.

PubMed

Li, Guangwen; Song, Yanyan; Shi, Mengqi; Du, Yuanhong; Wang, Wei; Zhang, Yumei

2017-02-01

Micro/nano-textured titanium surface topography promotes osteoblast differentiation and the Wnt/β-catenin signaling pathway. However, the response of rat bone mesenchymal stem cells (MSCs) to micro/nano-textured topography, and the underlying mechanisms of its effects, are not well understood. We hypothesized that cell division cycle 42 protein (Cdc42), a key member of the Rho GTPases family, may regulate rat MSCs morphology and osteogenic differentiation by micro/nano-textured topography, and that crosstalk between Cdc42 and Wnt/β-catenin is the underlying mechanism. To confirm the hypothesis, we first tested rat MSCs' morphology, cytoskeleton, and osteogenic differentiation on micro/nano-textured topography. We then examined the cells' Wnt pathway and Cdc42 signaling activity. The results show that micro/nano-textured topography enhances MSCs' osteogenic differentiation. In addition, the cells' morphology and cytoskeletal reorganization were dramatically different on smooth surfaces and micropitted/nanotubular topography. Ligands of the canonical Wnt pathway, as well as accumulation of β-catenin in the nucleus, were up-regulated by micro/nano-textured topography. Cdc42 protein expression was markedly increased under these conditions; conversely, Cdc42 silencing significantly depressed the enhancement of MSCs osteogenic differentiation by micro/nano-textured topography. Moreover, Cdc42si attenuated p-GSK3β activation and resulted in β-catenin cytoplasmic degradation on the micro/nano-textured topography. Our results indicate that Cdc42 is a key modulator of rat MSCs morphology and cytoskeletal reorganization, and that crosstalk between Cdc42 and Wnt/β-catenin signaling though GSK3β regulates MSCs osteogenic differentiation by implant topographical cues. Topographical modification at micro- and nanoscale is widely applied to enhance the tissue integration properties of biomaterials. However, the response of bone mesenchymal stem cells (MSCs) to the micro/nano-textured topography and the underlying mechanisms are not well understood. This study shows that the micropitted/nanotubular hierarchical topography produced by etching and anodic oxidation treatment drives fusiform cell morphology, cytoskeletal reorganization as well as better MSCs osteogenic differentiation. The cross-talk between Cdc42 pathway and Wnt/β-catenin pathway though GSK3β modulates the osteoinductive effect of the micro/nano-textured topography on MSCs. This finding sheds light on a novel mechanism involved in micro/nano-textured surface-mediated MSCs osteogenic differentiation and is a major step in the development of new surface modifications aiming to accelerate and enhance the process of osseointegration. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Multiway modeling and analysis in stem cell systems biology

PubMed Central

2008-01-01

Background Systems biology refers to multidisciplinary approaches designed to uncover emergent properties of biological systems. Stem cells are an attractive target for this analysis, due to their broad therapeutic potential. A central theme of systems biology is the use of computational modeling to reconstruct complex systems from a wealth of reductionist, molecular data (e.g., gene/protein expression, signal transduction activity, metabolic activity, etc.). A number of deterministic, probabilistic, and statistical learning models are used to understand sophisticated cellular behaviors such as protein expression during cellular differentiation and the activity of signaling networks. However, many of these models are bimodal i.e., they only consider row-column relationships. In contrast, multiway modeling techniques (also known as tensor models) can analyze multimodal data, which capture much more information about complex behaviors such as cell differentiation. In particular, tensors can be very powerful tools for modeling the dynamic activity of biological networks over time. Here, we review the application of systems biology to stem cells and illustrate application of tensor analysis to model collagen-induced osteogenic differentiation of human mesenchymal stem cells. Results We applied Tucker1, Tucker3, and Parallel Factor Analysis (PARAFAC) models to identify protein/gene expression patterns during extracellular matrix-induced osteogenic differentiation of human mesenchymal stem cells. In one case, we organized our data into a tensor of type protein/gene locus link × gene ontology category × osteogenic stimulant, and found that our cells expressed two distinct, stimulus-dependent sets of functionally related genes as they underwent osteogenic differentiation. In a second case, we organized DNA microarray data in a three-way tensor of gene IDs × osteogenic stimulus × replicates, and found that application of tensile strain to a collagen I substrate accelerated the osteogenic differentiation induced by a static collagen I substrate. Conclusion Our results suggest gene- and protein-level models whereby stem cells undergo transdifferentiation to osteoblasts, and lay the foundation for mechanistic, hypothesis-driven studies. Our analysis methods are applicable to a wide range of stem cell differentiation models. PMID:18625054

Influence of cantilevered sheet pile deflection on adjacent roadways.

DOT National Transportation Integrated Search

2009-06-01

Cantilevered sheet pile walls are often used adjacent roadways as temporary support during construction. Excess movement of these walls has led to excessive roadway distress causing additional repairs to be necessary. This study assessed the effects ...

Compared to the amniotic membrane, Wharton's jelly may be a more suitable source of mesenchymal stem cells for cardiovascular tissue engineering and clinical regeneration.

PubMed

Pu, Lei; Meng, Mingyao; Wu, Jian; Zhang, Jing; Hou, Zongliu; Gao, Hui; Xu, Hui; Liu, Boyu; Tang, Weiwei; Jiang, Lihong; Li, Yaxiong

2017-03-21

The success of developing cardiovascular tissue engineering (CTE) grafts greatly needs a readily available cell substitute for endothelial and interstitial cells. Perinatal annexes have been proposed as a valuable source of mesenchymal stem cells (MSCs) for tissue engineering and regenerative medicine. The objective of the present study is to evaluate the potential of human Wharton's jelly MSCs (WJ-MSCs) and amniotic membrane MSCs (AM-MSCs) as a seeding cell in CTE and cardiovascular regenerative medicine. WJ-MSCs/AM-MSCs were isolated and characterized in vitro according to their morphology, proliferation, self-renewal, phenotype, and multipotency. More importantly, the characteristics of hemocompatibility, extracellular matrix deposition, and gene expression and viability of both MSCs were investigated. Fibroblast-like human WJ-MSCs and AM-MSCs were successfully isolated and positively expressed the characteristic markers CD73, CD90, and CD105 but were negative for CD34, CD45, and HLA-DR. Both MSCs shared trilineage differentiation toward the adipogenic, osteogenic, and chondrogenic lineages. The proliferative and self-renewal capacity of WJ-MSCs was significantly higher than that of AM-MSCs (P < 0.001). WJ-MSCs provided comparable properties of antiplatelet adhesion and did not activate the coagulation cascade to endothelial cells. However, aggregated platelets were visualized on the surface of AM-MSCs-derived cell sheets and the intrinsic pathway was activated. Furthermore, WJ-MSCs have superior properties of collagen deposition and higher viability than AM-MSCs during cell sheet formation. This study highlights that WJ-MSCs could act as a functional substitute of endothelial and interstitial cells, which could serve as an appealing and practical single-cell source for CTE and regenerative therapy.

Cross-talk between EGF and BMP9 signalling pathways regulates the osteogenic differentiation of mesenchymal stem cells.

PubMed

Liu, Xing; Qin, Jiaqiang; Luo, Qing; Bi, Yang; Zhu, Gaohui; Jiang, Wei; Kim, Stephanie H; Li, Mi; Su, Yuxi; Nan, Guoxin; Cui, Jing; Zhang, Wenwen; Li, Ruidong; Chen, Xiang; Kong, Yuhan; Zhang, Jiye; Wang, Jinhua; Rogers, Mary Rose; Zhang, Hongyu; Shui, Wei; Zhao, Chen; Wang, Ning; Liang, Xi; Wu, Ningning; He, Yunfeng; Luu, Hue H; Haydon, Rex C; Shi, Lewis L; Li, Tingyu; He, Tong-Chuan; Li, Ming

2013-09-01

Mesenchymal stem cells (MSCs) are multipotent progenitors, which give rise to several lineages, including bone, cartilage and fat. Epidermal growth factor (EGF) stimulates cell growth, proliferation and differentiation. EGF acts by binding with high affinity to epidermal growth factor receptor (EGFR) on the cell surface and stimulating the intrinsic protein tyrosine kinase activity of its receptor, which initiates a signal transduction cascade causing a variety of biochemical changes within the cell and regulating cell proliferation and differentiation. We have identified BMP9 as one of the most osteogenic BMPs in MSCs. In this study, we investigate if EGF signalling cross-talks with BMP9 and regulates BMP9-induced osteogenic differentiation. We find that EGF potentiates BMP9-induced early and late osteogenic markers of MSCs in vitro, which can be effectively blunted by EGFR inhibitors Gefitinib and Erlotinib or receptor tyrosine kinase inhibitors AG-1478 and AG-494 in a dose- and time-dependent manner. Furthermore, EGF significantly augments BMP9-induced bone formation in the cultured mouse foetal limb explants. In vivo stem cell implantation experiment reveals that exogenous expression of EGF in MSCs can effectively potentiate BMP9-induced ectopic bone formation, yielding larger and more mature bone masses. Interestingly, we find that, while EGF can induce BMP9 expression in MSCs, EGFR expression is directly up-regulated by BMP9 through Smad1/5/8 signalling pathway. Thus, the cross-talk between EGF and BMP9 signalling pathways in MSCs may underline their important roles in regulating osteogenic differentiation. Harnessing the synergy between BMP9 and EGF should be beneficial for enhancing osteogenesis in regenerative medicine. © 2013 The Authors. Journal of Cellular and Molecular Medicine Published by Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

Cross-talk between EGF and BMP9 signalling pathways regulates the osteogenic differentiation of mesenchymal stem cells

PubMed Central

Liu, Xing; Qin, Jiaqiang; Luo, Qing; Bi, Yang; Zhu, Gaohui; Jiang, Wei; Kim, Stephanie H; Li, Mi; Su, Yuxi; Nan, Guoxin; Cui, Jing; Zhang, Wenwen; Li, Ruidong; Chen, Xiang; Kong, Yuhan; Zhang, Jiye; Wang, Jinhua; Rogers, Mary Rose; Zhang, Hongyu; Shui, Wei; Zhao, Chen; Wang, Ning; Liang, Xi; Wu, Ningning; He, Yunfeng; Luu, Hue H; Haydon, Rex C; Shi, Lewis L; Li, Tingyu; He, Tong-Chuan; Li, Ming

2013-01-01

Mesenchymal stem cells (MSCs) are multipotent progenitors, which give rise to several lineages, including bone, cartilage and fat. Epidermal growth factor (EGF) stimulates cell growth, proliferation and differentiation. EGF acts by binding with high affinity to epidermal growth factor receptor (EGFR) on the cell surface and stimulating the intrinsic protein tyrosine kinase activity of its receptor, which initiates a signal transduction cascade causing a variety of biochemical changes within the cell and regulating cell proliferation and differentiation. We have identified BMP9 as one of the most osteogenic BMPs in MSCs. In this study, we investigate if EGF signalling cross-talks with BMP9 and regulates BMP9-induced osteogenic differentiation. We find that EGF potentiates BMP9-induced early and late osteogenic markers of MSCs in vitro, which can be effectively blunted by EGFR inhibitors Gefitinib and Erlotinib or receptor tyrosine kinase inhibitors AG-1478 and AG-494 in a dose- and time-dependent manner. Furthermore, EGF significantly augments BMP9-induced bone formation in the cultured mouse foetal limb explants. In vivo stem cell implantation experiment reveals that exogenous expression of EGF in MSCs can effectively potentiate BMP9-induced ectopic bone formation, yielding larger and more mature bone masses. Interestingly, we find that, while EGF can induce BMP9 expression in MSCs, EGFR expression is directly up-regulated by BMP9 through Smad1/5/8 signalling pathway. Thus, the cross-talk between EGF and BMP9 signalling pathways in MSCs may underline their important roles in regulating osteogenic differentiation. Harnessing the synergy between BMP9 and EGF should be beneficial for enhancing osteogenesis in regenerative medicine. PMID:23844832

Osteogenic Differentiation of Three-Dimensional Bioprinted Constructs Consisting of Human Adipose-Derived Stem Cells In Vitro and In Vivo.

PubMed

Wang, Xiao-Fei; Song, Yang; Liu, Yun-Song; Sun, Yu-Chun; Wang, Yu-Guang; Wang, Yong; Lyu, Pei-Jun

2016-01-01

Here, we aimed to investigate osteogenic differentiation of human adipose-derived stem cells (hASCs) in three-dimensional (3D) bioprinted tissue constructs in vitro and in vivo. A 3D Bio-plotter dispensing system was used for building 3D constructs. Cell viability was determined using live/dead cell staining. After 7 and 14 days of culture, real-time quantitative polymerase chain reaction (PCR) was performed to analyze the expression of osteogenesis-related genes (RUNX2, OSX, and OCN). Western blotting for RUNX2 and immunofluorescent staining for OCN and RUNX2 were also performed. At 8 weeks after surgery, osteoids secreted by osteogenically differentiated cells were assessed by hematoxylin-eosin (H&E) staining, Masson trichrome staining, and OCN immunohistochemical staining. Results from live/dead cell staining showed that most of the cells remained alive, with a cell viability of 89%, on day 1 after printing. In vitro osteogenic induction of the 3D construct showed that the expression levels of RUNX2, OSX, and OCN were significantly increased on days 7 and 14 after printing in cells cultured in osteogenic medium (OM) compared with that in normal proliferation medium (PM). Fluorescence microscopy and western blotting showed that the expression of osteogenesis-related proteins was significantly higher in cells cultured in OM than in cells cultured in PM. In vivo studies demonstrated obvious bone matrix formation in the 3D bioprinted constructs. These results indicated that 3D bioprinted constructs consisting of hASCs had the ability to promote mineralized matrix formation and that hASCs could be used in 3D bioprinted constructs for the repair of large bone tissue defects.

Differences in the calcification of preosteoblast cultured on sputter-deposited titanium, zirconium, and gold.

PubMed

Chen, Peng; Nagai, Akiko; Tsutsumi, Yusuke; Ashida, Maki; Doi, Hisashi; Hanawa, Takao

2016-03-01

In this study, osteogenic differentiation and calcification of preosteoblast (MC3T3-E1) cultured on sputter-deposited titanium (Ti), zirconium (Zr), and gold (Au) on cover glasses were evaluated to understand the differences in bone formation ability among these three metals; these metals show the same high corrosion resistance, but Ti and Zr are covered by surface passive oxide film while Au is not covered by the oxide film. Ti and Zr promoted cellular proliferation without osteogenic differentiation. Cells cultured on Ti and Zr expressed higher levels of Runx2, Col1α1, and Akp2 at an earlier stage, which indicated faster promotion of osteogenic differentiation, as compared to those cultured on Au. Moreover, after 21 days of culture, the Bglap1 and Ifitm5 expression peaks in cells cultured on Ti and Zr were higher than those in cells cultured on Au, which indicated faster promotion of calcification. Cells cultured on Ti showed an advantage in osteogenic differentiation at an early stage, while cells on Zr showed better calcification promotion with a long-term culture. The amount of extracellular calcified deposits was in good agreement with the gene expression results. On the other hand, the intracellular calcium content of cells on Au specimens was higher than that of cells on Ti and Zr specimens. The results indicate that preosteoblasts on Ti and Zr showed faster osteogenic differentiation and calcification than those on Au, whereas Au improved the intracellular calcium content. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 639-651, 2016. © 2015 Wiley Periodicals, Inc.

Rapamycin inhibits BMP-7-induced osteogenic and lipogenic marker expressions in fetal rat calvarial cells.

PubMed

Yeh, Lee-Chuan C; Ma, Xiuye; Ford, Jeffery J; Adamo, Martin L; Lee, John C

2013-08-01

Bone morphogenetic proteins (BMPs) promote osteoblast differentiation and bone formation in vitro and in vivo. BMPs canonically signal through Smad transcription factors, but BMPs may activate signaling pathways traditionally stimulated by growth factor tyrosine kinase receptors. Of these, the mTOR pathway has received considerable attention because BMPs activate P70S6K, a downstream effector of mTOR, suggesting that BMP-induced osteogenesis is mediated by mTOR activation. However, contradictory effects of the mTOR inhibitor rapamycin (RAPA) on bone formation have been reported. Since bone formation is thought to be inversely related to lipid accumulation and mTOR is also important for lipid synthesis, we postulated that BMP-7 may stimulate lipogenic enzyme expression in a RAPA-sensitive mechanism. To test this hypothesis, we determined the effects of RAPA on BMP-7-stimulated expression of osteogenic and lipogenic markers in cultured fetal rat calvarial cells. Our study showed that BMP-7 promoted the expression of osteogenic and lipogenic markers. The effect of BMP-7 on osteogenic markers was greater in magnitude than on lipogenic markers and was temporally more sustained. RAPA inhibited basal and BMP-7-stimulated osteogenic and lipogenic marker expression and bone nodule mineralization. The acetyl CoA carboxylase inhibitor TOFA stimulated the expression of osteoblast differentiation markers, whereas palmitate suppressed their expression. We speculate that the BMP-7-stimulated adipogenesis is part of the normal anabolic response to BMPs, but that inappropriate activation of the lipid biosynthetic pathway by mTOR could have deleterious effects on bone formation and could explain paradoxical effects of RAPA to promote bone formation. Copyright © 2013 Wiley Periodicals, Inc.

Effect of surface modification of nanofibres with glutamic acid peptide on calcium phosphate nucleation and osteogenic differentiation of marrow stromal cells.

PubMed

Karaman, Ozan; Kumar, Ankur; Moeinzadeh, Seyedsina; He, Xuezhong; Cui, Tong; Jabbari, Esmaiel

2016-02-01

Biomineralization is mediated by extracellular matrix (ECM) proteins with amino acid sequences rich in glutamic acid. The objective of this study was to investigate the effect of calcium phosphate deposition on aligned nanofibres surface-modified with a glutamic acid peptide on osteogenic differentiation of rat marrow stromal cells. Blend of EEGGC peptide (GLU) conjugated low molecular weight polylactide (PLA) and high molecular weight poly(lactide-co-glycolide) (PLGA) was electrospun to form aligned nanofibres (GLU-NF). The GLU-NF microsheets were incubated in a modified simulated body fluid for nucleation of calcium phosphate crystals on the fibre surface. To achieve a high calcium phosphate to fibre ratio, a layer-by-layer approach was used to improve diffusion of calcium and phosphate ions inside the microsheets. Based on dissipative particle dynamics simulation of PLGA/PLA-GLU fibres, > 80% of GLU peptide was localized to the fibre surface. Calcium phosphate to fibre ratios as high as 200%, between those of cancellous (160%) and cortical (310%) bone, was obtained with the layer-by-layer approach. The extent of osteogenic differentiation and mineralization of marrow stromal cells seeded on GLU-NF microsheets was directly related to the amount of calcium phosphate deposition on the fibres prior to cell seeding. Expression of osteogenic markers osteopontin, alkaline phosphatase (ALP), osteocalcin and type 1 collagen increased gradually with calcium phosphate deposition on GLU-NF microsheets. Results demonstrate that surface modification of aligned synthetic nanofibres with EEGGC peptide dramatically affects nucleation and growth of calcium phosphate crystals on the fibres leading to increased osteogenic differentiation of marrow stromal cells and mineralization. Copyright © 2013 John Wiley & Sons, Ltd.

Origins of endothelial and osteogenic cells in the subcutaneous collagen gel implant.

PubMed

Bilic-Curcic, I; Kalajzic, Z; Wang, L; Rowe, D W

2005-11-01

The interdependent relationship between vascular endothelial cells and osteoblasts during bone formation and fracture healing has been long appreciated. This paper reports a heterotopic implant model using FGF-2-expanded bone marrow stromal cells (BMSC) derived from Tie2eGFP (endothelial marker) and pOBCol3.6GFPcyan or topaz (early osteoblast marker) transgenic mice to appreciate the host/donor relationships of cells participating in the process of heterotopic bone formation. The study included various combinations of Tie2eGFP and pOBCol3.6GFPcyan and topaz transgenics as BMSC or whole bone marrow (WBM) donors and also as recipients. Rat tail collagen was used as a carrier of donor cells and implantation was done in lethally irradiated mice rescued with WBM injection. Development of ossicles in the implants was followed weekly during the 4- to 5-week long post-implantation period. By 4-5 weeks after total body irradiation (TBI) and implantation, a well-formed bone spicule had developed that was invested with bone marrow. Experiments showed absolute dominance of donor-derived cells in the formation of endothelial-lined vessels inside the implants as well as the marrow stromal-derived osteogenic cells. Host-derived fibroblasts and osteogenic cells were confined to the fibrous capsule surrounding the implant. In addition, cells lining the endosteal surface of newly formed marrow space carrying a pOBCol3.6GFP marker were observed that were contributed by WBM donor cells and the host. Thus, FGF-2-expanded BMSC appear to be a source of endothelial and osteogenic progenitor cells capable of eliciting heterotopic bone formation independent of cells from the host. This model should be useful for understanding the interactions between these two cell types that control osteogenic differentiation in vivo.

Restoration of miR-1305 relieves the inhibitory effect of nicotine on periodontal ligament-derived stem cell proliferation, migration, and osteogenic differentiation.

PubMed

Chen, Zhuo; Liu, Hui-Li

2017-04-01

Nicotine hinders the regenerative potentials of human periodontal ligament-derived stem cells (PDLSCs) and delays the healing process of periodontal diseases, but the underlying mechanism remains unclear. miR-1305 upregulation and its potential target RUNX2 downregulation exist in the PDLSCs exposed to nicotine. In this study, we aimed to investigate whether nicotine inhibits PDLSC proliferation, migration, and osteogenic differentiation by increasing miR-1305 level and decreasing RUNX2 level. Quantitative real-time PCR (qRT-PCR) and Western blot assays were performed to detect the expression levels of miR-1305 and RUNX2 in the PDLSCs exposed to nicotine, respectively. PDLSCs with miR-1305 overexpression, low expression, or RUNX2 overexpression were constructed by lipofectin transfection. MTT, migration, and Western blot assays were applied to assess the effect of miR-1305 on PDLSC proliferation, migration, and osteogenic differentiation, respectively. Target prediction and luciferase reporter assays were performed to investigate the targets of miR-1305. Nicotine promoted miR-1305 expression and inhibited RUNX2 expression in PDLSCs. Cell proliferation, migration, and differentiation detection showed that nicotine suppressed proliferation, migration, and osteogenic differentiation of PDLSCs, and restoration of miR-1305 relieved the inhibitory effect of nicotine on PDLSCs. Moreover, we identified and validated that RUNX2 was a direct target of miR-1305, and upregulation of RUNX2 had similar effects with the downregulation of miR-1305 on relieving the inhibitory effect of nicotine on PDLSCs. Nicotine suppresses proliferation, migration, and osteogenic differentiation of PDLSCs, and restoration of miR-1305 relieves the inhibitory effect of nicotine on PDLSCs depending on its target RUNX2. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Aspirin Enhances Osteogenic Potential of Periodontal Ligament Stem Cells (PDLSCs) and Modulates the Expression Profile of Growth Factor-Associated Genes in PDLSCs.

PubMed

Abd Rahman, Fazliny; Mohd Ali, Johari; Abdullah, Mariam; Abu Kasim, Noor Hayaty; Musa, Sabri

2016-07-01

This study investigates the effects of aspirin (ASA) on the proliferative capacity, osteogenic potential, and expression of growth factor-associated genes in periodontal ligament stem cells (PDLSCs). Mesenchymal stem cells (MSCs) from PDL tissue were isolated from human premolars (n = 3). The MSCs' identity was confirmed by immunophenotyping and trilineage differentiation assays. Cell proliferation activity was assessed through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Polymerase chain reaction array was used to profile the expression of 84 growth factor-associated genes. Pathway analysis was used to identify the biologic functions and canonic pathways activated by ASA treatment. The osteogenic potential was evaluated through mineralization assay. ASA at 1,000 μM enhances osteogenic potential of PDLSCs. Using a fold change (FC) of 2.0 as a threshold value, the gene expression analyses indicated that 19 genes were differentially expressed, which includes 12 upregulated and seven downregulated genes. Fibroblast growth factor 9 (FGF9), vascular endothelial growth factor A (VEGFA), interleukin-2, bone morphogenetic protein-10, VEGFC, and 2 (FGF2) were markedly upregulated (FC range, 6 to 15), whereas pleotropin, FGF5, brain-derived neurotrophic factor, and Dickkopf WNT signaling pathway inhibitor 1 were markedly downregulated (FC 32). Of the 84 growth factor-associated genes screened, 35 showed high cycle threshold values (≥35). ASA modulates the expression of growth factor-associated genes and enhances osteogenic potential in PDLSCs. ASA upregulated the expression of genes that could activate biologic functions and canonic pathways related to cell proliferation, human embryonic stem cell pluripotency, tissue regeneration, and differentiation. These findings suggest that ASA enhances PDLSC function and may be useful in regenerative dentistry applications, particularly in the areas of periodontal health and regeneration.

Long noncoding RNA H19 mediates LCoR to impact the osteogenic and adipogenic differentiation of mBMSCs in mice through sponging miR-188.

PubMed

Wang, Yijun; Liu, Wentao; Liu, Yadong; Cui, Jianli; Zhao, Zhiwei; Cao, Hui; Fu, Zhuo; Liu, Bin

2018-04-16

The research aimed to examine the expression of lncRNA H19, miR-188, and LCoR in mouse bone marrow stromal stem cells (mBMSCs), and to investigate the regulatory mechanism of lncRNA H19/miR-188/LCoR in osteogenic and adipogenic differentiation of mBMSCs. The expression of miR-188 in mBMSCs and osteogenesis induced mBMSCs was detected by stem-loop RT-PCR, while the expression of H19 and LCoR in mBMSCs and adipogenesis induced mBMSCs was examined by qRT-PCR. Luciferase reporter assay verified the targeted relationship between miR-188 and H19 or LCoR. Cell proliferation ability was determined by MTT assay, while cell surface markers of mBMSCs were analyzed via flow cytometry. Alkaline phosphatase staining and Alizarin red staining was utilized to detect the osteogenic differentiation capability of mBMSCs, whereas Oil red O staining was applied to examine the ability of adipogenic differentiation of mBMSCs. The expression of miR-188 was lower in osteogenesis induced mBMSCs compared with normal mBMSCs, while H19 and LCoR were downregulated in adipogenic induced mBMSCs. Si-H19 could significantly increase the mRNA level of miR-188. Meanwhile, miR-188 directly regulated LCoR in mBMSCs. Overexpression of miR-188 and knockdown of LCoR suppressed osteogenic differentiation and induced adipogenic differentiation in mBMSCs. Long noncoding RNA H19 mediates LCoR to regulate the balance between osteogenic and adipogenic differentiation of mBMSCs in mice through sponging miR-188. © 2018 Wiley Periodicals, Inc.

IGF-1/IGF-1R/hsa-let-7c axis regulates the committed differentiation of stem cells from apical papilla

PubMed Central

Ma, Shu; Liu, Genxia; Jin, Lin; Pang, Xiyao; Wang, Yanqiu; Wang, Zilu; Yu, Yan; Yu, Jinhua

2016-01-01

Insulin-like growth factor-1 (IGF-1) and its receptor IGF-1R play a paramount role in tooth/bone formation while hsa-let-7c actively participates in the osteogenic differentiation of mesenchymal stem cells. However, the interaction between IGF-1/IGF-1R and hsa-let-7c on the committed differentiation of stem cells from apical papilla (SCAPs) remains unclear. In this study, human SCAPs were isolated and treated with IGF-1 and hsa-let-7c over/low-expression viruses. The odonto/osteogenic differentiation of these stem cells and the involvement of mitogen-activated protein kinase (MAPK) pathway were subsequently investigated. Alizarin red staining showed that hsa-let-7c low-expression can significantly promote the mineralization of IGF-1 treated SCAPs, while hsa-let-7c over-expression can decrease the calcium deposition of IGF-1 treated SCAPs. Western blot assay and real-time reverse transcription polymerase chain reaction further demonstrated that the expression of odonto/osteogenic markers (ALP, RUNX2/RUNX2, OSX/OSX, OCN/OCN, COL-I/COL-I, DSPP/DSP, and DMP-1/DMP-1) in IGF-1 treated SCAPs were significantly upregulated in Let-7c-low group. On the contrary, hsa-let-7c over-expression could downregulate the expression of these odonto/osteogenic markers. Moreover, western blot assay showed that the JNK and p38 MAPK signaling pathways were activated in Let-7c-low SCAPs but inhibited in Let-7c-over SCAPs. Together, the IGF-1/IGF-1R/hsa-let-7c axis can control the odonto/osteogenic differentiation of IGF-1-treated SCAPs via the regulation of JNK and p38 MAPK signaling pathways. PMID:27833148

Epigenetic Plasticity Drives Adipogenic and Osteogenic Differentiation of Marrow-derived Mesenchymal Stem Cells*

PubMed Central

Meyer, Mark B.; Benkusky, Nancy A.; Sen, Buer; Rubin, Janet; Pike, J. Wesley

2016-01-01

Terminal differentiation of multipotent stem cells is achieved through a coordinated cascade of activated transcription factors and epigenetic modifications that drive gene transcription responsible for unique cell fate. Within the mesenchymal lineage, factors such as RUNX2 and PPARγ are indispensable for osteogenesis and adipogenesis, respectively. We therefore investigated genomic binding of transcription factors and accompanying epigenetic modifications that occur during osteogenic and adipogenic differentiation of mouse bone marrow-derived mesenchymal stem cells (MSCs). As assessed by ChIP-sequencing and RNA-sequencing analyses, we found that genes vital for osteogenic identity were linked to RUNX2, C/EBPβ, retinoid X receptor, and vitamin D receptor binding sites, whereas adipocyte differentiation favored PPARγ, retinoid X receptor, C/EBPα, and C/EBPβ binding sites. Epigenetic marks were clear predictors of active differentiation loci as well as enhancer activities and selective gene expression. These marrow-derived MSCs displayed an epigenetic pattern that suggested a default preference for the osteogenic pathway; however, these patterns were rapidly altered near the Adipoq, Cidec, Fabp4, Lipe, Plin1, Pparg, and Cebpa genes during adipogenic differentiation. Surprisingly, we found that these cells also exhibited an epigenetic plasticity that enabled them to trans-differentiate from adipocytes to osteoblasts (and vice versa) after commitment, as assessed by staining, gene expression, and ChIP-quantitative PCR analysis. The osteogenic default pathway may be subverted during pathological conditions, leading to skeletal fragility and increased marrow adiposity during aging, estrogen deficiency, and skeletal unloading. Taken together, our data provide an increased mechanistic understanding of the epigenetic programs necessary for multipotent differentiation of MSCs that may prove beneficial in the development of therapeutic strategies. PMID:27402842

Engineered Fibrin Gels for Parallel Stimulation of Mesenchymal Stem Cell Proangiogenic and Osteogenic Potential

PubMed Central

Murphy, Kaitlin C.; Hughbanks, Marissa L.; Binder, Bernard Y.K.; Vissers, Caroline B.; Leach, J. Kent

2014-01-01

Mesenchymal stem/stromal cells (MSCs) are under examination for use in cell therapies to repair bone defects resulting from trauma or disease. MSCs secrete proangiogenic cues and can be induced to differentiate into bone-forming osteoblasts, yet there is limited evidence that these events can be achieved in parallel. Manipulation of the cell delivery vehicle properties represents a candidate approach for directing MSC function in bone healing. We hypothesized that the biophysical properties of a fibrin gel could simultaneously regulate the proangiogenic and osteogenic potential of entrapped MSCs. Fibrin gels were formed by supplementation with NaCl (1.2, 2.3, and 3.9% w/v) to modulate gel biophysical properties without altering protein concentrations. MSCs entrapped in 1.2% w/v NaCl gels were the most proangiogenic in vitro, yet cells in 3.9% w/v gels exhibited the greatest osteogenic response. Compared to the other groups, MSCs entrapped in 2.3% w/v gels provided the best balance between proangiogenic potential, osteogenic potential, and gel contractility. The contribution of MSCs to bone repair was then examined when deployed in 2.3% w/v NaCl gels and implanted into an irradiated orthotopic bone defect. Compared to acellular gels after 3 weeks of implantation, defects treated with MSC-loaded fibrin gels exhibited significant increases in vessel density, early osteogenesis, superior morphology, and increased cellularity of repair tissue. Defects treated with MSC-loaded gels exhibited increased bone formation after 12 weeks compared to blank gels. These results confirm that fibrin gel properties can be modulated to simultaneously promote both the proangiogenic and osteogenic potential of MSCs, and fibrin gels modified by supplementation with NaCl are promising carriers for MSCs to stimulate bone repair in vivo. PMID:25527322

Influence of Partial O₂ Pressure on the Adhesion, Proliferation, and Osteogenic Differentiation of Human Dental Pulp Stem Cells on β-Tricalcium Phosphate Scaffold.

PubMed

Viña-Almunia, Jose; Mas-Bargues, Cristina; Borras, Consuelo; Gambini, Juan; El Alami, Marya; Sanz-Ros, Jorge; Peñarrocha, Miguel; Vina, Jose

To analyze, in vitro, the influence of O₂ pressure on the adhesion, proliferation, and osteogenic differentiation of human dental pulp stem cells (DPSC) on β-tricalcium phosphate (β-TCP) scaffold. DPSC, positive for the molecular markers CD133, Oct4, Nestin, Stro-1, and CD34, and negative for CD45, were isolated from extracted third molars. Experiments were started by seeding 200,000 cells on β-TCP cultured under 3% or 21% O₂ pressure. No osteogenic medium was used. Eight different cultures were performed at each time point under each O₂ pressure condition. Cell adhesion, proliferation, and differentiation over the biomaterial were evaluated at 7, 13, 18, and 23 days of culture. Cell adhesion was determined by light microscopy, proliferation by DNA quantification, and osteogenic differentiation by alkaline phosphatase (ALP) activity analysis. DPSC adhered to β-TCP with both O₂ conditions. Cell proliferation was found from day 7 of culture. Higher values were recorded at 3% O₂ in each time point. Statistically significant differences were recorded at 23 days of culture (P = .033). ALP activity was not detectable at 7 days. There was, however, an increase in ALP activity over time in both groups. At 13, 18, and 23 days of culture, higher ALP activity was recorded under 3% O₂ pressure. Statistical differences were found at day 23 (P = .014). DPSC display capacity of adhering to β-TCP under 3% or 21% O₂ pressure conditions. Cell proliferation on β-TCP phosphate is significantly higher at 3% than at 21% O₂ pressure, the most frequently used O₂ tension. β-TCP can itself promote osteogenic differentiation of DPSC and is enhanced under 3% O₂ compared with 21%.

Novel oxysterols have pro-osteogenic and anti-adipogenic effects in vitro and induce spinal fusion in vivo.

PubMed

Johnson, Jared S; Meliton, Vicente; Kim, Woo Kyun; Lee, Kwang-Bok; Wang, Jeffrey C; Nguyen, Khanhlinh; Yoo, Dongwon; Jung, Michael E; Atti, Elisa; Tetradis, Sotirios; Pereira, Renata C; Magyar, Clara; Nargizyan, Taya; Hahn, Theodore J; Farouz, Francine; Thies, Scott; Parhami, Farhad

2011-06-01

Stimulation of bone formation by osteoinductive materials is of great clinical importance in spinal fusion surgery, repair of bone fractures, and in the treatment of osteoporosis. We previously reported that specific naturally occurring oxysterols including 20(S)-hydroxycholesterol (20S) induce the osteogenic differentiation of pluripotent mesenchymal cells, while inhibiting their adipogenic differentiation. Here we report the characterization of two structural analogues of 20S, Oxy34 and Oxy49, which induce the osteogenic and inhibit the adipogenic differentiation of bone marrow stromal cells (MSC) through activation of Hedgehog (Hh) signaling. Treatment of M2-10B4 MSC with Oxy34 or Oxy49 induced the expression of osteogenic differentiation markers Runx2, Osterix (Osx), alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OCN), as well as ALP enzymatic activity and robust mineralization. Treatment with oxysterols together with PPARγ activator, troglitazone (Tro), inhibited mRNA expression for adipogenic genes PPARγ, LPL, and aP2, and inhibited the formation of adipocytes. Efficacy of Oxy34 and Oxy49 in stimulating bone formation in vivo was assessed using the posterolateral intertransverse process rat spinal fusion model. Rats receiving collagen implants with Oxy 34 or Oxy49 showed comparable osteogenic efficacy to BMP2/collagen implants as measured by radiography, MicroCT, and manual inspection. Histological analysis showed trabecular and cortical bone formation by oxysterols and rhBMP2 within the fusion mass, with robust adipogenesis in BMP2-induced bone and significantly less adipocytes in oxysterol-induced bone. These data suggest that Oxy34 and Oxy49 are effective novel osteoinductive molecules and may be suitable candidates for further development and use in orthopedic indications requiring local bone formation. Copyright © 2011 Wiley-Liss, Inc.

[Osteogenic potential of bone marrow mesenchymal stem cells from ovariectomied osteoporotic rat].

PubMed

Li, Dong-ju; Ge, Dong-xia; Wu, Wen-chao; Wu, Jiang; Li, Liang

2005-05-01

To investigate the difference of osteogenic potential of bone marrow mesenchymal stem cells (MSCs) between healthy rats and osteoporotic rats. We established the animal model of osteoporosis by performing ovariectom on the 3-month-old female Sprague-Dawley rats. Bone marrow mesenchymal stem cells(MSCs) were isolated from the rats of control group and of ovariectomized (ovx) group by means of the density-gradient centrifugation method, and the 3rd-4th passage MSCs were used in all the experiments. The experiments comprised 4 groups: (1) Marrow mesenchymal stem cells control group (MSCs control group); (2) Marrow mesenchymal stem cells ovx group (MSCs ovx group); (3) Osteogenesis induction control group (OSI control group); (4) Osteogenesis induction ovx group (OSI ovx group). Cell cycle and proliferation index (PI) of MSCs were detected by flow cytometry. The expression of alkaline phosphatase (ALP) was detected by dynamics method with substrate of phosphoric acid para-Nitro benzene. The levels of osteocalcin were detected with the isotope labelling method. (1) PI of MSCs was lower in MSCs ovx group than in MSCs control group. (2) The expression of alkaline phosphatase (ALP) was much higher in OSI control group than in the MSCs control group; the expression of alkaline phosphatase (ALP) was much higher in the OSI control group than in OSI ovx group after 7-day and 14-day osteogenic induction. (3) The level of osteocalcin was much higher in the OSI control group than in the MSCs control group after 14-day, 21-day, 28-day osteogenic induction. The level of osteocalcin was much higher in the OSI control group than in the OSI ovx group. Both the proliferative potential and the osteogenic potential of bone marrow mesenchymal stem cells (MSCs) from the ovariectomized osteoporotic rat are decreased.

A new platelet cryoprecipitate glue promoting bone formation after ectopic mesenchymal stromal cell-loaded biomaterial implantation in nude mice.

PubMed

Trouillas, Marina; Prat, Marie; Doucet, Christelle; Ernou, Isabelle; Laplace-Builhé, Corinne; Blancard, Patrick Saint; Holy, Xavier; Lataillade, Jean-Jacques

2013-01-04

This study investigated the promising effect of a new Platelet Glue obtained from Cryoprecipitation of Apheresis Platelet products (PGCAP) used in combination with Mesenchymal Stromal Cells (MSC) loaded on ceramic biomaterials to provide novel strategies enhancing bone repair. PGCAP growth factor content was analyzed by ELISA and compared to other platelet and plasma-derived products. MSC loaded on biomaterials (65% hydroxyapatite/35% beta-TCP or 100% beta-TCP) were embedded in PGCAP and grown in presence or not of osteogenic induction medium for 21 days. Biomaterials were then implanted subcutaneously in immunodeficient mice for 28 days. Effect of PGCAP on MSC was evaluated in vitro by proliferation and osteoblastic gene expression analysis and in vivo by histology and immunohistochemistry. We showed that PGCAP, compared to other platelet-derived products, allowed concentrating large amount of growth factors and cytokines which promoted MSC and osteoprogenitor proliferation. Next, we found that PGCAP improves the proliferation of MSC and osteogenic-induced MSC. Furthermore, we demonstrated that PGCAP up-regulates the mRNA expression of osteogenic markers (Collagen type I, Osteonectin, Osteopontin and Runx2). In vivo, type I collagen expressed in ectopic bone-like tissue was highly enhanced in biomaterials embedded in PGCAP in the absence of osteogenic pre-induction. Better results were obtained with 65% hydroxyapatite/35% beta-TCP biomaterials as compared to 100% beta-TCP. We have demonstrated that PGCAP is able to enhance in vitro MSC proliferation, osteoblastic differentiation and in vivo bone formation in the absence of osteogenic pre-induction. This clinically adaptable platelet glue could be of interest for improving bone repair.

Intrafibrillar-silicified collagen scaffolds enhance the osteogenic capacity of human dental pulp stem cells.

PubMed

Niu, Li-na; Sun, Jia-qi; Li, Qi-hong; Jiao, Kai; Shen, Li-juan; Wu, Dan; Tay, Franklin; Chen, Ji-hua

2014-07-01

The present study investigated the effects of intrafibrillar-silicified collagen scaffolds (ISCS) on the osteogenic differentiation of human dental pulp stem cells (hDPSCs) in vitro and in vivo. The hDPSCs were co-cultured with ISCS or nonsilicified collagen scaffolds (NCS) in control medium (CM) or osteogenic differentiation medium (ODM). Cell cycle and cell apoptosis were analyzed with flow cytometry to measure the viability of hDPSCs. Reverse transcription-polymerase chain reaction (RT-PCR) and western blotting were used to evaluate the expression levels of osteogenic marker genes and proteins of hDPSCs. Alkaline phosphatase (ALP) staining and alizarin red S assay were used to evaluate the ALP activity of hDPSCs and their calcium deposition potential. In addition, hDPSCs and scaffolds were implanted subcutaneously in nude mice for 8 weeks. Harvested tissues were immunohistochemically stained for osteocalcin (OCN) expression from hDPSCs, and stained with alizarin red S for examination of their calcium deposition in vivo. The ISCS had no adverse effect on hDPSCs, promoted their proliferation, and significantly up-regulated the expression of osteogenesis-related genes and proteins. The hDPSCs co-cultured with ISCS in ODM exhibited the highest ALP activity and calcium deposition in vitro. The ISCS promoted the OCN expression and calcium deposition of hDPSCs after ectopic transplantation in vivo. Intrafibrillar-silicified collagen scaffolds significantly promoted the proliferation, osteogenic differentiation and mineralization of hDPSCs, when compared with NCS. This study demonstrates combining the use of hDPSCs and ISCS to promote bone-like tissue formation is a promising approach for clinical bone repair and regeneration. Copyright © 2014 Elsevier Ltd. All rights reserved.

Osteosarcoma cells promote the production of pro-tumor cytokines in mesenchymal stem cells by inhibiting their osteogenic differentiation through the TGF-β/Smad2/3 pathway.

PubMed

Tu, Bing; Peng, Zhao-Xiang; Fan, Qi-Ming; Du, Lin; Yan, Wei; Tang, Ting-Ting

2014-01-01

Mesenchymal stem cells (MSCs) are among the most important components of the osteosarcoma microenvironment and are reported to promote tumor progression. However, the means by which osteosarcoma cells modulate MSC behavior remains unclear. The aim of this study was to determine the effects of osteosarcoma cells on both the production of pro-tumor cytokines by mesenchymal stem cells (MSCs) and the osteogenic differentiation of MSCs. High level of transforming growth factor-β (TGF-β) was detected in three osteosarcoma cell lines. Conditioned media (CM) from the osteosarcoma cell lines Saos-2 and U2-OS were used to stimulate the cultured MSCs. We found that osteosarcoma cells promoted the production of IL-6 and VEGF in MSCs by inhibiting their osteogenic differentiation. Furthermore, TGF-β in tumor CM was proved to be an important factor. The TGF-β neutralizing antibody antagonized the effects induced by osteosarcoma CM. The inhibition of Smad2/3 by siRNA significantly decreased the production of IL-6 and VEGF in MSCs and induced their osteogenic differentiation. We also found that Smad2/3 enhanced the expression of β-catenin in MSCs by decreasing the level of Dickkopf-1 (DKK1). Although the inhibition of β-catenin did not affect the production of IL-6 or VEGF, or the gene expression of the early osteogenic markers Runx2 and ALP, it did enhance the gene expression of osteocalcin. Taken together, our data indicate that osteosarcoma cells secrete TGF-β to maintain the stemness of MSCs and promote the production of pro-tumor cytokines by these cells. © 2013 Published by Elsevier Inc.

A new platelet cryoprecipitate glue promoting bone formation after ectopic mesenchymal stromal cell-loaded biomaterial implantation in nude mice

PubMed Central

2013-01-01

Introduction This study investigated the promising effect of a new Platelet Glue obtained from Cryoprecipitation of Apheresis Platelet products (PGCAP) used in combination with Mesenchymal Stromal Cells (MSC) loaded on ceramic biomaterials to provide novel strategies enhancing bone repair. Methods PGCAP growth factor content was analyzed by ELISA and compared to other platelet and plasma-derived products. MSC loaded on biomaterials (65% hydroxyapatite/35% beta-TCP or 100% beta-TCP) were embedded in PGCAP and grown in presence or not of osteogenic induction medium for 21 days. Biomaterials were then implanted subcutaneously in immunodeficient mice for 28 days. Effect of PGCAP on MSC was evaluated in vitro by proliferation and osteoblastic gene expression analysis and in vivo by histology and immunohistochemistry. Results We showed that PGCAP, compared to other platelet-derived products, allowed concentrating large amount of growth factors and cytokines which promoted MSC and osteoprogenitor proliferation. Next, we found that PGCAP improves the proliferation of MSC and osteogenic-induced MSC. Furthermore, we demonstrated that PGCAP up-regulates the mRNA expression of osteogenic markers (Collagen type I, Osteonectin, Osteopontin and Runx2). In vivo, type I collagen expressed in ectopic bone-like tissue was highly enhanced in biomaterials embedded in PGCAP in the absence of osteogenic pre-induction. Better results were obtained with 65% hydroxyapatite/35% beta-TCP biomaterials as compared to 100% beta-TCP. Conclusions We have demonstrated that PGCAP is able to enhance in vitro MSC proliferation, osteoblastic differentiation and in vivo bone formation in the absence of osteogenic pre-induction. This clinically adaptable platelet glue could be of interest for improving bone repair. PMID:23290259

Naringin protects human adipose-derived mesenchymal stem cells against hydrogen peroxide-induced inhibition of osteogenic differentiation.

PubMed

Wang, Lei; Zhang, Yu-Ge; Wang, Xiu-Mei; Ma, Long-Fei; Zhang, Yuan-Min

2015-12-05

Extensive evidence indicates that oxidative stress plays a pivotal role in the development of osteoporosis. We show that naringin, a natural antioxidant and anti-inflammatory compound, effectively protects human adipose-derived mesenchymal stem cells (hADMSCs) against hydrogen peroxide (H2O2)-induced inhibition of osteogenic differentiation. Naringin increased viability of hAMDSCs and attenuated H2O2-induced cytotoxicity. Naringin also reversed H2O2-induced oxidative stress. Oxidative stress induced by H2O2 inhibits osteogenic differentiation by decreasing alkaline phosphatase (ALP) activity, calcium content and mRNA expression levels of osteogenesis marker genes RUNX2 and OSX in hADMSCs. However, addition of naringin leads to a significant recovery, suggesting the protective effects of naringin against H2O2-induced inhibition of osteogenic differentiation. Furthermore, the H2O2-induced decrease of protein expressions of β-catenin and clyclin D1, two important transcriptional regulators of Wnt-signaling, was successfully rescued by naringin treatment. Also, in the presence of Wnt inhibitor DKK-1, naringin is no longer effective in stimulating ALP activity, increasing calcium content and mRNA expression levels of RUNX2 and OSX in H2O2-exposed hADMSCs. These data clearly demonstrates that naringin protects hADMSCs against oxidative stress-induced inhibition of osteogenic differentiation, which may involve Wnt signaling pathway. Our work suggests that naringin may be a useful addition to the treatment armamentarium for osteoporosis and activation of Wnt signaling may represent attractive therapeutic strategy for the treatment of degenerative disease of bone tissue. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Gene expression profiling of bone marrow mesenchymal stem cells from Osteogenesis Imperfecta patients during osteoblast differentiation.

PubMed

Kaneto, Carla Martins; Pereira Lima, Patrícia S; Prata, Karen Lima; Dos Santos, Jane Lima; de Pina Neto, João Monteiro; Panepucci, Rodrigo Alexandre; Noushmehr, Houtan; Covas, Dimas Tadeu; de Paula, Francisco José Alburquerque; Silva, Wilson Araújo

2017-06-01

Mesenchymal stem cells (MSCs) are precursors present in adult bone marrow that are able to differentiate into osteoblasts, adipocytes and chondroblasts that have gained great importance as a source for cell therapy. Recently, a number of studies involving the analysis of gene expression of undifferentiated MSCs and of MSCs in the differentiation into multiple lineage processes were observed but there is no information concerning the gene expression of MSCs from Osteogenesis Imperfecta (OI) patients. Osteogenesis Imperfecta is characterized as a genetic disorder in which a generalized osteopenia leads to excessive bone fragility and severe bone deformities. The aim of this study was to analyze gene expression profile during osteogenic differentiation from BMMSCs (Bone Marrow Mesenchymal Stem Cells) obtained from patients with Osteogenesis Imperfecta and from control subjects. Bone marrow samples were collected from three normal subjects and five patients with OI. Mononuclear cells were isolated for obtaining mesenchymal cells that had been expanded until osteogenic differentiation was induced. RNA was harvested at seven time points during the osteogenic differentiation period (D0, D+1, D+2, D+7, D+12, D+17 and D+21). Gene expression analysis was performed by the microarray technique and identified several differentially expressed genes. Some important genes for osteoblast differentiation had lower expression in OI patients, suggesting a smaller commitment of these patient's MSCs with the osteogenic lineage. Other genes also had their differential expression confirmed by RT-qPCR. An increase in the expression of genes related to adipocytes was observed, suggesting an increase of adipogenic differentiation at the expense osteogenic differentiation. Copyright © 2017. Published by Elsevier Masson SAS.

In vitro osteogenic/dentinogenic potential of an experimental calcium aluminosilicate cement

PubMed Central

Eid, Ashraf A.; Niu, Li-na; Primus, Carolyn M.; Opperman, Lynne A.; Watanabe, Ikuya; Pashley, David H.; Tay, Franklin R.

2013-01-01

Introduction Calcium aluminosilicate cements are fast-setting, acid-resistant, bioactive cements that may be used as root-repair materials. This study examined the osteogenic/dentinogenic potential of an experimental calcium aluminosilicate cement (Quick-Set) using a murine odontoblast-like cell model. Methods Quick-Set and white ProRoot MTA (WMTA) were mixed with the proprietary gel or deionized water, allowed to set completely in 100% relative humidity and aged in complete growth medium for 2 weeks until rendered non-cytotoxic. Similarly-aged Teflon discs were used as negative control. The MDPC-23 cell-line was used for evaluating changes in mRNA expressions of genes associated with osteogenic/dentinogenic differentiation and mineralization (qRT-PCR) alkaline phosphatase enzyme production and extracellular matrix mineralization (Alizarin red-S staining). Results After MDPC-23 cells were incubated with the materials in osteogenic differentiation medium for 1 week, both cements showed upregulation in ALP and DSPP expression. Fold increases in these two genes were not significantly different between Quick-Set and WMTA. Both cements showed no statistically significant upregulation/downregulation in RUNX2, OCN, BSP and DMP1 gene expression compared with Teflon. Alkaline phosphatase activity of cells cultured on Quick-Set and WMTA were not significantly different at 1 week or 2 weeks, but were significantly higher (p<0.05) than Teflon in both weeks. Both cements showed significantly higher calcium deposition compared with Teflon after 3 weeks of incubation in mineralizing medium (p<0.001). Differences between Quick-Set and WMTA were not statistically significant. Conclusions The experimental calcium aluminosilicate cement exhibits similar osteogenic/dentinogenic properties to WMTA and may be a potential substitute for commercially-available tricalcium silicate cements. PMID:23953291

Icaritin, a novel plant-derived osteoinductive agent, enhances the osteogenic differentiation of human bone marrow- and human adipose tissue-derived mesenchymal stem cells.

PubMed

Wu, Tao; Shu, Tao; Kang, Le; Wu, Jinhui; Xing, Jianzhou; Lu, Zhiqin; Chen, Shuxiang; Lv, Jun

2017-04-01

For the treatment of diseases affecting bones using bone regenerative medicine, there is an urgent need to develop safe, inexpensive drugs that can strongly induce bone formation. In the present study, we systematically investigated the effects of icaritin, a metabolic product of icariin, on the osteogenic differentiation of human bone marrow‑derived mesenchymal stem cells (hBMSCs) and human adipose tissue‑derived stem cells (hADSCs) in vitro. After treatment with icaritin at concentrations of 10‑8-10‑5 M, hBMSCs and hADSCs were examined for alkaline phosphatase activity, osteocalcin (OC) secretion, matrix mineralization and expression levels of bone‑related mRNA and proteins. Data showed that icaritin at concentrations 10‑7-10‑5 M significantly increased alkaline phosphatase activity, OC secretion at different time points, and calcium deposition at day 21. In addition, icaritin upregulated the mRNA expression of genes for bone morphogenetic proteins (BMP‑2, ‑4 and ‑7), bone transcription factors (Runx2 and Dlx5) and bone matrix proteins (ALP, OC and Col‑1). Moreover, icaritin increased the protein levels of BMPs, Runx2 and OC, as detected by western blot analysis. These findings suggest that icaritin enhances the osteogenic differentiation of hBMSCS and hADSCs. Icaritin exerts its potent osteogenic effect possibly by directly stimulating the production of BMPs. Although the osteogenic activity of icaritin in vitro was inferior to that of rhBMP‑2, icaritin displayed better results than icariin. Moreover, the low cost, simple extraction procedure, and an abundance of icaritin make it appealing as a bone regenerative medicine.

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

Osteogenic Differentiation of Three-Dimensional Bioprinted Constructs Consisting of Human Adipose-Derived Stem Cells In Vitro and In Vivo

PubMed Central

Liu, Yun-Song; Sun, Yu-chun; Wang, Yu-guang; Wang, Yong; Lyu, Pei-Jun

2016-01-01

Here, we aimed to investigate osteogenic differentiation of human adipose-derived stem cells (hASCs) in three-dimensional (3D) bioprinted tissue constructs in vitro and in vivo. A 3D Bio-plotter dispensing system was used for building 3D constructs. Cell viability was determined using live/dead cell staining. After 7 and 14 days of culture, real-time quantitative polymerase chain reaction (PCR) was performed to analyze the expression of osteogenesis-related genes (RUNX2, OSX, and OCN). Western blotting for RUNX2 and immunofluorescent staining for OCN and RUNX2 were also performed. At 8 weeks after surgery, osteoids secreted by osteogenically differentiated cells were assessed by hematoxylin-eosin (H&E) staining, Masson trichrome staining, and OCN immunohistochemical staining. Results from live/dead cell staining showed that most of the cells remained alive, with a cell viability of 89%, on day 1 after printing. In vitro osteogenic induction of the 3D construct showed that the expression levels of RUNX2, OSX, and OCN were significantly increased on days 7 and 14 after printing in cells cultured in osteogenic medium (OM) compared with that in normal proliferation medium (PM). Fluorescence microscopy and western blotting showed that the expression of osteogenesis-related proteins was significantly higher in cells cultured in OM than in cells cultured in PM. In vivo studies demonstrated obvious bone matrix formation in the 3D bioprinted constructs. These results indicated that 3D bioprinted constructs consisting of hASCs had the ability to promote mineralized matrix formation and that hASCs could be used in 3D bioprinted constructs for the repair of large bone tissue defects. PMID:27332814

Tyrosine kinase receptor c-ros-oncogene 1 inhibition alleviates aberrant bone formation of TWIST-1 haploinsufficient calvarial cells from Saethre-Chotzen syndrome patients.

PubMed

Camp, Esther; Anderson, Peter J; Zannettino, Andrew C W; Glackin, Carlotta A; Gronthos, Stan

2018-09-01

Saethre-Chotzen syndrome (SCS), associated with TWIST-1 mutations, is characterized by premature fusion of cranial sutures. TWIST-1 haploinsufficiency, leads to alterations in suture mesenchyme cellular gene expression patterns, resulting in aberrant osteogenesis and craniosynostosis. We analyzed the expression of the TWIST-1 target, Tyrosine kinase receptor c-ros-oncogene 1 (C-ROS-1) in TWIST-1 haploinsufficient calvarial cells derived from SCS patients and calvaria of Twist-1 del/+ mutant mice and found it to be highly expressed when compared to TWIST-1 wild-type controls. Knock-down of C-ROS-1 expression in TWIST-1 haploinsufficient calvarial cells derived from SCS patients was associated with decreased capacity for osteogenic differentiation in vitro. Furthermore, treatment of human SCS calvarial cells with the tyrosine kinase chemical inhibitor, Crizotinib, resulted in reduced C-ROS-1 activity and the osteogenic potential of human SCS calvarial cells with minor effects on cell viability or proliferation. Cultured human SCS calvarial cells treated with Crizotinib exhibited a dose-dependent decrease in alkaline phosphatase activity and mineral deposition, with an associated decrease in expression levels of Runt-related transcription factor 2 and OSTEOPONTIN, with reduced PI3K/Akt signalling in vitro. Furthermore, Crizotinib treatment resulted in reduced BMP-2 mediated bone formation potential of whole Twist-1 del/+ mutant mouse calvaria organotypic cultures. Collectively, these results suggest that C-ROS-1 promotes osteogenic differentiation of TWIST-1 haploinsufficient calvarial osteogenic progenitor cells. Furthermore, the aberrant osteogenic potential of these cells is inhibited by the reduction of C-ROS-1. Therefore, targeting C-ROS-1 with a pharmacological agent, such as Crizotinib, may serve as a novel therapeutic strategy to alleviate craniosynostosis associated with aberrant TWIST-1 function. © 2018 Wiley Periodicals, Inc.

Human Embryonic Stem Cells Undergo Osteogenic Differentiation in Human Bone Marrow Stromal Cell Microenvironments

PubMed Central

Tong, Wilbur; Brown, Shelley E.; Krebsbach, Paul H.

2009-01-01

Human embryonic stem cells (hESCs) may offer an unlimited supply of cells that can be directed to differentiate into all cell types within the body and used in regenerative medicine for tissue and cell replacement therapies. Previous work has shown that exposing hESCs to exogenous factors such as dexamethasone, ascorbic acid and β-glycerophosphate can induce osteogenesis. The specific factors that induce osteogenic differentiation of hESCs have not been identified yet, however, it is possible that differentiated human bone marrow stromal cells (hMBSCs) may secrete factors within the local microenvironment that promote osteogenesis. Here we report that the lineage progression of hESCs to osteoblasts is achieved in the presence of soluble signaling factors derived from differentiated hBMSCs. For 28 days, hESCs were grown in a transwell co-culture system with hBMSCs that had been previously differentiated in growth medium containing defined osteogenic supplements for 7-24 days. As a control. hESCs were co-cultured with undifferentiated hBMSCs and alone. Von Kossa and Alizarin Red staining as well as immunohistochemistry confirmed that the hESCs co-cultured with differentiated hBMSCs formed mineralized bone nodules and secreted extracellular matrix protein osteocalcin (OCN). Quantitative Alizarin Red assays showed increased mineralization as compared to the control with undifferentiated hBMSCs. RT-PCR revealed the loss of pluripotent hESC markers with the concomitant gain of osteoblastic markers such as collagen type I, runx2, and osterix. We demonstrate that osteogenic growth factors derived from differentiated hBMSCs within the local microenvironment may help to promote hESC osteogenic differentiation. PMID:20671800

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.

Combined use of leptin and mechanical stress has osteogenic effects on ossification of the posterior longitudinal ligament.

PubMed

Chen, Shuai; Zhu, Haifeng; Wang, Gangliang; Xie, Ziang; Wang, Jiying; Chen, Jian

2018-06-16

To evaluate the effects of leptin/leptin receptor (LepR) combined with mechanical stress on the development of ossification of the posterior longitudinal ligament (OPLL), which is a disease characterized by ectopic bone formation of the posterior longitudinal ligament (PLL) and can lead to radiculopathy and myelopathy. Six human samples of the PLL were analyzed for the expression of leptin and LepR by RT-PCR and western blotting. PLL cells were stimulated with leptin and mechanical stress delivered via a Flexcell tension system, and osteogenic differentiation was evaluated by RT-PCR and western blotting analysis of osteogenic marker expression as well as by alkaline phosphatase (ALP) staining and alizarin red S staining. Activation of mitogen-activated protein kinase (MAPK), Janus kinase (JAK) 2-signal transducer, activator of transcription (STAT) 3 and phosphatidylinositol 3-kinase (PI3K)-Akt was evaluated by western blotting. Samples from the OPLL group had higher LepR mRNA and protein levels and lower leptin levels than those from healthy controls. Exposure to leptin and Flexcell increased the number of ALP-positive cells and calcium nodules in a dose-dependent manner; this effect was accompanied by upregulation of the osteogenic markers osteocalcin, runt-related transcription factor 2 (RUNX2) and osteopontin. Extracellular signal-regulated kinase, P38 MAPK, JAK2, STAT3, PI3K and Akt signaling, was also activated by the combined effects of leptin and mechanical stress. Leptin and LepR are differentially expressed in OPLL tissues, and the combined use of leptin/LepR and mechanical stress promotes osteogenic differentiation of PLL cells via MAPK, JAK2-STAT3 and PI3K/Akt signaling. These slides can be retrieved under Electronic Supplementary Material.

On the Application of Science Systems Engineering and Uncertainty Quantification for Ice Sheet Science and Sea Level Projections

NASA Astrophysics Data System (ADS)

Schlegel, Nicole-Jeanne; Boening, Carmen; Larour, Eric; Limonadi, Daniel; Schodlok, Michael; Seroussi, Helene; Watkins, Michael

2017-04-01

Research and development activities at the Jet Propulsion Laboratory (JPL) currently support the creation of a framework to formally evaluate the observational needs within earth system science. One of the pilot projects of this effort aims to quantify uncertainties in global mean sea level rise projections, due to contributions from the continental ice sheets. Here, we take advantage of established uncertainty quantification tools embedded within the JPL-University of California at Irvine Ice Sheet System Model (ISSM). We conduct sensitivity and Monte-Carlo style sampling experiments on forward simulations of the Greenland and Antarctic ice sheets. By varying internal parameters and boundary conditions of the system over both extreme and credible worst-case ranges, we assess the impact of the different parameter ranges on century-scale sea level rise projections. The results inform efforts to a) isolate the processes and inputs that are most responsible for determining ice sheet contribution to sea level; b) redefine uncertainty brackets for century-scale projections; and c) provide a prioritized list of measurements, along with quantitative information on spatial and temporal resolution, required for reducing uncertainty in future sea level rise projections. Results indicate that ice sheet mass loss is dependent on the spatial resolution of key boundary conditions - such as bedrock topography and melt rates at the ice-ocean interface. This work is performed at and supported by the California Institute of Technology's Jet Propulsion Laboratory. Supercomputing time is also supported through a contract with the National Aeronautics and Space Administration's Cryosphere program.

Influence of alternative silviculture on small mammals

USGS Publications Warehouse

Waldien, David L.; Hayes, John P.

2006-01-01

HIGHLIGHT: A variety of harvest methods promote diversity within forests while still generating income. For example, recent studies have shown that when dead wood is left on the forest floor during harvest, biodiversity increases. A new Cooperative Forest Ecosystem Research (CFER) program fact sheet summarizes how small mammals respond to dead wood in forests that are harvested with alternative methods. CFER is developing a series of fact sheets about responses to changes in young western Oregon forests. The fact sheets are designed to help resource managers balance management needs, including timber and wildlife. The USGS provides a primary source of financial support for CFER, a consortium of federal and state partners conducting research in support of the Northwest Forest Plan.

Modular heat exchanger

DOEpatents

Giardina, A.R.

1981-03-03

A shell and tube heat exchanger is described having a plurality of individually removable tube bundle modules. A lattice of structural steel forming rectangular openings therein is placed at each end of a cylindrical shell. Longitudinal structural members are placed in the shell between corners of the rectangular openings situated on opposite ends of the shell. Intermediate support members interconnect the longitudinal supports so as to increase the longitudinal supports rigidity. Rectangular parallelepiped tube bundle modules occupy the space defined by the longitudinal supports and end supports and each include a rectangular tube sheet situated on each end of a plurality of tubes extending there through, a plurality of rectangular tube supports located between the tube sheets, and a tube bundle module stiffening structure disposed about the bundle's periphery and being attached to the tube sheets and tube supports. The corners of each tube bundle module have longitudinal framework members which are mateable with and supported by the longitudinal support members. Intermediate support members constitute several lattices, each of which is situated in a plane between the end support members. The intermediate support members constituting the several lattices extend horizontally and vertically between longitudinal supports of adjacent tube module voids. An alternative embodiment for intermediate support members constitute a series of structural plates situated at the corners of the module voids and having recesses therein for receiving the respective longitudinal support members adjacent thereto, protrusions separating the recesses, and a plurality of struts situated between protrusions of adjacent structural plates. 12 figs.

Modular heat exchanger

DOEpatents

Giardina, Angelo R. [Marple Township, Delaware County, PA

1981-03-03

A shell and tube heat exchanger having a plurality of individually removable tube bundle modules. A lattice of structural steel forming rectangular openings therein is placed at each end of a cylindrical shell. Longitudinal structural members are placed in the shell between corners of the rectangular openings situated on opposite ends of the shell. Intermediate support members interconnect the longitudinal supports so as to increase the longitudinal supports rigidity. Rectangular parallelpiped tube bundle moldules occupy the space defined by the longitudinal supports and end supports and each include a rectangular tube sheet situated on each end of a plurality of tubes extending therethrough, a plurality of rectangular tube supports located between the tube sheets, and a tube bundle module stiffening structure disposed about the bundle's periphery and being attached to the tube sheets and tube supports. The corners of each tube bundle module have longitudinal framework members which are mateable with and supported by the longitudinal support members. Intermediate support members constitute several lattice, each of which is situate d in a plane between the end support members. The intermediate support members constituting the several lattice extend horizontally and vertically between longitudinal supports of adjacent tube module voids. An alternative embodiment for intermediate support members constitute a series of structural plates situated at the corners of the module voids and having recesses therein for receiving the respective longitudinal support members adjacent thereto, protrusions separating the recesses, and a plurality of struts situated between protrusions of adjacent structural plates.

The Onset of Magnetic Reconnection in Tail-Like Equilibria

NASA Technical Reports Server (NTRS)

Hesse, Michael; Birn, Joachim; Kuznetsova, Masha

1999-01-01

Magnetic reconnection is a fundamental mode of dynamics in the magnetotail, and is recognized as the basic mechanisms converting stored magnetic energy into kinetic energy of plasma particles. The effects of the reconnection process are well documented by spacecraft observations of plasmoids in the distant magnetotail, or bursty bulk flows, and magnetic field dipolarizations in the near Earth region. Theoretical and numerical analyses have, in recent years, shed new light on the way reconnection operates, and, in particular, which microscopic mechanism supports the dissipative electric field in the associated diffusion region. Despite this progress, however. the question of how magnetic reconnection initiates in a tail-like magnetic field with finite flux threading the current i.sheet remains unanswered. Instead, theoretical studies supported by numerical simulations support the point-of-view that such plasma and current sheets are stable with respect to collisionless tearing mode. In this paper, we will further investigate this conclusion, with emphasis on the question whether it remains valid in plasma sheets with embedded thin current sheets. For this purpose, we perform particle-in-cell simulations of the driven formation of thin current sheets, and their subsequent evolution either to equilibrium or to instability of a tearing-type mode. In the latter case we will pay particular attention to the nature of the electric field contribution which unmagnetizes the electrons.

Staphylococcus aureus Biofilms Decrease Osteoblast Viability, Inhibits Osteogenic Differentiation, and Increases Bone Resorption in vitro

DTIC Science & Technology

2013-06-01

severe and often debilitating disease characterized by inflammatory destruction of bone. Despite treatment, chronic infection often develops which is...biofilms may contribute to bone loss during chronic osteomyelitis simultaneously by: (1) reducing osteoblast viability and osteogenic potential thereby...accounting for more than half of all cases [1]. Despite treatment and surgical intervention up to 30% of osteomyelitis cases progress into a chronic

Vitamin K2 promotes mesenchymal stem cell differentiation by inhibiting miR‑133a expression.

PubMed

Zhang, Yuelei; Weng, Shiyang; Yin, Junhui; Ding, Hao; Zhang, Changqing; Gao, Youshui

2017-05-01

Vitamin K2 has been demonstrated to promote the osteogenic differentiation of mesenchymal stem cells; however, the mechanisms underlying this effect remain unclear. As microRNA (miR)‑133a has been identified as a negative regulator of osteogenic differentiation, the present study hypothesized that vitamin K2 promoted osteogenesis by inhibiting miR‑133a. Using human bone marrow stromal cells (hBMSCs) overexpressing miR‑133a, or a control, the expression levels of osteogenesis‑associated proteins, including runt‑related transcription factor 2, alkaline phosphatase and osteocalcin, were analyzed. miR‑133a significantly suppressed the osteogenic differentiation of hBMSCs. To determine the effect of vitamin K2 on miR‑133a expression and osteogenesis, hBMSCs were treated with vitamin K2. Vitamin K2 inhibited miR‑133a expression, which was accompanied by enhanced osteogenic differentiation. Furthermore, the expression levels of vitamin K epoxide reductase complex subunit 1, the key protein in γ‑carboxylation, were downregulated by miR‑133a overexpression and upregulated by vitamin K2 treatment, indicating a positive feedback on γ‑carboxylation. The results of the present study suggested that vitamin K2 targets miR‑133a to regulate osteogenesis.

[The effect of osteogenic inducer on healing of tooth extraction sockets].

PubMed

Chen, Junliang; Shan, Chuncheng; He, Yun; Xia, Delin

2012-06-01

To study the effect of osteogenic inducer (dexamethasone, beta-sodium glycerophosphate and Vitamin C) carried by gelatin sponge on healing and remodeling of tooth extraction sockets. Fifty rabbits were selected. After extracting the first premolars of bilateral maxillary, the right side tooth extraction sockets were filled with gelatin sponge containing osteogenic inducer as experimental side, tooth extraction sockets on left side were filled with gelatin sponge as control. Every ten rabbits were executed at the end of 1, 2, 4, 8, 12 weeks after tooth extraction. Bone density was measured through digital X-ray images. The specimens were examined by histology. The absorption height of alveolar bone at 12 weeks was measured. X-ray measurement showed that the bone density of experimental side was higher than that of control side at 2, 4, 8, 12 weeks, the difference had statistical significance (P<0.01). The histology examination showed that new bone formation in tooth extraction sockets of experimental side was earlier than that in control side. The absorptional height of alveolar bone had significant difference between experimental side and control side (P<0.01), of which experimental side was less. Filling the osteogenic inducer in tooth extraction sockets can promote the healing and new bone formation and prevent from alveolar bone absorption.

The use of a novel bone allograft wash process to generate a biocompatible, mechanically stable and osteoinductive biological scaffold for use in bone tissue engineering.

PubMed

Smith, C A; Richardson, S M; Eagle, M J; Rooney, P; Board, T; Hoyland, J A

2015-05-01

Fresh-frozen biological allograft remains the most effective substitute for the 'gold standard' autograft, sharing many of its osteogenic properties but, conversely, lacking viable osteogenic cells. Tissue engineering offers the opportunity to improve the osseointegration of this material through the addition of mesenchymal stem cells (MSCs). However, the presence of dead, immunogenic and potentially harmful bone marrow could hinder cell adhesion and differentiation, graft augmentation and incorporation, and wash procedures are therefore being utilized to remove the marrow, thereby improving the material's safety. To this end, we assessed the efficiency of a novel wash technique to produce a biocompatible, biological scaffold void of cellular material that was mechanically stable and had osteoinductive potential. The outcomes of our investigations demonstrated the efficient removal of marrow components (~99.6%), resulting in a biocompatible material with conserved biomechanical stability. Additionally, the scaffold was able to induce osteogenic differentiation of MSCs, with increases in osteogenic gene expression observed following extended culture. This study demonstrates the efficiency of the novel wash process and the potential of the resultant biological material to serve as a scaffold in bone allograft tissue engineering. © 2014 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons Ltd.

Human dental pulp stem cells produce mineralized matrix in 2D and 3D cultures

PubMed Central

Riccio, M.; Resca, E.; Maraldi, T.; Pisciotta, A.; Ferrari, A.; Bruzzesi, G.; De Pol, A.

2010-01-01

The aim of this study was to characterize the in vitro osteogenic differentiation of dental pulp stem cells (DPSCs) in 2D cultures and 3D biomaterials. DPSCs, separated from dental pulp by enzymatic digestion, and isolated by magnetic cell sorting were differentiated toward osteogenic lineage on 2D surface by using an osteogenic medium. During differentiation process, DPSCs express specific bone proteins like Runx-2, Osx, OPN and OCN with a sequential expression, analogous to those occurring during osteoblast differentiation, and produce extracellular calcium deposits. In order to differentiate cells in a 3D space that mimes the physiological environment, DPSCs were cultured in two distinct bioscaffolds, Matrigel™ and Collagen sponge. With the addition of a third dimension, osteogenic differentiation and mineralized extracellular matrix production significantly improved. In particular, in Matrigel™ DPSCs differentiated with osteoblast/osteocyte characteristics and connected by gap junction, and therefore formed calcified nodules with a 3D intercellular network. Furthermore, DPSCs differentiated in collagen sponge actively secrete human type I collagen micro-fibrils and form calcified matrix containing trabecular-like structures. These neo-formed DPSCs-scaffold devices may be used in regenerative surgical applications in order to resolve pathologies and traumas characterized by critical size bone defects. PMID:21263745

Indirect immobilized Jagged1 suppresses cell cycle progression and induces odonto/osteogenic differentiation in human dental pulp cells.

PubMed

Manokawinchoke, Jeeranan; Nattasit, Praphawi; Thongngam, Tanutchaporn; Pavasant, Prasit; Tompkins, Kevin A; Egusa, Hiroshi; Osathanon, Thanaphum

2017-08-31

Notch signaling regulates diverse biological processes in dental pulp tissue. The present study investigated the response of human dental pulp cells (hDPs) to the indirect immobilized Notch ligand Jagged1 in vitro. The indirect immobilized Jagged1 effectively activated Notch signaling in hDPs as confirmed by the upregulation of HES1 and HEY1 expression. Differential gene expression profiling using an RNA sequencing technique revealed that the indirect immobilized Jagged1 upregulated genes were mainly involved in extracellular matrix organization, disease, and signal transduction. Downregulated genes predominantly participated in the cell cycle, DNA replication, and DNA repair. Indirect immobilized Jagged1 significantly reduced cell proliferation, colony forming unit ability, and the number of cells in S phase. Jagged1 treated hDPs exhibited significantly higher ALP enzymatic activity, osteogenic marker gene expression, and mineralization compared with control. Pretreatment with a γ-secretase inhibitor attenuated the Jagged1-induced ALP activity and mineral deposition. NOTCH2 shRNA reduced the Jagged1-induced osteogenic marker gene expression, ALP enzymatic activity, and mineral deposition. In conclusion, indirect immobilized Jagged1 suppresses cell cycle progression and induces the odonto/osteogenic differentiation of hDPs via the canonical Notch signaling pathway.

Effects of water extract of Cajanus cajan leaves on the osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells and the adipocytic trans-differentiation of mouse primary osteoblasts.

PubMed

Zhang, Jinchao; Liu, Cuilian; Sun, Jing; Liu, Dandan; Wang, Peng

2010-01-01

The effects of water extract of Cajanus cajan (Linn.) Millsp. (Leguminosae) leaves (WECML) on the osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells (BMSCs) and the adipocytic trans-differentiation of mouse primary osteoblasts (OBs) were studied. The results indicated that WECML promoted the proliferation of BMSCs and OBs at most concentrations. WECML promoted the osteogenic differentiation and formation of mineralized matrix nodules of BMSCs at concentrations of 0.1, 1, and 10 microg/mL, but inhibited the osteogenic differentiation and formation of mineralized matrix nodules of BMSCs at concentration of 0.01 microg/mL. WECML inhibited the adipogenic differentiation of BMSCs and adipocytic trans-differentiation of OBs at concentrations of 0.001, 0.1, 1, 10, and 100 microg/mL, but had no effects at concentration of 0.01 microg/mL. The results suggest that WECML has protective effects on bone and these protective effects may be mediated by decreasing adipocytic cell formation from BMSCs, which may promote the proliferation, differentiation, and mineralization function of OBs. The defined active ingredients in the WECML and the active mechanism need to be further studied.

Proliferation and osteoblastic differentiation of human bone marrow-derived stromal cells on akermanite-bioactive ceramics.

PubMed

Sun, Hongli; Wu, Chengtie; Dai, Kerong; Chang, Jiang; Tang, Tingting

2006-11-01

In the present study, the effects of a calcium magnesium silicate bioactive ceramic (akermanite) on proliferation and osteoblastic differentiation of human bone marrow stromal cells (hBMSC) have been investigated and compared with the classical ceramic (beta-tricalcium phosphate, beta-TCP). Akermanite and beta-TCP disks were seeded with hBMSC and kept in growth medium or osteogenic medium for 10 days. Proliferation and osteoblastic differentiation were evaluated on day 1, 4, 7 and 10. The data from the Alamar Blue assay and lactic acid production assay showed that hBMSC proliferated more significantly on akermanite than on beta-TCP. The analysis of osteoblast-related genes, including alkaline phosphatase (ALP), osteopontin (OPN), bone sialoprotein (BSP) and osteocalcin (OC), indicated that akermanite ceramics enhanced the expression of osteoblast-related genes, but type I collagen (COL I) showed no noticeable difference among akermanite and beta-TCP ceramics. Furthermore, this stimulatory effect was observed not only in osteogenic medium, but also in normal growth medium without osteogenic reagents such as l-ascorbic acid, glycerophosphate and dexamethasone. This result suggests that akermanite can promote osteoblastic differentiation of hBMSC in vitro even without osteogenic reagents, and may be used as a bioactive material for bone regeneration and tissue engineering applications.

DENTAL PULP STEM CELLS AND HUMAN PERIAPICAL CYST MESENCHYMAL STEM CELLS IN BONE TISSUE REGENERATION: COMPARISON OF BASAL AND OSTEOGENIC DIFFERENTIATED GENE EXPRESSION OF A NEWLY DISCOVERED MESENCHYMAL STEM CELL LINEAGE.

PubMed

Tatullo, M; Falisi, G; Amantea, M; Rastelli, C; Paduano, F; Marrelli, M

2015-01-01

Bone regeneration is an interesting field of biomedicine. The most recent studies are aimed to achieve a bone regeneration using mesenchymal stem cells (MSCs) taken from more accessible sites: oral and dental tissues have been widely investigated as a rich accessible source of MSCs. Dental Pulp Stem Cells (DPSCs) and human Periapical Cysts Mesenchymal Stem Cells (hPCy-MSCs) represent the new generation MSCs. The aim of this study is to compare the gene expression of these two innovative cell types to highlight the advantages of their use in bone regeneration. The harvesting, culturing and differentiating of cells isolated from dental pulp as well as from periapical cystic tissue were carried out as described in previously published reports. qRT-PCR analyses were performed on osteogenic genes in undifferentiated and osteogenic differentiated cells of DPSC and hPCy-MSC lineage. Real-time RT-PCR data suggested that both DPSCs and hPCy-MSCs cultured in osteogenic media are able to differentiate into osteoblast/odontoblast-like cells: however, some differences indicated that DPSCs seem to be directed more towards dentinogenesis, while hPCy-MSCs seem to be directed more towards osteogenesis.

Enhanced differentiation of mesenchymal stromal cells by three-dimensional culture and azacitidine

PubMed Central

Bae, Yoo-Jin; Kwon, Yong-Rim; Kim, Hye Joung; Lee, Seok

2017-01-01

Background Mesenchymal stromal cells (MSCs) are useful for cell therapy because of their potential for multilineage differentiation. However, MSCs that are expanded in traditional two-dimensional (2D) culture systems eventually lose their differentiation abilities. Therefore, we investigated whether azacitidine (AZA) supplementation and three-dimensional culture (3D) could improve the differentiation properties of MSCs. Methods 2D- or 3D-cultured MSCs which were prepared according to the conventional or hanging-drop culture method respectively, were treated with or without AZA (1 µM for 72 h), and their osteogenic and adipogenic differentiation potential were determined and compared. Results AZA treatment did not affect the cell apoptosis or viability in both 2D- and 3D-cultured MSCs. However, compared to conventionally cultured 2D-MSCs, AZA-treated 2D-MSCs showed marginally increased differentiation abilities. In contrast, 3D-MSCs showed significantly increased osteogenic and adipogenic differentiation ability. When 3D culture was performed in the presence of AZA, the osteogenic differentiation ability was further increased, whereas adipogenic differentiation was not affected. Conclusion 3D culture efficiently promoted the multilineage differentiation of MSCs, and in combination with AZA, it could help MSCs to acquire greater osteogenic differentiation ability. This optimized culture method can enhance the therapeutic potential of MSCs. PMID:28401097

Proliferation and osteogenic differentiation of rat BMSCs on a novel Ti/SiC metal matrix nanocomposite modified by friction stir processing

NASA Astrophysics Data System (ADS)

Zhu, Chenyuan; Lv, Yuting; Qian, Chao; Qian, Haixin; Jiao, Ting; Wang, Liqiang; Zhang, Fuqiang

2016-12-01

The aims of this study were to fabricate a novel titanium/silicon carbide (Ti/SiC) metal matrix nanocomposite (MMNC) by friction stir processing (FSP) and to investigate its microstructure and mechanical properties. In addition, the adhesion, proliferation and osteogenic differentiation of rat bone marrow stromal cells (BMSCs) on the nanocomposite surface were investigated. The MMNC microstructure was observed by both scanning and transmission electron microscopy. Mechanical properties were characterized by nanoindentation and Vickers hardness testing. Integrin β1 immunofluorescence, cell adhesion, and MTT assays were used to evaluate the effects of the nanocomposite on cell adhesion and proliferation. Osteogenic and angiogenic differentiation were evaluated by alkaline phosphatase (ALP) staining, ALP activity, PCR and osteocalcin immunofluorescence. The observed microstructures and mechanical properties clearly indicated that FSP is a very effective technique for modifying Ti/SiC MMNC to contain uniformly distributed nanoparticles. In the interiors of recrystallized grains, characteristics including twins, fine recrystallized grains, and dislocations formed concurrently. Adhesion, proliferation, and osteogenic and angiogenic differentiation of rat BMSCs were all enhanced on the novel Ti/SiC MMNC surface. In conclusion, nanocomposites modified using FSP technology not only have superior mechanical properties under stress-bearing conditions but also provide improved surface and physicochemical properties for cell attachment and osseointegration.

Effects of fructose-induced metabolic syndrome on rat skeletal cells and tissue, and their responses to metformin treatment.

PubMed

Felice, Juan Ignacio; Schurman, León; McCarthy, Antonio Desmond; Sedlinsky, Claudia; Aguirre, José Ignacio; Cortizo, Ana María

2017-04-01

Deleterious effects of metabolic syndrome (MS) on bone are still controversial. In this study we evaluated the effects of a fructose-induced MS, and/or an oral treatment with metformin on the osteogenic potential of bone marrow mesenchymal stromal cells (MSC), as well as on bone formation and architecture. 32 male 8week-old Wistar rats were assigned to four groups: control (C), control plus oral metformin (CM), rats receiving 10% fructose in drinking water (FRD), and FRD plus metformin (FRDM). Samples were collected to measure blood parameters, and to perform pQCT analysis and static and dynamic histomorphometry. MSC were isolated to determine their osteogenic potential. Metformin improved blood parameters in FRDM rats. pQCT and static and dynamic histomorphometry showed no significant differences in trabecular and cortical bone parameters among groups. FRD reduced TRAP expression and osteocyte density in trabecular bone and metformin only normalized osteocyte density. FRD decreased the osteogenic potential of MSC and metformin administration could revert some of these parameters. FRD-induced MS shows reduction in MSC osteogenic potential, in osteocyte density and in TRAP activity. Oral metformin treatment was able to prevent trabecular osteocyte loss and the reduction in extracellular mineralization induced by FRD-induced MS. Copyright © 2017 Elsevier B.V. All rights reserved.

Photostimulation of osteogenic differentiation on silk scaffolds by plasma arc light source.

PubMed

Çakmak, Anıl Sera; Çakmak, Soner; Vatansever, H Seda; Gümüşderelioğlu, Menemşe

2018-05-01

Low-level laser therapy (LLLT) has been used for more than 30 years to heal wounds. In recent years, LLLT or photostimulation has been indicated as an effective tool for regenerative and dental medicine by using monochromatic light. The aim of this study is to indicate the usability of plasma arc light source for bone regeneration. This is why we used polychromatic light source providing effective wavelengths in the range of 590-1500 nm for cellular response and investigated photostimulation effects on osteogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on 3D silk scaffolds. Cellular responses were examined by using cell culture methods in terms of proliferation, differentiation, and morphological analyses. The results showed that photostimulation with a polychromatic light source (applied for 5 min from the 3rd day after seeding up to the 28th day in 2-day intervals with 92-mW/cm 2 power from 10-cm distance to the cells) enhanced osteogenic differentiation of hMSCs according to higher alkaline phosphatase (ALP) activity, collagen and calcium content, osteogenic gene expressions, and matrix mineralization. In conclusion, we suggest that the plasma arc light source that was used here has a great potential for bone regeneration.

The Promiscuity of [beta]-Strand Pairing Allows for Rational Design of [beta]-Sheet Face Inversion

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

Makabe, Koki; Koide, Shohei

2009-06-17

Recent studies suggest the dominant role of main-chain H-bond formation in specifying {beta}-sheet topology. Its essentially sequence-independent nature implies a large degree of freedom in designing {beta}-sheet-based nanomaterials. Here we show rational design of {beta}-sheet face inversions by incremental deletions of {beta}-strands from the single-layer {beta}-sheet of Borrelia outer surface protein A. We show that a {beta}-sheet structure can be maintained when a large number of native contacts are removed and that one can design large-scale conformational transitions of a {beta}-sheet such as face inversion by exploiting the promiscuity of strand-strand interactions. High-resolution X-ray crystal structures confirmed the success ofmore » the design and supported the importance of main-chain H-bonds in determining {beta}-sheet topology. This work suggests a simple but effective strategy for designing and controlling nanomaterials based on {beta}-rich peptide self-assemblies.« less

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.

Platelet lysate supports the in vitro expansion of human periodontal ligament stem cells for cytotherapeutic use.

PubMed

Wu, Rui-Xin; Yu, Yang; Yin, Yuan; Zhang, Xi-Yu; Gao, Li-Na; Chen, Fa-Ming

2017-08-01

Human platelet lysate (PL) produced under optimal conditions of standardization and safety has been increasingly suggested as the future 'gold standard' supplement to replace fetal bovine serum (FBS) for the ex vivo propagation of mesenchymal stem cells for translational medicine and cell therapy applications. However, the multifaceted effects of PL on tissue-specific stem cells remain largely unexplored. In the present study, we investigated the stem cell behaviours of human periodontal ligament stem cells (PDLSCs) in media with or without PL. Our data indicate that human PL, either as an adjuvant for culture media or as a substitute for FBS, supports the proliferation and expansion of human PDLSCs derived from either 'young' or 'old' donors to the same extent as FBS, without interfering with their immunomodulatory capacities. Although PL appears to inhibit the in vitro differentiation of 'young' or 'old' PDLSCs, their decreased osteogenic potential may be restored to similar or higher levels compared with FBS-expanded cells. PL- and FBS-expanded PDLSCs exhibited a similar potential to form mineralized nodules and expressed similar levels of osteogenic genes. Our data indicate that large clinically relevant quantities of PDLSCs may be yielded by the use of human PL; however, further analysis of its precise composition and function will pave the way for determining optimized, defined culture conditions. In addition to the potential increase in patient safety, our findings highlight the need for further research to develop the potential of PL-expanded PDLSCs for clinical use. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Sheet Probability Index (SPI): Characterizing the geometrical organization of the white matter with diffusion MRI

PubMed Central

Tax, Chantal M.W.; Haije, Tom Dela; Fuster, Andrea; Westin, Carl-Fredrik; Viergever, Max A.; Florack, Luc; Leemans, Alexander

2017-01-01

The question whether our brain pathways adhere to a geometric grid structure has been a popular topic of debate in the diffusion imaging and neuroscience society. Wedeen et al. (2012a b) proposed that the brain’s white matter is organized like parallel sheets of interwoven pathways. Catani et al. (2012) concluded that this grid pattern is most likely an artifact, resulting from methodological biases that cause the tractography pathways to cross in orthogonal angles. To date, ambiguities in the mathematical conditions for a sheet structure to exist (e.g. its relation to orthogonal angles) combined with the lack of extensive quantitative evidence have prevented wide acceptance of the hypothesis. In this work, we formalize the relevant terminology and recapitulate the condition for a sheet structure to exist. Note that this condition is not related to the presence or absence of orthogonal crossing fibers, and that sheet structure is defined formally as a surface formed by two sets of interwoven pathways intersecting at arbitrary angles within the surface. To quantify the existence of sheet structure, we present a novel framework to compute the sheet probability index (SPI), which reflects the presence of sheet structure in discrete orientation data (e.g. fiber peaks derived from diffusion MRI). With simulation experiments we investigate the effect of spatial resolution, curvature of the fiber pathways, and measurement noise on the ability to detect sheet structure. In real diffusion MRI data experiments we can identify various regions where the data supports sheet structure (high SPI values), but also areas where the data does not support sheet structure (low SPI values) or where no reliable conclusion can be drawn. Several areas with high SPI values were found to be consistent across subjects, across multiple data sets obtained with different scanners, resolutions, and degrees of diffusion weighting, and across various modeling techniques. Under the strong assumption that the diffusion MRI peaks reflect true axons, our results would therefore indicate that pathways do not form sheet structures at every crossing fiber region but instead at well-defined locations in the brain. With this framework, sheet structure location, extent, and orientation could potentially serve as new structural features of brain tissue. The proposed method can be extended to quantify sheet structure in directional data obtained with techniques other than diffusion MRI, which is essential for further validation. PMID:27456538

How Fast? How Far? How Much? What We Need to Learn from the Past Behavior of Ice Sheets, and What They Might Not Tell Us

NASA Astrophysics Data System (ADS)

Alley, R. B.

2016-12-01

Paleoclimatic data support physical understanding that changes in ice sheets are primarily caused by changes in ocean temperature and in melting from above. With interesting qualifications, ice sheets tend to grow as accumulation rate in central regions drops into an ice age, and to shrink as accumulation rate rises. Changes in sea level may be influential but generally are too small and slow to be of primary importance. Thus, future atmospheric warming, oceanic warming and changes in oceanic circulation are especially important to future ice-sheet behavior. Paleoclimatic data support models and physical understanding that sustained warming beyond thresholds will cause progressively larger sea-level rise, up to quite high values, although the thresholds remain poorly quantified. Several indirect lines of evidence indicate great shrinkage or loss of parts or all of the Greenland ice sheet and marine sectors of the Antarctic ice sheet under warmth corresponding to CO2 levels similar to the modern or committed level. Despite this evidence, the state of the ice sheets during the most recent times warmer than today, including MIS 5e, remains unclear. The Greenland ice sheet did survive MIS 5e, but that may reflect warmth sufficient to remove the ice sheet but not sustained long enough to do so; greater warming in the future could cause much faster sea-level rise than generated in the past. Several indirect lines of evidence indicate that the marine basins of the West Antarctic Ice Sheet deglaciated during MIS 5e, and targeted field data could clarify this greatly. Physical understanding suggests, however, that even if this deglaciation did occur, it may have been slower than is possible in an even warmer future world; past rates of sea-level rise may define minimum rather than likely future rates.

Human fallopian tube mesenchymal stromal cells enhance bone regeneration in a xenotransplanted model.

PubMed

Jazedje, Tatiana; Bueno, Daniela F; Almada, Bruno V P; Caetano, Heloisa; Czeresnia, Carlos E; Perin, Paulo M; Halpern, Silvio; Maluf, Mariangela; Evangelista, Lucila P; Nisenbaum, Marcelo G; Martins, Marília T; Passos-Bueno, Maria R; Zatz, Mayana

2012-06-01

We have recently reported that human fallopian tubes, which are discarded during surgical procedures of women submitted to sterilization or hysterectomies, are a rich source of human fallopian tube mesenchymal stromal cells (htMSCs). It has been previously shown that human mesenchymal stromal cells may be useful in enhancing the speed of bone regeneration. This prompted us to investigate whether htMSCs might be useful for the treatment of osteoporosis or other bone diseases, since they present a pronounced capacity for osteogenic differentiation in vitro. Based on this prior knowledge, our aim was to evaluate, in vivo, the osteogenic capacity of htMSCs to regenerate bone through an already described xenotransplantation model: nonimmunosuppressed (NIS) rats with cranial defects. htMSCs were obtained from five 30-50 years old healthy women and characterized by flow cytometry and for their multipotenciality in vitro capacity (osteogenic, chondrogenic and adipogenic differentiations). Two symmetric full-thickness cranial defects on each parietal region of seven NIS rats were performed. The left side (LS) of six animals was covered with CellCeram (Scaffdex)-a bioabsorbable ceramic composite scaffold that contains 60% hydroxyapatite and 40% β-tricalciumphosphate-only, and the right side (RS) with the CellCeram and htMSCs (10(6) cells/scaffold). The animals were euthanized at 30, 60 and 90 days postoperatively and cranial tissue samples were taken for histological analysis. After 90 days we observed neobone formation in both sides. However, in animals euthanized 30 and 60 days after the procedure, a mature bone was observed only on the side with htMSCs. PCR and immunofluorescence analysis confirmed the presence of human DNA and thus that human cells were not rejected, which further supports the imunomodulatory property of htMSCs. In conclusion, htMSCs can be used successfully to enhance bone regeneration in vivo, opening a new field for future treatments of osteoporosis and bone reconstruction.

Rough titanium alloys regulate osteoblast production of angiogenic factors.

PubMed

Olivares-Navarrete, Rene; Hyzy, Sharon L; Gittens, Rolando A; Schneider, Jennifer M; Haithcock, David A; Ullrich, Peter F; Slosar, Paul J; Schwartz, Zvi; Boyan, Barbara D

2013-11-01

Polyether-ether-ketone (PEEK) and titanium-aluminum-vanadium (titanium alloy) are used frequently in lumbar spine interbody fusion. Osteoblasts cultured on microstructured titanium generate an environment characterized by increased angiogenic factors and factors that inhibit osteoclast activity mediated by integrin α2β1 signaling. It is not known if this is also true of osteoblasts on titanium alloy or PEEK. The purpose of this study was to determine if osteoblasts generate an environment that supports angiogenesis and reduces osteoclastic activity when grown on smooth titanium alloy, rough titanium alloy, or PEEK. This in vitro study compared angiogenic factor production and integrin gene expression of human osteoblast-like MG63 cells cultured on PEEK or titanium-aluminum-vanadium (titanium alloy). MG63 cells were grown on PEEK, smooth titanium alloy, or rough titanium alloy. Osteogenic microenvironment was characterized by secretion of osteoprotegerin and transforming growth factor beta-1 (TGF-β1), which inhibit osteoclast activity and angiogenic factors including vascular endothelial growth factor A (VEGF-A), fibroblast growth factor 2 (FGF-2), and angiopoietin-1 (ANG-1). Expression of integrins, transmembrane extracellular matrix recognition proteins, was measured by real-time polymerase chain reaction. Culture on titanium alloy stimulated osteoprotegerin, TGF-β1, VEGF-A, FGF-2, and angiopoietin-1 production, and levels were greater on rough titanium alloy than on smooth titanium alloy. All factors measured were significantly lower on PEEK than on smooth or rough titanium alloy. Culture on titanium alloy stimulated expression of messenger RNA for integrins that recognize Type I collagen in comparison with PEEK. Rough titanium alloy stimulated cells to create an osteogenic-angiogenic microenvironment. The osteogenic-angiogenic responses to titanium alloy were greater than PEEK and greater on rough titanium alloy than on smooth titanium alloy. Surface features regulated expression of integrins important in collagen recognition. These factors may increase bone formation, enhance integration, and improve implant stability in interbody spinal fusions. Copyright © 2013 Elsevier Inc. All rights reserved.

Tissue engineering approaches for studying the effect of biochemical and physiological stimuli on cell behavior

NASA Astrophysics Data System (ADS)

Jimenez Vergara, Andrea Carolina

Tissue engineering (TE) approaches have emerged as an alternative to traditional tissue and organ replacements. The aim of this work was to contribute to the understanding of the effects of cell-material and endothelial cell (EC) paracrine signaling on cell responses using poly(ethylene glycol) diacrylate (PEGDA) hydrogels as a material platform. Three TE applications were explored. First, the effect of glycosaminoglycan (GAG) identity was evaluated for vocal fold restoration. Second, the influence of GAG identity was explored and a novel approach for stable endothelialization was developed for vascular graft applications. Finally, EC paracrine signaling in the presence of cyclic stretch, and hydrophobicity and inorganic content were studied for osteogenic applications. In terms of vocal fold restoration, it was found that vocal fold fibroblast (VFF) phenotype and extracellular matrix (ECM) production were impacted by GAG identity. VFF phenotype was preserved in long-term cultured hydrogels containing high molecular weight hyaluronan (HAHMW). Furthermore, collagen I deposition, fibronectin production and smooth muscle α-actin (SM-α-actin) expression in PEG-HA, PEG-chondroitin sulfate C and PEG-heparan sulfate (HS) gels suggest that CSC and HS may be undesirable for vocal fold implants. Regarding vascular graft applications, the impact of GAG identity on smooth muscle cell (SMC) foam cell formation was explored. Results support the increasing body of literature that suggests a critical role for dermatan sulfate (DS)-bearing proteoglycans in early atherosclerosis. In addition, an approach for fabricating bi-layered tissue engineering vascular grafts (TEVGs) with stable endothelialization was validated using PEGDA as an intercellular “cementing” agent between adjacent endothelial cells (ECs). Finally, mesenchymal stem cell (MSC) differentiation toward osteogenic like cells was evaluated. ECM and cell phenotypic data showed that elevated scaffold inorganic content and hydrophobicity were indeed correlated with increased osteogenic differentiation. Moreover, the present results suggest that EC paracrine signaling enhances MSC osteogenesis in the presence of cyclic stretch.

A Dual Role of Graphene Oxide Sheet Deposition on Titanate Nanowire Scaffolds for Osteo-implantation: Mechanical Hardener and Surface Activity Regulator

NASA Astrophysics Data System (ADS)

Dong, Wenjun; Hou, Lijuan; Li, Tingting; Gong, Ziqiang; Huang, Huandi; Wang, Ge; Chen, Xiaobo; Li, Xiaoyun

2015-12-01

Scaffold biomaterials with open pores and channels are favourable for cell growth and tissue regeneration, however the inherent poor mechanical strength and low surface activity limit their applications as load-bearing bone grafts with satisfactory osseointegration. In this study, macro-porous graphene oxide (GO) modified titanate nanowire scaffolds with desirable surface chemistry and tunable mechanical properties were prepared through a simple hydrothermal process followed by electrochemical deposition of GO nanosheets. The interconnected and porous structure of the GO/titanate nanowire scaffolds provides a large surface area for cellular attachment and migration and displays a high compressive strength of approximately 81.1 MPa and a tunable Young’s modulus over the range of 12.4-41.0 GPa, which satisfies site-specific requirements for implantation. Surface chemistry of the scaffolds was modulated by the introduction of GO, which endows the scaffolds flexibility in attaching and patterning bioactive groups (such as -OH, -COOH and -NH2). In vitro cell culture tests suggest that the GO/titanate nanowire scaffolds act as a promising biomaterial candidate, in particular the one terminated with -OH groups, which demonstrates improved cell viability, and proliferation, differentiation and osteogenic activities.

A Dual Role of Graphene Oxide Sheet Deposition on Titanate Nanowire Scaffolds for Osteo-implantation: Mechanical Hardener and Surface Activity Regulator.

PubMed

Dong, Wenjun; Hou, Lijuan; Li, Tingting; Gong, Ziqiang; Huang, Huandi; Wang, Ge; Chen, Xiaobo; Li, Xiaoyun

2015-12-21

Scaffold biomaterials with open pores and channels are favourable for cell growth and tissue regeneration, however the inherent poor mechanical strength and low surface activity limit their applications as load-bearing bone grafts with satisfactory osseointegration. In this study, macro-porous graphene oxide (GO) modified titanate nanowire scaffolds with desirable surface chemistry and tunable mechanical properties were prepared through a simple hydrothermal process followed by electrochemical deposition of GO nanosheets. The interconnected and porous structure of the GO/titanate nanowire scaffolds provides a large surface area for cellular attachment and migration and displays a high compressive strength of approximately 81.1 MPa and a tunable Young's modulus over the range of 12.4-41.0 GPa, which satisfies site-specific requirements for implantation. Surface chemistry of the scaffolds was modulated by the introduction of GO, which endows the scaffolds flexibility in attaching and patterning bioactive groups (such as -OH, -COOH and -NH2). In vitro cell culture tests suggest that the GO/titanate nanowire scaffolds act as a promising biomaterial candidate, in particular the one terminated with -OH groups, which demonstrates improved cell viability, and proliferation, differentiation and osteogenic activities.

Assessment of the Genetic Variation in Bone Fracture Healing

DTIC Science & Technology

2004-10-01

decrease in the expression of the major mRNA markers of differentiated osteogenic cells (osteocalcin type I collagen and bone sialoprotein ) (Kon et al., 9...and higher levels of bone sialoprotein which are seen both during cartilage hypertrophy and as a marker of early to mid osteogenic differentiation...Biochem 89(2):401-26. Barnes GL, Della Torre T, Sommer B, Young MF, Gerstenfeld LC. 2002. Transcriptional regulation restricting bone sialoprotein gene

Injectable Sources of Locally Controlled Electrical Fields to Facilitate Tissue Repair

DTIC Science & Technology

2001-10-25

craniofacial and somatic developmental anomalies for which the current gold standard is highly invasive distraction osteogenesis. Shown here is a...representation of "Developmental Osteogenic Stimulation" [2] being used to direct and promote maxillary and palatal shelf growth following cleft lip...Tessier to distraction ," Childs Nerv Syst, vol. 15:11-12, pp. 681-694 , Nov. 1999. [2] H.M. Kaplan, "’Developmental Osteogenic Stimulation’ – An

YY1 and HDAC9c transcriptionally regulate p38-mediated mesenchymal stem cell differentiation into osteoblasts

PubMed Central

Chen, Ya-Huey; Chung, Chiao-Chen; Liu, Yu-Chia; Lai, Wei-Chen; Lin, Zong-Shin; Chen, Tsung-Ming; Li, Long-Yuan; Hung, Mien-Chie

2018-01-01

Mesenchymal stem cells (MSCs) have a high self-renewal potential and can differentiate into various types of cells, including adipocytes, osteoblasts, and chondrocytes. Previously, we reported that the enhancer of zeste homolog 2 (EZH2), the catalytic component of the Polycomb-repressive complex 2, and HDAC9c mediate the osteogenesis and adipogenesis of MSCs. In the current study, we identify the role of p38 in osteogenic differentiation from a MAPK antibody array screen and investigate the mechanisms underlying its transcriptional regulation. Our data show that YY1, a ubiquitously expressed transcription factor, and HDAC9c coordinate p38 transcriptional activity to promote its expression to facilitate the osteogenic potential of MSCs. Our results show that p38 mediates osteogenic differentiation, and this has significant implications in bone-related diseases, bone tissue engineering, and regenerative medicine. PMID:29637005

Morinda citrifolia leaves enhance osteogenic differentiation and mineralization of human periodontal ligament cells.

PubMed

Boonanantanasarn, Kanitsak; Janebodin, Kajohnkiart; Suppakpatana, Prapan; Arayapisit, Tawepong; Rodsutthi, Jit-aree; Chunhabundit, Panjit; Boonanuntanasarn, Surintorn; Sripairojthikoon, Wanida

2012-01-01

This present study investigated the potential of Morinda citrifolia leaf aqueous extract to induce osteogenic differentiation and matrix mineralization of human periodontal ligament (hPDL) cells. Human periodontal ligament cells were cultured in complete medium, ascorbic acid with β-glycerophosphate, or Morinda citrifolia leaf aqueous extract. Morinda citrifolia leaf aqueous extract significantly increased alkaline phosphatase activity compared to culturing in complete medium or ascorbic acid with β-glycerophosphate. Matrixcontaining mineralized nodules were formed only when the cells were cultured in the presence of Morinda citrifolia leaf aqueous extract. These nodules showed positive alizarin red S staining and were rich in calcium and phosphorus according to energy dispersive X-ray analysis. In conclusion, Morinda citrifolia leaf extract promoted osteogenic differentiation and matrix mineralization in human periodontal ligament cells, a clear indication of the therapeutic potential of Morinda citrifolia leaves in bone and periodontal tissue regeneration.

Morinda citrifolia leaves enhance osteogenic differentiation and mineralization of human periodontal ligament cells.

PubMed

Boonanantanasarn, Kanitsak; Janebodin, Kajohnkiart; Suppakpatana, Prapan; Arayapisit, Tawepong; Rodsutthi, Jit-aree; Chunhabundit, Panjit; Boonanuntanasarn, Surintorn; Sripairojthikoon, Wanida

2014-01-01

This present study investigated the potential of Morinda citrifolia leaf aqueous extract to induce osteogenic differentiation and matrix mineralization of human periodontal ligament (hPDL) cells. Human periodontal ligament cells were cultured in complete medium, ascorbic acid with β-glycerophosphate, or Morinda citrifolia leaf aqueous extract. Morinda citrifolia leaf aqueous extract significantly increased alkaline phosphatase activity compared to culturing in complete medium or ascorbic acid with β-glycerophosphate. Matrixcontaining mineralized nodules were formed only when the cells were cultured in the presence of Morinda citrifolia leaf aqueous extract. These nodules showed positive alizarin red S staining and were rich in calcium and phosphorus according to energy dispersive X-ray analysis. In conclusion, Morinda citrifolia leaf extract promoted osteogenic differentiation and matrix mineralization in human periodontal ligament cells, a clear indication of the therapeutic potential of Morinda citrifolia leaves in bone and periodontal tissue regeneration.

Case report 673: Telangiectic osteogenic sarcoma.

PubMed

Liu, S K; Thacher, C

1991-01-01

A case is presented of a telangiectatic osteogenic sarcoma involving the left third metatarsal of a 4-year-old male Great Dane dog. Radiographs revealed a diaphyseal, expanding, lytic lesion with minimal intralesional sclerosis and a sclerotic rim in the proximal portion. The lesion contained a large amount of blood. The biopsy specimens consisted of spaces which were outlined by fibrous osteoid or granulation tissue. There were islands of multinuclear giant cells and/or fibrous osteoid tissue. A diagnosis of aneurysmal bone cyst was made. The lesion was treated by curettage and insertion of cancellous bone graft but was progressive 10 weeks after treatment. The lesion was further curetted, and these biopsy specimens consisted of aneurysmally dilated spaces and areas of anaplastic sarcomatous cells with mitotic figures and osteoid production, characteristic of telangiectatic osteogenic sarcoma. The dog was euthanized as the owner requested; an autopsy was not performed.

A High Throughput Phenotypic Screening reveals compounds that counteract premature osteogenic differentiation of HGPS iPS-derived mesenchymal stem cells

PubMed Central

Lo Cicero, Alessandra; Jaskowiak, Anne-Laure; Egesipe, Anne-Laure; Tournois, Johana; Brinon, Benjamin; Pitrez, Patricia R.; Ferreira, Lino; de Sandre-Giovannoli, Annachiara; Levy, Nicolas; Nissan, Xavier

2016-01-01

Hutchinson-Gilford progeria syndrome (HGPS) is a rare fatal genetic disorder that causes systemic accelerated aging in children. Thanks to the pluripotency and self-renewal properties of induced pluripotent stem cells (iPSC), HGPS iPSC-based modeling opens up the possibility of access to different relevant cell types for pharmacological approaches. In this study, 2800 small molecules were explored using high-throughput screening, looking for compounds that could potentially reduce the alkaline phosphatase activity of HGPS mesenchymal stem cells (MSCs) committed into osteogenic differentiation. Results revealed seven compounds that normalized the osteogenic differentiation process and, among these, all-trans retinoic acid and 13-cis-retinoic acid, that also decreased progerin expression. This study highlights the potential of high-throughput drug screening using HGPS iPS-derived cells, in order to find therapeutic compounds for HGPS and, potentially, for other aging-related disorders. PMID:27739443

A High Throughput Phenotypic Screening reveals compounds that counteract premature osteogenic differentiation of HGPS iPS-derived mesenchymal stem cells.

PubMed

Lo Cicero, Alessandra; Jaskowiak, Anne-Laure; Egesipe, Anne-Laure; Tournois, Johana; Brinon, Benjamin; Pitrez, Patricia R; Ferreira, Lino; de Sandre-Giovannoli, Annachiara; Levy, Nicolas; Nissan, Xavier

2016-10-14

Hutchinson-Gilford progeria syndrome (HGPS) is a rare fatal genetic disorder that causes systemic accelerated aging in children. Thanks to the pluripotency and self-renewal properties of induced pluripotent stem cells (iPSC), HGPS iPSC-based modeling opens up the possibility of access to different relevant cell types for pharmacological approaches. In this study, 2800 small molecules were explored using high-throughput screening, looking for compounds that could potentially reduce the alkaline phosphatase activity of HGPS mesenchymal stem cells (MSCs) committed into osteogenic differentiation. Results revealed seven compounds that normalized the osteogenic differentiation process and, among these, all-trans retinoic acid and 13-cis-retinoic acid, that also decreased progerin expression. This study highlights the potential of high-throughput drug screening using HGPS iPS-derived cells, in order to find therapeutic compounds for HGPS and, potentially, for other aging-related disorders.

Bioprocess Forces and Their Impact on Cell Behavior: Implications for Bone Regeneration Therapy

PubMed Central

Brindley, David; Moorthy, Kishaani; Lee, Jae-Ho; Mason, Chris; Kim, Hae-Won; Wall, Ivan

2011-01-01

Bioprocess forces such as shear stress experienced during routine cell culture are considered to be harmful to cells. However, the impact of physical forces on cell behavior is an area of growing interest within the tissue engineering community, and it is widely acknowledged that mechanical stimulation including shear stress can enhance osteogenic differentiation. This paper considers the effects of bioprocess shear stress on cell responses such as survival and proliferation in several contexts, including suspension-adapted cells used for recombinant protein and monoclonal antibody manufacture, adherent cells for therapy in suspension, and adherent cells attached to their growth substrates. The enhanced osteogenic differentiation that fluid flow shear stress is widely found to induce is discussed, along with the tissue engineering of mineralized tissue using perfusion bioreactors. Recent evidence that bioprocess forces produced during capillary transfer or pipetting of cell suspensions can enhance osteogenic responses is also discussed. PMID:21904661

Effects of dexamethasone, ascorbic acid and β-glycerophosphate on the osteogenic differentiation of stem cells in vitro.

PubMed

Langenbach, Fabian; Handschel, Jörg

2013-01-01

The standard procedure for the osteogenic differentiation of multipotent stem cells is treatment of a confluent monolayer with a cocktail of dexamethasone (Dex), ascorbic acid (Asc) and β-glycerophosphate (β-Gly). This review describes the effects of these substances on intracellular signaling cascades that lead to osteogenic differentiation of bone marrow stroma-derived stem cells. We conclude that Dex induces Runx2 expression by FHL2/β-catenin-mediated transcriptional activation and that Dex enhances Runx2 activity by upregulation of TAZ and MKP1. Asc leads to the increased secretion of collagen type I (Col1), which in turn leads to increased Col1/α2β1 integrin-mediated intracellular signaling. The phosphate from β-Gly serves as a source for the phosphate in hydroxylapatite and in addition influences intracellular signaling molecules. In this context we give special attention to the differences between dystrophic and bone-specific mineralization.

Fact Sheet: Documenting Ground-Water Models Selection at Site Contaminated with Radioactive Substance

EPA Pesticide Factsheets

This fact sheet summarizes the report by a joint Interagency Environmental Pathway Modeling Working Group. It was designed to be used by technical staff responsible for identifying and implementing flow and transport models to support cleanup decisions.

Focusing solar collector and method for manufacturing same

DOEpatents

Murphy, Lawrence M.

1984-01-01

Disclosed is a solar collector comprising an annular-shaped frame and a composite membrane member for concentrating and focusing sun radiation. The composite membrane member is supported and tensioned by the frame and consists of first and second differentially pretensioned sheet members which are integrally bonded to one another. The frame and one of the two sheet members are adapted to allow tensions in both of the sheets to be adjusted. Subsequent to bonding and upon adjusting a tension in one of the two sheet members, both of the two bonded sheet members react with one another so as to cause the composite membrane member to have a contoured configuration, which enables the membrane member to be focusable. Additionally, adjusting the tension in one of the two sheet members provides a reciprocal adjustment in a focus provided by the membrane member.

Focusing solar collector and method for manufacturing same

DOEpatents

Murphy, L.M.

1984-01-01

Disclosed is a solar collector comprising an annular-shaped frame and a composite membrane member for concentrating and focusing sun radiation. The composite membrane member is supported and tensioned by the frame and consists of first and second differentially pretensioned sheet members which are integrally bonded to one another. The frame and one of the two sheet members are adapted to allow tensions in both of the two sheets to be adjusted. Subsequent to bonding and upon adjusting a tension in one of the two sheet members, both of the two bonded sheet members react with one another so as to cause the composite membrane member to have a contoured configuration, which enables the membrane member to be focusable. Additionally, adjusting the tension in one of the two sheet members provides a reciprocal adjustment in a focus provided by the membrane member.

Optimal Design of Sheet Pile Wall Embedded in Clay

NASA Astrophysics Data System (ADS)

Das, Manas Ranjan; Das, Sarat Kumar

2015-09-01

Sheet pile wall is a type of flexible earth retaining structure used in waterfront offshore structures, river protection work and temporary supports in foundations and excavations. Economy is an essential part of a good engineering design and needs to be considered explicitly in obtaining an optimum section. By considering appropriate embedment depth and sheet pile section it may be possible to achieve better economy. This paper describes optimum design of both cantilever and anchored sheet pile wall penetrating clay using a simple optimization tool Microsoft Excel ® Solver. The detail methodology and its application with examples are presented for cantilever and anchored sheet piles. The effects of soil properties, depth of penetration and variation of ground water table on the optimum design are also discussed. Such a study will help professional while designing the sheet pile wall penetrating clay.

Osteogenic response of human MSCs and osteoblasts to hydrophilic and hydrophobic nanostructured titanium implant surfaces.

PubMed

Lotz, Ethan M; Olivares-Navarrete, Rene; Berner, Simon; Boyan, Barbara D; Schwartz, Zvi

2016-12-01

Microstructured implant surfaces created by grit blasting and acid etching titanium (Ti) support osseointegration. This effect is further enhanced by storing in aqueous solution to retain hydrophilicity, but this also leads to surface nanostructure formation. The purpose of this study was to assess the contributions of nanostructures on the improved osteogenic response of osteoblast lineage cells to hydrophilic microstructured Ti. Human mesenchymal stem cells (MSCs) and normal human osteoblasts (NHOsts) were cultured separately on non-nanostructured/hydrophobic (SLA), nanostructured/hydrophilic (modSLA), or nanostructured/hydrophobic (SLAnano) Ti surfaces. XPS showed elevated carbon levels on SLA and SLAnano compared to modSLA. Contact angle measurements indicated only modSLA was hydrophilic. Confocal laser microscopy revealed minor differences in mean surface roughness. SEM showed the presence of nanostructures on modSLA and SLAnano. MSCs and NHOst cells exhibited similar morphology on the substrates and osteoblastic differentiation and maturation were greatest on modSLA. These results suggest that when the appropriate microstructure is present, hydrophilicity may play a greater role in stimulating MSC and NHOst osteoblastic differentiation and maturation than the presence of nanostructures generated during storage in an aqueous environment. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3137-3148, 2016. © 2016 Wiley Periodicals, Inc.

Stem cells applications in bone and tooth repair and regeneration: New insights, tools, and hopes.

PubMed

Abdel Meguid, Eiman; Ke, Yuehai; Ji, Junfeng; El-Hashash, Ahmed H K

2018-03-01

The exploration of stem and progenitor cells holds promise for advancing our understanding of the biology of tissue repair and regeneration mechanisms after injury. This will also help in the future use of stem cell therapy for the development of regenerative medicine approaches for the treatment of different tissue-species defects or disorders such as bone, cartilages, and tooth defects or disorders. Bone is a specialized connective tissue, with mineralized extracellular components that provide bones with both strength and rigidity, and thus enable bones to function in body mechanical supports and necessary locomotion process. New insights have been added to the use of different types of stem cells in bone and tooth defects over the last few years. In this concise review, we briefly describe bone structure as well as summarize recent research progress and accumulated information regarding the osteogenic differentiation of stem cells, as well as stem cell contributions to bone repair/regeneration, bone defects or disorders, and both restoration and regeneration of bones and cartilages. We also discuss advances in the osteogenic differentiation and bone regeneration of dental and periodontal stem cells as well as in stem cell contributions to dentine regeneration and tooth engineering. © 2017 Wiley Periodicals, Inc.

Design and Assessment of a Dynamic Perfusion Bioreactor for Large Bone Tissue Engineering Scaffolds.

PubMed

Bhaskar, Birru; Owen, Robert; Bahmaee, Hossein; Rao, Parcha Sreenivasa; Reilly, Gwendolen C

2018-06-01

Bioreactors can be used to apply fluid flow in vitro to scaffolds to improve mass transport of media and apply mechanical forces to cells. In this study, we developed and tested an autoclavable, modular perfusion bioreactor suitable for large scaffolds. We investigated the effects of fluid flow induced shear stress (FFSS) on osteogenic differentiation of human embryonic stem cell-derived mesenchymal progenitors (hES-MP cells) cultured on large polyurethane (PU) scaffolds (30 mm diameter × 5 mm thickness) in osteogenesis induction media (OIM). After seeding, scaffolds were either maintained in static conditions or transferred to the bioreactor 3 days post-seeding and a continuous flow rate of 3.47 mL/min was applied. Alkaline phosphatase activity (ALP) was used to evaluate osteogenic differentiation and resazurin salt reduction (RR) to measure metabolic activity after 10 days. Cultures subjected to flow contained significantly more metabolically active cells and higher total DNA content, as well as significantly higher ALP activity compared to scaffolds grown in static culture. These results confirm the responsiveness of hES-MP cells to fluid flow stimuli, and present a cost-effective, user-friendly bioreactor capable of supporting the growth and differentiation of mesenchymal progenitor cells within scaffolds capable of filling large bone defects.

Fact Sheet: Environmental Characteristics of EPA, NRC, and DOE Sites Contaminated with Radioactive Substances

EPA Pesticide Factsheets

This fact sheet summarizes the findings of a report by a joint Interagency Environmental Pathway Modeling Working Group. It was designed to be used by technical staff responsible for implementing flow and transport models to support cleanup decisions.

Processing experiments on non-Czochralski silicon sheet

NASA Technical Reports Server (NTRS)

Pryor, R. A.; Grenon, L. A.; Sakiotis, N. G.; Pastirik, E. M.; Sparks, T. O.; Legge, R. N.

1981-01-01

A program is described which supports and promotes the development of processing techniques which may be successfully and cost-effectively applied to low-cost sheets for solar cell fabrication. Results are reported in the areas of process technology, cell design, cell metallization, and production cost simulation.