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Sample records for enhances osteogenic differentiation

  1. Osteogenic differentiation of mesenchymal stem cells could be enhanced by strontium.

    PubMed

    Yang, Fan; Tu, Jie; Yang, Dazhi; Li, Guanglin; Cai, Lintao; Wang, Liping

    2010-01-01

    Strontium is a newly developed drug for decreasing risks of hip or vertebral fractures in postmenopausal women. Experimental studies have suggested that Strontium could enhance new bone formation and decrease the bone resorption. Mesenchymal stem cell (MSC) is an important category of stem cells which possess the osteogenic differentiation potential and could be used in the bone and cartilage tissue engineering. Here we investigated the effects of strontium on the osteogenic differentiation process of MSC. We found that strontium could enhance the calcium deposition process and promote bone repair, through enhancing the osteogenic differentiation of MSC. This study could help to develop a new strategy to induce the MSC to differentiate into the osteogenic lineage. PMID:21096310

  2. 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.

  3. Inhibition of Histone Deacetylases Enhances the Osteogenic Differentiation of Human Periodontal Ligament Cells.

    PubMed

    Huynh, Nam Cong-Nhat; Everts, Vincent; Pavasant, Prasit; Ampornaramveth, Ruchanee Salingcarnboriboon

    2016-06-01

    One of the characteristics of periodontal ligament (PDL) cells is their plasticity. Yet, the underlying mechanisms responsible for this phenomenon are unknown. One possible mechanism might be related to epigenetics, since histone deacetylases (HDACs) have been shown to play a role in osteoblast differentiation. This study was aimed to investigate the role of HDACs in osteogenic differentiation of human PDL (hPDL) cells. HDAC inhibitor trichostatin A (TSA) had no effect on cell viability as was assessed by MTT assay. Osteogenic and adipogenic differentiation was analyzed by gene expression, ALP activity and mineral deposition. Western blotting was used to investigate the effect of TSA on histone acetylation and protein expression. In the presence of the HDAC inhibitor osteogenic differentiation was induced; osteoblast-related gene expression was increased significantly. ALP activity and mineral nodule formation were also enhanced. Inhibition of HDACs did not induce differentiation into the adipocyte lineage. hPDL highly expressed HDACs of both class I (HDAC 1, 2, 3) and class II (HDAC 4, 6). During osteogenic differentiation HDAC 3 expression gradually decreased. This was apparent in the absence and presence of the inhibitor. The level of acetylated Histone H3 was increased during osteogenic differentiation. Inhibition of HDAC activity induced hyperacetylation of Histone H3, therefore, demonstrating Histone H3 as a candidate target molecule for HDAC inhibition. In conclusion, hPDL cells express a distinguished series of HDACs and these enzymes appear to be involved in osteogenic differentiation. This finding suggests a potential application of TSA for bone regeneration therapy by hPDL cells. PMID:27043246

  4. 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.

  5. Enhancing proliferation and osteogenic differentiation of HMSCs on casein/chitosan multilayer films.

    PubMed

    Li, Yan; Zheng, Zebin; Cao, Zhinan; Zhuang, Liangting; Xu, Yong; Liu, Xiaozhen; Xu, Yue; Gong, Yihong

    2016-05-01

    Creating a bioactive surface is important in tissue engineering. Inspired by the natural calcium binding property of casein (CA), multilayer films ((CA/CS)n) with chitosan (CS) as polycation were fabricated to enhance biomineralization, cell adhesion and differentiation. LBL self-assembly technique was used and the assembly process was intensively studied based on changes of UV absorbance, zeta potential and water contact angle. The increasing content of chitosan and casein with bilayers was further confirmed with XPS and TOF-SIMS analysis. To improve the biocompatibility, gelatin was surface grafted. In vitro mineralization test demonstrated that multilayer films had more hydroxyapatite crystal deposition. Human mesenchymal stem cells (HMSCs) were seeded onto these films. According to fluorescein diacetate (FDA) and cell cytoskeleton staining, MTT assay, expression of osteogenic marker genes, ALP activity, and calcium deposition quantification, it was found that these multilayer films significantly promoted HMSCs attachment, proliferation and osteogenic differentiation than TCPS control. PMID:26895501

  6. Hierarchical scaffolds enhance osteogenic differentiation of human Wharton's jelly derived stem cells.

    PubMed

    Canha-Gouveia, Analuce; Rita Costa-Pinto, Ana; Martins, Albino M; Silva, Nuno A; Faria, Susana; Sousa, Rui A; Salgado, António J; Sousa, Nuno; Reis, Rui L; Neves, Nuno M

    2015-09-01

    Hierarchical structures, constituted by polymeric nano and microfibers, have been considered promising scaffolds for tissue engineering strategies, mainly because they mimic, in some way, the complexity and nanoscale detail observed in real organs. The chondrogenic potential of these scaffolds has been previously demonstrated, but their osteogenic potential is not yet corroborated. In order to assess if a hierarchical structure, with nanoscale details incorporated, is an improved scaffold for bone tissue regeneration, we evaluate cell adhesion, proliferation, and osteogenic differentiation of human Wharton's jelly derived stem cells (hWJSCs), seeded into hierarchical fibrous scaffolds. Biological data corroborates that hierarchical fibrous scaffolds show an enhanced cell entrapment when compared to rapid prototyped scaffolds without nanofibers. Furthermore, upregulation of bone specific genes and calcium phosphate deposition confirms the successful osteogenic differentiation of hWJSCs on these scaffolds. These results support our hypothesis that a scaffold with hierarchical structure, in conjugation with hWJSCs, represents a possible feasible strategy for bone tissue engineering applications. PMID:26335618

  7. Osteogenic Differentiation of Human Mesenchymal Stem Cells Synergistically Enhanced by Biomimetic Peptide Amphiphiles Combined with Conditioned Media

    PubMed Central

    Anderson, Joel M.; Vines, Jeremy B.; Patterson, Jessica L.; Chen, Haiyan; Javed, Amjad; Jun, Ho-Wook

    2010-01-01

    An attractive strategy for bone tissue engineering is the use of extracellular matrix (ECM) analogous biomaterials capable of governing biological response based on synthetic cell-ECM interactions. In this study, peptide amphiphiles (PAs) were investigated as an ECM-mimicking biomaterial to provide an instructive microenvironment for human mesenchymal stem cells (hMSCs) in an effort to guide osteogenic differentiation. PAs were biologically functionalized with ECM isolated ligand sequences (i.e. RGDS, DGEA), and the osteoinductive potential was studied with or without conditioned media, containing the supplemental factors of dexamethasone, β-glycerol phosphate, and ascorbic acid. It was hypothesized that the ligand-functionalized PAs would synergistically enhance osteogenic differentiation in combination with conditioned media. Concurrently, comparative evaluations independent of osteogenic supplements investigated the differentiating potential of the functionalized PA scaffolds as promoted exclusively by the inscribed ligand signals, thus offering the potential for therapeutic effectiveness under physiological conditions. Osteoinductivity was assessed by histochemical staining for alkaline phosphatase (ALP) and quantitative real-time PCR analysis of key osteogenic markers. Both of the ligand-functionalized PAs were found to synergistically enhance the level of visualized ALP activity and osteogenic gene expression compared to the control surfaces lacking biofunctionality. Guided osteoinduction was also observed without supplemental aid on the PA scaffolds, but at a delayed response and not to the same phenotypic levels. Thus, the biomimetic PAs foster a symbiotic enhancement of osteogenic differentiation, demonstrating the potential of ligand functionalized biomaterials for future bone tissue repair. PMID:20728586

  8. In Vitro Osteogenic Differentiation Enhanced by Zirconia Coated with Nano-Layered Growth and Differentiation Factor-5.

    PubMed

    Yoon, Sun Jung; Yang, Dae Hyeok; Kim, Eun-Cheol; Noh, Kwantae; Lee, Deok-Won

    2016-01-01

    Zirconia (Zr) is also known as a biocompatible material with favorable mechanical properties as well as low plaque adhesion. In this study, we examined the efficacy of Zr coated with growth and differentiation factor-5 (GDF-5) bonded via click reaction as a substrate to support osteogenic differentiation of MC3T3-E1 cells. Pristine and surface-modified Zr surfaces were characterized by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS), resulting that GDF-5 was successfully coated to the pristine Zr surface. GDF-5 coated to Zr surfaces was released for 28 days in a sustained manner. New bone formation onto GDF-5 coated Zr (Zr/GDF-5) surface was confirmed by in vitro test including cell proliferation, alkaline phosphatase activity and calcium deposition assays, and in vivo test including real-time polymerase chain reaction (qPCR) assay including osterix (OSX), runt-related transcription factor 2 (Runx 2), COL 1 (type I collagen) and osteocalcin (OC). Cell proliferation, alkaline phosphatase activity, and calcium deposition of MC3T3- E1 cells were significantly enhanced when the cells were cultured on Zr/GDF-5. Additionally, the results of qPCR revealed that genes related with osteogenic differentiation were up regulated when the cells were cultured on Zr/GDF-5. Our findings demonstrate that Zr/GDF-5 could be used as a material for enhancing the efficacy of osteogenic differentiation. PMID:27398455

  9. 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. PMID:25766516

  10. FTY720 enhances osteogenic differentiation of bone marrow mesenchymal stem cells in ovariectomized rats.

    PubMed

    Huang, Chuang; Ling, Rui; Li, Fei-Jiang; Li, Er-Cui; Huang, Qi-Ke; Liu, Bao-Gang; Ding, Yin; You, Si-Wei

    2016-07-01

    Sphingosine-1-phosphate and its structural analog FTY720 (fingolimod) are important in the inhibition of osteoclast differentiation and bone resorption, however, it remains unknown whether they enhance osteogenic differentiation of the bone marrow mesenchymal stem cells (BM‑MSCs). The present study investigated the effect of FTY720 on the osteogenic differentiation of BM‑MSCs from the femurs of the ovariectomized (OVX) rats. Three different concentrations (1, 10 and 100 nM) of FTY720 were demonstrated to markedly upregulate mRNA expression levels of Runt‑related transcription factor 2 (Runx2) and Sp7 transcription factor (Sp7) at 2 weeks, and alkaline phosphatase (ALP) at 3 weeks. The osteocalcin (OCN) expression was similar at weeks 2 and 3. The protein expression levels of Runx2, Sp7, OCN and ALP induced by three different concentrations of FTY720 were higher than those in the control groups at 3 weeks in the OVX and sham groups. The findings of the current study suggested a beneficial effect of FTY720 on bone formation in OVX rats, and provided a potential therapeutic method of FTY720 to prevent alveolar bone resorption in patients with post‑menopausal osteoporosis. PMID:27220612

  11. Dynamic piezoelectric stimulation enhances osteogenic differentiation of human adipose stem cells.

    PubMed

    Ribeiro, Clarisse; Pärssinen, Jenita; Sencadas, Vítor; Correia, Vítor; Miettinen, S; Hytönen, Vesa P; Lanceros-Méndez, Senentxu

    2015-06-01

    This work reports on the influence of the substrate polarization of electroactive β-poly(vinylidene fluoride) (β-PVDF) on human adipose stem cells (hASCs) differentiation under static and dynamic conditions. hASCs were cultured on different β-PVDF surfaces (non-poled and "poled -") adsorbed with fibronectin and osteogenic differentiation was determined using a quantitative alkaline phosphatase assay. "Poled -" β-PVDF samples promote higher osteogenic differentiation, which is even higher under dynamic conditions. It is thus demonstrated that electroactive membranes can provide the necessary electromechanical stimuli for the differentiation of specific cells and therefore will support the design of suitable tissue engineering strategies, such as bone tissue engineering. PMID:25370596

  12. Structural and Biochemical Modification of a Collagen Scaffold to Selectively Enhance MSC Tenogenic, Chondrogenic, and Osteogenic Differentiation

    PubMed Central

    Caliari, Steven R.

    2014-01-01

    Biomaterial approaches for engineering orthopedic interfaces such as the tendon-bone junction (TBJ) are limited by a lack of understanding of how insoluble (microstructure, composition) and soluble regulators of stem cell fate work in concert to promote bioactivity and differentiation. One strategy for regenerating the interface is to design biomaterials containing spatially-graded structural properties sufficient to induce divergent mesenchymal stem cell (MSC) differentiation into multiple interface-specific phenotypes. This work explores the hypothesis that selective structural modification to a 3D collagen-glycosaminoglycan (CG) scaffold combined with biochemical supplementation can drive human bone marrow-derived MSC differentiation down tenogenic, osteogenic, and chondrogenic lineages. Tenogenic differentiation is enhanced in geometrically anisotropic scaffolds versus a standard isotropic control. Notably, blebbistatin treatment abrogates this microstructurally-driven effect. Further, enhanced osteogenic differentiation and new mineral synthesis is achieved by incorporation of a calcium phosphate mineral phase within the CG scaffold along with the use of osteogenic induction media. Finally, chondrogenic differentiation is optimally driven by combining chondrogenic induction media with a reduced density scaffold that promotes increased cellular condensation, significantly higher expression of chondrogenic genes, and increased GAG deposition. Together these data provide critical insight regarding design rules for elements of an integrated biomaterial platform for orthopedic interface regeneration. PMID:24574180

  13. 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. PMID:24682022

  14. Enhancement of adhesion and promotion of osteogenic differentiation of human adipose stem cells by poled electroactive poly(vinylidene fluoride).

    PubMed

    Pärssinen, Jenita; Hammarén, Henrik; Rahikainen, Rolle; Sencadas, Vitor; Ribeiro, Clarisse; Vanhatupa, Sari; Miettinen, Susanna; Lanceros-Méndez, Senentxu; Hytönen, Vesa P

    2015-03-01

    Poly(vinylidene fluoride) (PVDF) is a biocompatible material with excellent electroactive properties. Nonelectroactive α-PVDF and electroactive β-PVDF were used to investigate the substrate polarization and polarity influence on the focal adhesion (FA) size and number as well as on human adipose stem cells (hASCs) differentiation. hASCs were cultured on different PVDF surfaces adsorbed with fibronectin and FA size and number, total adhesion area, cell size, cell aspect ratio and FA density were estimated using cells expressing vinculin fused to enhanced green fluorescent protein. Osteogenic differentiation was also determined using a quantitative alkaline phosphatase assay. The surface charge of the poled PVDF films (positive or negative) influenced the hydrophobicity of the samples, leading to variations in the conformation of adsorbed extracellular matrix proteins, which ultimately modulated the stem cell adhesion on the films and induced their osteogenic differentiation. PMID:24838756

  15. Human mesenchymal stromal cell-enhanced osteogenic differentiation by contact interaction with polyethylene terephthalate nanogratings.

    PubMed

    Antonini, Sara; Meucci, Sandro; Parchi, Paolo; Pacini, Simone; Montali, Marina; Poggetti, Andrea; Lisanti, Michele; Cecchini, Marco

    2016-01-01

    Among the very large number of polymeric materials that have been proposed in the field of orthopedics, polyethylene terephthalate (PET) is one of the most attractive thanks to its flexibility, thermal resistance, mechanical strength and durability. Several studies have been proposed that interface nano- or micro-structured surfaces with mesenchymal stromal cells (MSCs), demonstrating the potential of this technology for promoting osteogenesis. All these studies were carried out on biomaterials other than PET, which remains almost uninvestigated in terms of cell shaping, alignment and differentiation. Here, we study the effect of PET 350-depth nanogratings (NGs) with a ridge and lateral groove size of 500 nm (T1) or 1 μm (T2), on bone marrow-derived human MSC (hMSC) differentiation in relation to the osteogenic fate. We demonstrate that these substrates, especially T2, can promote the osteogenic phenotype more efficiently than standard flat surfaces and that this effect is more marked if cells are cultured in osteogenic medium than in basal medium. Finally, we show that the shape and disposition of calcium hydroxyapatite granules on the different substrates was influenced by the substrate symmetry, being more elongated and spatially organized on NGs than on flat surfaces. PMID:27388559

  16. IGF1 potentiates BMP9-induced osteogenic differentiation in mesenchymal stem cells through the enhancement of BMP/Smad signaling

    PubMed Central

    Chen, Liang; Zou, Xiang; Zhang, Ran-Xi; Pi, Chang-Jun; Wu, Nian; Yin, Liang-Jun; Deng, Zhong-Liang

    2016-01-01

    Engineered bone tissue is thought to be the ideal alternative for bone grafts in the treatment of related bone diseases. BMP9 has been demonstrated as one of the most osteogenic factors, and enhancement of BMP9-induced osteogenesis will greatly accelerate the development of bone tissue engineering. Here, we investigated the effect of insulin-like growth factor 1 (IGF1) on BMP9-induced osteogenic differentiation, and unveiled a possible molecular mechanism underling this process. We found that IGF1 and BMP9 are both detectable in mesenchymal stem cells (MSCs). Exogenous expression of IGF1 potentiates BMP9-induced alkaline phosphatase (ALP), matrix mineralization, and ectopic bone formation. Similarly, IGF1 enhances BMP9-induced endochondral ossification. Mechanistically, we found that IGF1 increases BMP9-induced activation of BMP/Smad signaling in MSCs. Our findings demonstrate that IGF1 can enhance BMP9-induced osteogenic differentiation in MSCs, and that this effect may be mediated by the enhancement of the BMP/Smad signaling transduction triggered by BMP9. [BMB Reports 2016; 49(2): 122-127] PMID:26645636

  17. MicroRNA-200c Represses IL-6, IL-8, and CCL-5 Expression and Enhances Osteogenic Differentiation

    PubMed Central

    Sharp, Thad; Khorsand, Behnoush; Fischer, Carol; Eliason, Steven; Salem, Ali; Akkouch, Adil; Brogden, Kim; Amendt, Brad A.

    2016-01-01

    MicroRNAs (miRs) regulate inflammation and BMP antagonists, thus they have potential uses as therapeutic reagents. However, the molecular function of miR-200c in modulating proinflammatory and bone metabolic mediators and osteogenic differentiation is not known. After miR-200c was transduced into a human embryonic palatal mesenchyme (HEPM) (a cell line of preosteoblasts), using lentiviral vectors, the resulting miR-200c overexpression increased osteogenic differentiation biomarkers, including osteocalcin (OCN) transcripts and calcium content. miR-200c expression also down-regulated interleukin (IL)-6, IL-8, and chemokine (C-C motif) ligand (CCL)-5 under lipopolysaccharide (LPS) stimulation and increased osteoprotegerin (OPG) in these cells. miR-200c directly regulates the expression of IL-6, IL-8 and CCL-5 transcripts by binding to their 3’UTRs. A plasmid-based miR-200c inhibitor effectively reduces their binding activities. Additionally, miR-200c delivered using polyethylenimine (PEI) nanoparticles effectively inhibits IL-6, IL-8 and CCL-5 in primary human periodontal ligament fibroblasts and increases the biomarkers of osteogenic differentiation in human bone marrow mesenchymal stem cells (MSCs), including calcium content, ALP, and Runx2. These data demonstrate that miR-200c represses IL-6, IL-8 and CCL-5 and improves osteogenic differentiation. miR-200c may potentially be used as an effective means to prevent periodontitis-associated bone loss by arresting inflammation and osteoclastogenesis and enhancing bone regeneration. PMID:27529418

  18. MicroRNA-200c Represses IL-6, IL-8, and CCL-5 Expression and Enhances Osteogenic Differentiation.

    PubMed

    Hong, Liu; Sharp, Thad; Khorsand, Behnoush; Fischer, Carol; Eliason, Steven; Salem, Ali; Akkouch, Adil; Brogden, Kim; Amendt, Brad A

    2016-01-01

    MicroRNAs (miRs) regulate inflammation and BMP antagonists, thus they have potential uses as therapeutic reagents. However, the molecular function of miR-200c in modulating proinflammatory and bone metabolic mediators and osteogenic differentiation is not known. After miR-200c was transduced into a human embryonic palatal mesenchyme (HEPM) (a cell line of preosteoblasts), using lentiviral vectors, the resulting miR-200c overexpression increased osteogenic differentiation biomarkers, including osteocalcin (OCN) transcripts and calcium content. miR-200c expression also down-regulated interleukin (IL)-6, IL-8, and chemokine (C-C motif) ligand (CCL)-5 under lipopolysaccharide (LPS) stimulation and increased osteoprotegerin (OPG) in these cells. miR-200c directly regulates the expression of IL-6, IL-8 and CCL-5 transcripts by binding to their 3'UTRs. A plasmid-based miR-200c inhibitor effectively reduces their binding activities. Additionally, miR-200c delivered using polyethylenimine (PEI) nanoparticles effectively inhibits IL-6, IL-8 and CCL-5 in primary human periodontal ligament fibroblasts and increases the biomarkers of osteogenic differentiation in human bone marrow mesenchymal stem cells (MSCs), including calcium content, ALP, and Runx2. These data demonstrate that miR-200c represses IL-6, IL-8 and CCL-5 and improves osteogenic differentiation. miR-200c may potentially be used as an effective means to prevent periodontitis-associated bone loss by arresting inflammation and osteoclastogenesis and enhancing bone regeneration. PMID:27529418

  19. Adiponectin enhances osteogenic differentiation in human adipose-derived stem cells by activating the APPL1-AMPK signaling pathway

    SciTech Connect

    Chen, Tong; Wu, Yu-wei; Lu, Hui; Guo, Yuan; Tang, Zhi-hui

    2015-05-29

    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 regulatory 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

  20. Vascular endothelial growth factor enhances in vitro proliferation and osteogenic differentiation of human dental pulp stem cells.

    PubMed

    D' Alimonte, I; Nargi, E; Mastrangelo, F; Falco, G; Lanuti, P; Marchisio, M; Miscia, S; Robuffo, I; Capogreco, M; Buccella, S; Caputi, S; Caciagli, F; Tetè, S; Ciccarelli, R

    2011-01-01

    Mesenchymal stem cells (MSC), isolated from dental tissues, are largely studied for future application in regenerative dentistry. In this study, we used MSC obtained from human dental pulp (DPSC) of normal impacted third molars that, when cultured in lineage-specific inducing media, differentiate into osteoblasts and adipocytes (evaluated by Alizarin Red S and Red Oil O stainings, respectively), thus showing a multipotency. We confirmed that DPSC, grown under undifferentiating conditions, are negative for hematopoietic (CD45, CD31, CD34, CD144) and positive for mesenchymal (CD29, CD90, CD105, CD166, CD146, STRO-1) markers, that underwent down-regulation when cells were grown in osteogenic medium for 3 weeks. In this condition, they also exhibit an increase in the expression of osteogenic markers (RUNX-2, alkaline phosphatase) and extracellular calcium deposition, whereas the expression of receptors (VEGFR-1 and -2) for vascular endothelial growth factors (VEGF) and related VEGF binding proteins was similar to that found in undifferentiated DPSC. Exposure of DPSC growing under undifferentiating or osteogenic conditions to VEGF-A165 peptide (10-40 ng/ml) for 8 days dose- and time-dependently increased the number of proliferating cells without inducing differentiation towards endothelial lineage, as evaluated by the lack of expression of specific markers (CD31, CD34, CD144). Additionally, exposure of DPSC cultured in osteogenic medium to VEGF-A165 for a similar period enhanced cell differentiation towards osteoblasts as evaluated after 14 and 21 days by Alizarin Red S staining and alkaline phosphatase activity quantification. These findings may have clinical implications possibly facilitating tissue repair and remodeling. PMID:21382274

  1. 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

    2015-12-01

    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. PMID:26586668

  2. Enhanced gene delivery by chitosan-disulfide-conjugated LMW-PEI for facilitating osteogenic differentiation.

    PubMed

    Zhao, Xiaoli; Li, Zhaoyang; Pan, Haobo; Liu, Wenguang; Lv, Minmin; Leung, Frankie; Lu, William W

    2013-05-01

    Chitosan-disulfide-conjugated LMW-PEI (CS-ss-PEI) was designed to combine the biocompatibility of chitosan and the gene delivery ability of polyethylenimine (PEI) using bio-reducible disulfide for bone morphogenetic protein (BMP2) gene delivery in mediating osteogenic differentiation. It was prepared by conjugating low molecular weight PEI (LMW-PEI) to chitosan through oxidization of thiols introduced for the formation of disulfide linkage. The structure, molecular weight and buffer capacity were characterized by Fourier transform infrared (FTIR), light scattering and acid-base titration, respectively. The reduction in molecular weight of CS-ss-PEI by the reducing agent indicated its bio-reducible property. With the increment in the LMW-PEI component, the copolymer showed increased DNA binding ability and formed denser nanocomplexes. CS-ss-PEI exhibited low cytotoxicity in COS-1, HepG2 and 293T cells over the different weight ratios. The transfection efficiency of CS-ss-PEI4 was significantly higher than that of PEI 25k and comparable with Lipofectamine in mediating luciferase expression. Its application for BMP2 gene delivery was confirmed in C2C12 cells by BMP2 expression. For inducing in vitro osteogenic differentiation, CS-ss-PEI4 mediated BMP2 gene delivery showed a stronger effect in MG-63 osteoblast cells and stem cells in terms of alkaline phosphatase activity and mineralization compared with PEI25k and Lipofectamine. This study provides a potential gene delivery system for orthopedic-related disease. PMID:23395816

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

    PubMed

    Maroni, Paola; Brini, Anna Teresa; Arrigoni, Elena; de Girolamo, Laura; Niada, Stefania; Matteucci, Emanuela; Bendinelli, Paola; Desiderio, Maria Alfonsina

    2012-11-16

    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) γ. These key osteogenic and adipogenic transcription factors are oppositely 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 PPARγ 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 PPARγ/ALP activity diminutions mostly after HDAC3 silencing. HDAC1 and 3 genetic blockade increased and decreased Runx2 and PPARγ 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. PMID:23085045

  4. 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

  5. Sustained presentation of BMP-2 enhances osteogenic differentiation of human adipose-derived stem cells in gelatin hydrogels.

    PubMed

    Samorezov, Julia E; Headley, Emma B; Everett, Christopher R; Alsberg, Eben

    2016-06-01

    Human adipose-derived stem cells (hASCs) show great potential for healing bone defects. Bone morphogenetic protein-2 (BMP-2) has been reported to stimulate their osteogenic differentiation both in vitro and in vivo. Here, methacrylated gelatin (GelMA) hydrogels were evaluated as a system to deliver BMP-2 to encapsulated hASCs from two different donors, and BMP-2 delivered from the hydrogels was compared to BMP-2 presented exogenously in culture media. GelMA hydrogels were shown to provide sustained, localized presentation of BMP-2 due to electrostatic interactions between the growth factor and biomaterial after an initial burst release. Both donors exhibited similar responses to the loaded and exogenous growth factor; BMP-2 from the hydrogels had a statistically significant effect on hASC osteogenic differentiation compared to exogenous BMP-2. Expression of alkaline phosphatase was accelerated, and cells in hydrogels with loaded BMP-2 deposited more calcium at one, two, and four weeks than cells without BMP-2 or with the growth factor presented in the media. There were no statistically significant differences in calcium content between groups with 25, 50, or 100 µg/mL loaded BMP-2, suggesting that using a lower growth factor dose may be as effective as a higher loading amount in this system. Taken together, these findings suggest that controlled delivery of BMP-2 from the GelMA enhances its osteogenic bioactivity compared to free growth factor presented in the media. Thus, the GelMA system is a promising biomaterial for BMP-2-mediated hASC osteogenesis. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1387-1397, 2016. PMID:26822338

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

    SciTech Connect

    Maroni, Paola; Brini, Anna Teresa; Arrigoni, Elena; Girolamo, Laura de; Niada, Stefania; Matteucci, Emanuela; Bendinelli, Paola; Desiderio, Maria Alfonsina

    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 oppositely 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

  7. Enhanced Osteogenic and Vasculogenic Differentiation Potential of Human Adipose Stem Cells on Biphasic Calcium Phosphate Scaffolds in Fibrin Gels.

    PubMed

    van Esterik, Fransisca A S; Zandieh-Doulabi, Behrouz; Kleverlaan, Cornelis J; Klein-Nulend, Jenneke

    2016-01-01

    For bone tissue engineering synthetic biphasic calcium phosphate (BCP) with a hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) ratio of 60/40 (BCP60/40) is successfully clinically applied, but the high percentage of HA may hamper efficient scaffold remodelling. Whether BCP with a lower HA/β-TCP ratio (BCP20/80) is more desirable is still unclear. Vascular development is needed before osteogenesis can occur. We aimed to test the osteogenic and/or vasculogenic differentiation potential as well as degradation of composites consisting of human adipose stem cells (ASCs) seeded on BCP60/40 or BCP20/80 incorporated in fibrin gels that trigger neovascularization for bone regeneration. ASC attachment to BCP60/40 and BCP20/80 within 30 min was similar (>93%). After 11 days of culture BCP20/80-based composites showed increased alkaline phosphatase activity and DMP1 gene expression, but not RUNX2 and osteonectin expression, compared to BCP60/40-based composites. BCP20/80-based composites also showed enhanced expression of the vasculogenic markers CD31 and VEGF189, but not VEGF165 and endothelin-1. Collagen-1 and collagen-3 expression was similar in both composites. Fibrin degradation was increased in BCP20/80-based composites at day 7. In conclusion, BCP20/80-based composites showed enhanced osteogenic and vasculogenic differentiation potential compared to BCP60/40-based composites in vitro, suggesting that BCP20/80-based composites might be more promising for in vivo bone augmentation than BCP60/40-based composites. PMID:27547223

  8. Enhanced Osteogenic and Vasculogenic Differentiation Potential of Human Adipose Stem Cells on Biphasic Calcium Phosphate Scaffolds in Fibrin Gels

    PubMed Central

    2016-01-01

    For bone tissue engineering synthetic biphasic calcium phosphate (BCP) with a hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) ratio of 60/40 (BCP60/40) is successfully clinically applied, but the high percentage of HA may hamper efficient scaffold remodelling. Whether BCP with a lower HA/β-TCP ratio (BCP20/80) is more desirable is still unclear. Vascular development is needed before osteogenesis can occur. We aimed to test the osteogenic and/or vasculogenic differentiation potential as well as degradation of composites consisting of human adipose stem cells (ASCs) seeded on BCP60/40 or BCP20/80 incorporated in fibrin gels that trigger neovascularization for bone regeneration. ASC attachment to BCP60/40 and BCP20/80 within 30 min was similar (>93%). After 11 days of culture BCP20/80-based composites showed increased alkaline phosphatase activity and DMP1 gene expression, but not RUNX2 and osteonectin expression, compared to BCP60/40-based composites. BCP20/80-based composites also showed enhanced expression of the vasculogenic markers CD31 and VEGF189, but not VEGF165 and endothelin-1. Collagen-1 and collagen-3 expression was similar in both composites. Fibrin degradation was increased in BCP20/80-based composites at day 7. In conclusion, BCP20/80-based composites showed enhanced osteogenic and vasculogenic differentiation potential compared to BCP60/40-based composites in vitro, suggesting that BCP20/80-based composites might be more promising for in vivo bone augmentation than BCP60/40-based composites. PMID:27547223

  9. Instructive Conductive 3D Silk Foam-Based Bone Tissue Scaffolds Enable Electrical Stimulation of Stem Cells for Enhanced Osteogenic Differentiation.

    PubMed

    Hardy, John G; Geissler, Sydney A; Aguilar, David; Villancio-Wolter, Maria K; Mouser, David J; Sukhavasi, Rushi C; Cornelison, R Chase; Tien, Lee W; Preda, R Carmen; Hayden, Rebecca S; Chow, Jacqueline K; Nguy, Lindsey; Kaplan, David L; Schmidt, Christine E

    2015-11-01

    Stimuli-responsive materials enabling the behavior of the cells that reside within them to be controlled are vital for the development of instructive tissue scaffolds for tissue engineering. Herein, we describe the preparation of conductive silk foam-based bone tissue scaffolds that enable the electrical stimulation of human mesenchymal stem cells (HMSCs) to enhance their differentiation toward osteogenic outcomes. PMID:26033953

  10. Cartilage-derived morphogenetic proteins enhance the osteogenic protein-1-induced osteoblastic cell differentiation of C2C12 cells.

    PubMed

    Yeh, Lee-Chuan C; Tsai, Alicia D; Zavala, Michelle C; Lee, John C

    2004-12-01

    Previous studies have shown that osteogenic protein-1 (OP-1; also known as BMP-7) induces differentiation of the pluripotent mesenchymal cell line C2C12 into osteoblastic cells. OP-1 also alters the steady-state levels of messenger RNA (mRNA) encoding for the cartilage-derived morphogenetic proteins (CDMPs) in C2C12 cells. In the present study, the effects of exogenous CDMPs on bone cell differentiation induced by OP-1 in C2C12 cells were examined. Exogenous CDMP-1, -2, and -3 synergistically and dose-dependently enhanced OP-1 action in stimulating alkaline phosphatase (AP) activity and osteocalcin (OC) mRNA expression. AP staining studies revealed that the combination of OP-1 and CDMP enhanced OP-1 action by stimulating those cells that had responded to OP-1 and not by activating additional cells. The combination did not change the mRNA expression of the BMPs and their receptors. CDMP-1 enhanced the suppression of the OP-1-induced expression of the myogeneic differentiation regulator MyoD. CDMP-1 and OP-1 alone stimulated Smad5 protein expression, but the combination of OP-1 and CDMP-1 stimulated synergistically Smad5 protein expression. Thus, one mechanism of the observed synergy involved enhancement of the induced Smad5 protein expression. At the same protein concentration, CDMP-1 is most potent in enhancing OP-1 activity in inducing osteoblastic cell differentiation of C2C12 cells. CDMP-3 is about 80% as potent as CDMP-1, and CDMP-2 is the least potent (about 50% of CDMP-1). PMID:15389555

  11. 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. PMID:20569096

  12. Overexpression of HSPA1A enhances the osteogenic differentiation of bone marrow mesenchymal stem cells via activation of the Wnt/β-catenin signaling pathway

    PubMed Central

    Zhang, Wei; Xue, Deting; Yin, Houfa; Wang, Shengdong; Li, Chao; Chen, Erman; Hu, Dongcai; Tao, Yiqing; Yu, Jiawei; Zheng, Qiang; Gao, Xiang; Pan, Zhijun

    2016-01-01

    HSPA1A, which encodes cognate heat shock protein 70, plays important roles in various cellular metabolic pathways. To investigate its effects on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), its expression level was compared between undifferentiated and differentiated BMSCs. Rat HSPA1A overexpression in BMSCs increased osteoblast-specific gene expression, alkaline phosphatase activity, and mineral deposition in vitro. Moreover, it upregulated β-catenin and downregulated DKK1 and SOST. The enhanced osteogenesis due to HSPA1A overexpression was partly rescued by a Wnt/β-catenin inhibitor. Additionally, using a rat tibial fracture model, a sheet of HSPA1A-overexpressing BMSCs improved bone fracture healing, as determined by imaging and histological analysis. Taken together, these findings suggest that HSPA1A overexpression enhances osteogenic differentiation of BMSCs, partly through Wnt/β-catenin. PMID:27279016

  13. Exogenous polyamines promote osteogenic differentiation by reciprocally regulating osteogenic and adipogenic gene expression.

    PubMed

    Lee, Mon-Juan; Chen, Yuhsin; Huang, Yuan-Pin; Hsu, Yi-Chiang; Chiang, Lan-Hsin; Chen, Tzu-Yu; Wang, Gwo-Jaw

    2013-12-01

    Polyamines are naturally occurring organic polycations that are ubiquitous in all organisms, and are essential for cell proliferation and differentiation. Although polyamines are involved in various cellular processes, their roles in stem cell differentiation are relatively unexplored. In this study, we found that exogenous polyamines, putrescine, spermidine, and spermine, promoted osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) without inducing cell death or apoptosis. Alkaline phosphatase (ALP) activity and the mRNA level of osteogenic genes, including Runx2, ALP, osteopontin, and osteocalcin, were up-regulated by exogenous polyamines. When hBMSCs were cultured at high cell density favoring adipocyte formation, exogenous polyamines resulted in down-regulation of adipogenic genes such as PPARγ, aP2, and adipsin. Extracellular matrix mineralization, a marker for osteoblast maturation, was enhanced in the presence of exogenous polyamines, while lipid accumulation, an indication of adipogenic differentiation, was attenuated. Exogenous polyamines increased the mRNA expression of polyamine-modulated factor 1 (PMF-1) and its downstream effector, spermidine/spermine N(1)-acetyltransferase (SSAT), while that of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, was suppressed. These results lead to possible connections between polyamine metabolism and osteogenic differentiation pathways. To summarize, this study provides evidence for the involvement of polyamines in osteogenic differentiation of hBMSCs, and is the first to demonstrate that osteogenic and adipogenic differentiation are reciprocally regulated by exogenous polyamines. PMID:23794266

  14. Magnetically induced electrostimulation of human osteoblasts results in enhanced cell viability and osteogenic differentiation

    PubMed Central

    HIEMER, BETTINA; ZIEBART, JOSEFIN; JONITZ-HEINCKE, ANIKA; GRUNERT, PHILIP CHRISTIAN; SU, YUKUN; HANSMANN, DORIS; BADER, RAINER

    2016-01-01

    The application of electromagnetic fields to support the bone-healing processes is a therapeutic approach for patients with musculoskeletal disorders. The ASNIS-III s-series screw is a bone stimulation system providing electromagnetic stimulation; however, its influence on human osteoblasts (hOBs) has not been extensively investigated. Therefore, in the present study, the impact of this system on the viability and differentiation of hOBs was examined. We used the ASNIS-III s screw system in terms of a specific experimental test set-up. The ASNIS-III s screw system was used for the application of electromagnetic fields (EMF, 3 mT, 20 Hz) and electromagnetic fields combined with an additional alternating electric field (EMF + EF) (3 mT, 20 Hz, 700 mV). The stimulation of primary hOBs was conducted 3 times per day for 45 min over a period of 72 h. Unstimulated cells served as the controls. Subsequently, the viability, the gene expression of differentiation markers and pro-collagen type 1 synthesis of the stimulated osteoblasts and corresponding controls were investigated. The application of both EMF and EMF + EF using the ASNIS-III s screw system revealed a positive influence on bone cell viability and moderately increased the synthesis of pro-collagen type 1 compared to the unstimulated controls. Stimulation with EMF resulted in a slightly enhanced gene expression of type 1 collagen and osteocalcin; however, stimulation with EMF + EF resulted in a significant increase in alkaline phosphatase (1.4-fold) and osteocalcin (1.6-fold) levels, and a notable increase in the levels of runt-related transcription factor 2 (RUNX-2; 1.54-fold). Our findings demonstrate that stimulation with electromagnetic fields and an additional alternating electric field has a positive influence on hOBs as regards cell viability and the expression of osteoblastic differentiation markers. PMID:27220915

  15. Single-Layer Graphene Enhances the Osteogenic Differentiation of Human Mesenchymal Stem Cells In Vitro and In Vivo.

    PubMed

    Liu, Yunsong; Chen, Tong; Du, Feng; Gu, Ming; Zhang, Ping; Zhang, Xiao; Liu, Jianzhang; Lv, Longwei; Xiong, Chunyang; Zhou, Yongsheng

    2016-06-01

    In recent years, although several studies have demonstrated the potential of graphene-coated substrates in promoting attachment, proliferation and differentiation of osteoblasts and mesenchymal stem cells (MSCs), the effects of single-layer graphene on the osteogenic differentiation of human MSCs (hMSCs) remains unclear, especially in vivo. In this study, we transferred chemical vapor deposition (CVD) grown single-layer graphene to glass slides and observed its effects on adhesion, proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs) and human bone marrow mesenchymal stem cells (hBMMSCs) in vitro. Then, in vivo, we incubated hASCs and hBMMSCs on single-layer graphene-coated smooth titanium (Ti) disks before implanting them into the back subcutaneous area of nude mice. We found that single-layer graphene accelerated cell adhesion to the substrate without influencing cell proliferation of hMSCs. Moreover, we present the first study that explores the epigenetic role of single-layer graphene in determining stem cell fate. By utilizing epigenetic approaches, we reveal that single-layer graphene promotes osteogenic differentiation of hMSCs both in vitro and in vivo, potentially by upregulating methylation of H3K4 at the promoter regions of osteogenesis-associated genes. Overall, our results highlight the potential of this material in implants and injured tissues in clinical applications. PMID:27319220

  16. Porous tantalum coatings prepared by vacuum plasma spraying enhance bmscs osteogenic differentiation and bone regeneration in vitro and in vivo.

    PubMed

    Tang, Ze; Xie, Youtao; Yang, Fei; Huang, Yan; Wang, Chuandong; Dai, Kerong; Zheng, Xuebin; Zhang, Xiaoling

    2013-01-01

    Tantalum, as a potential metallic implant biomaterial, is attracting more and more attention because of its excellent anticorrosion and biocompatibility. However, its significantly high elastic modulus and large mechanical incompatibility with bone tissue make it unsuitable for load-bearing implants. In this study, porous tantalum coatings were first successfully fabricated on titanium substrates by vacuum plasma spraying (VPS), which would exert the excellent biocompatibility of tantalum and alleviate the elastic modulus of tantalum for bone tissue. We evaluated cytocompatibility and osteogenesis activity of the porous tantalum coatings using human bone marrow stromal cells (hBMSCs) and its ability to repair rabbit femur bone defects. The morphology and actin cytoskeletons of hBMSCs were observed via electron microscopy and confocal, and the cell viability, proliferation and osteogenic differentiation potential of hBMSCs were examined quantitatively by PrestoBlue assay, Ki67 immunofluorescence assay, real-time PCR technology and ALP staining. For in vivo detection, the repaired femur were evaluated by histomorphology and double fluorescence labeling 3 months postoperation. Porous tantalum coating surfaces promoted hBMSCs adhesion, proliferation, osteogenesis activity and had better osseointegration and faster new bone formation rate than titanium coating control. Our observation suggested that the porous tantalum coatings had good biocompatibility and could enhance osseoinductivity in vitro and promote new bone formation in vivo. The porous tantalum coatings prepared by VPS is a promising strategy for bone regeneration. PMID:23776648

  17. Porous Tantalum Coatings Prepared by Vacuum Plasma Spraying Enhance BMSCs Osteogenic Differentiation and Bone Regeneration In Vitro and In Vivo

    PubMed Central

    Tang, Ze; Xie, Youtao; Yang, Fei; Huang, Yan; Wang, Chuandong; Dai, Kerong; Zheng, Xuebin; Zhang, Xiaoling

    2013-01-01

    Tantalum, as a potential metallic implant biomaterial, is attracting more and more attention because of its excellent anticorrosion and biocompatibility. However, its significantly high elastic modulus and large mechanical incompatibility with bone tissue make it unsuitable for load-bearing implants. In this study, porous tantalum coatings were first successfully fabricated on titanium substrates by vacuum plasma spraying (VPS), which would exert the excellent biocompatibility of tantalum and alleviate the elastic modulus of tantalum for bone tissue. We evaluated cytocompatibility and osteogenesis activity of the porous tantalum coatings using human bone marrow stromal cells (hBMSCs) and its ability to repair rabbit femur bone defects. The morphology and actin cytoskeletons of hBMSCs were observed via electron microscopy and confocal, and the cell viability, proliferation and osteogenic differentiation potential of hBMSCs were examined quantitatively by PrestoBlue assay, Ki67 immunofluorescence assay, real-time PCR technology and ALP staining. For in vivo detection, the repaired femur were evaluated by histomorphology and double fluorescence labeling 3 months postoperation. Porous tantalum coating surfaces promoted hBMSCs adhesion, proliferation, osteogenesis activity and had better osseointegration and faster new bone formation rate than titanium coating control. Our observation suggested that the porous tantalum coatings had good biocompatibility and could enhance osseoinductivity in vitro and promote new bone formation in vivo. The porous tantalum coatings prepared by VPS is a promising strategy for bone regeneration. PMID:23776648

  18. Magnetically induced electrostimulation of human osteoblasts results in enhanced cell viability and osteogenic differentiation.

    PubMed

    Hiemer, Bettina; Ziebart, Josefin; Jonitz-Heincke, Anika; Grunert, Philip Christian; Su, Yukun; Hansmann, Doris; Bader, Rainer

    2016-07-01

    The application of electromagnetic fields to support the bone-healing processes is a therapeutic approach for patients with musculoskeletal disorders. The ASNIS-III s-series screw is a bone stimulation system providing electromagnetic stimulation; however, its influence on human osteoblasts (hOBs) has not been extensively investigated. Therefore, in the present study, the impact of this system on the viability and differentiation of hOBs was examined. We used the ASNIS-III s screw system in terms of a specific experimental test set-up. The ASNIS-III s screw system was used for the application of electromagnetic fields (EMF, 3 mT, 20 Hz) and electromagnetic fields combined with an additional alternating electric field (EMF + EF) (3 mT, 20 Hz, 700 mV). The stimulation of primary hOBs was conducted 3 times per day for 45 min over a period of 72 h. Unstimulated cells served as the controls. Subsequently, the viability, the gene expression of differentiation markers and pro-collagen type 1 synthesis of the stimulated osteoblasts and corresponding controls were investigated. The application of both EMF and EMF + EF using the ASNIS-III s screw system revealed a positive influence on bone cell viability and moderately increased the synthesis of pro-collagen type 1 compared to the unstimulated controls. Stimulation with EMF resulted in a slightly enhanced gene expression of type 1 collagen and osteocalcin; however, stimulation with EMF + EF resulted in a significant increase in alkaline phosphatase (1.4-fold) and osteocalcin (1.6-fold) levels, and a notable increase in the levels of runt-related transcription factor 2 (RUNX-2; 1.54-fold). Our findings demonstrate that stimulation with electromagnetic fields and an additional alternating electric field has a positive influence on hOBs as regards cell viability and the expression of osteoblastic differentiation markers. PMID:27220915

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

    PubMed Central

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

    2016-01-01

    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. PMID:26961859

  20. 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.

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

    PubMed

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

    2016-01-01

    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. PMID:26961859

  2. Synergistic enhancement of human bone marrow stromal cell proliferation and osteogenic differentiation on BMP-2-derived and RGD peptide concentration gradients.

    PubMed

    Moore, Nicole M; Lin, Nancy J; Gallant, Nathan D; Becker, Matthew L

    2011-05-01

    Rational design of bioactive tissue engineered scaffolds for directing bone regeneration in vivo requires a comprehensive understanding of cell interactions with the immobilized bioactive molecules. In the current study, substrates possessing gradient concentrations of immobilized peptides were used to measure the concentration-dependent proliferation and osteogenic differentiation of human bone marrow stromal cells. Two bioactive peptides, one derived from extracellular matrix protein (ECM), GRGDS, and one from bone morphogenic protein-2 (BMP-2), KIPKASSVPTELSAISTLYL, were found to synergistically enhance cell proliferation, up-regulate osteogenic mRNA markers bone sialoprotein (BSP) and Runt-related transcription factor 2, and produce mineralization at densities greater than 130 pmol cm(-2) (65 pmol cm(-2) for each peptide). In addition, COOH-terminated self-assembled monolayers alone led to up-regulated BSP mRNA levels at densities above 200 pmol cm(-2) and increased cell proliferation from day 3 to day 14. Taking further advantage of both the synergistic potentials and the concentration-dependent activities of ECM and growth-factor-derived peptides on proliferative activity and osteogenic differentiation, without the need for additional osteogenic supplements, will enable the successful incorporation of the bioactive species into biorelevant tissue engineering scaffolds. PMID:21272672

  3. Cyclic Tensile Stress During Physiological Occlusal Force Enhances Osteogenic Differentiation of Human Periodontal Ligament Cells via ERK1/2-Elk1 MAPK Pathway

    PubMed Central

    Li, Lu; Han, Minxuan; Li, Sheng

    2013-01-01

    Physiological occlusal force constitutively exists in the oral environment and is important for periodontal homeostasis and remodeling. Cyclic tensile stress (CTS) triggers the biological response of periodontal ligament (PDL). However, a few reports have studied the correlation between CTS during physiological occlusal force and PDL cell activities such as osteogenic differentiation. In the present study, human PDL cells (hPDLCs) were subjected to 10% elongation CTS loading at 0.5 Hz for 24 h, which represents the physiological conditions of occlusal force. Gene expression microarray was used to investigate the mechano-induced differential gene profile and pathway analysis in vitro. The osteogenic relative factors, that is, SPP1, RUNX2, and SP7, were assessed by real-time PCR and Western blot. The involvement of mitogen-activated protein kinase (MAPK) signaling pathways was investigated by Western blot with a specific inhibitor. The expressions of SPP1, RUNX2, SP7, p-ERK1/2, and p-Elk1 were up-regulated after 10% CTS exposure. However, these up-regulated expressions were prevented by ERK1/2 inhibitor U0126 in the physiological occlusal force-applied hPDLCs. These results showed that 10% CTS could enhance osteogenic differentiation of hPDLCs via ERK1/2-Elk1 MAPK pathway, indicating that CTS during physiological occlusal force is a potent agent for PDL remodeling. PMID:23781879

  4. 5-(Hydroxymethyl)-2-furaldehyde inhibits adipogenic and enhances osteogenic differentiation of rat bone mesenchymal stem cells.

    PubMed

    Tan, Xiang-Ling; Zhang, Yan-Hong; Cai, Jian-Ping; Zhu, Li-Hua; Ge, Wen-Jie; Zhang, Xian

    2014-04-01

    Eucommiae Cortex (Eucommia ulmoides Oliver Bark) has been used for anti-osteoporosis usually as an ethnic drug for hundred years in China. In this study, a bioactive compound, 5-(hydroxymethyl)-2-furaldehyde (5-HMF), was isolated from Eucommiae Cortex. We found that after rat bone mesenchymal stem cells (bMSCs) were induced by 5-HMF at the concentration of 0.05, 0.10 and 0.20 microg/mL in the normal medium for 7 and 14 days, the mRNA expression of ALP, COL1alpha1 (7 days only), OCN and OPN increased. However, in the adipogenic induction medium (AIM), the mRNA expression of PPARgamma, FABP4, C/EBPalpha and LPL decreased with the 5-HMF treatment. Mineralized nodule formations were enhanced after bMSCs were induced by 5-HMF for 14 and 21 days in normal medium. In the AIM medium, 5-HMF not only inhibited the formation of adipose cells obviously, but also stimulated the mineralized nodule formation after induced for 21 days. These results indicated that 5-HMF was a powerful inhibitor of adipogenesis and enhancer of osteoblastogenesis. It may be one of the constituents contributing to anti-osteoporosis in Eucommiae Cortex. PMID:24868876

  5. Enhanced Growth and Osteogenic Differentiation of Human Osteoblast-Like Cells on Boron-Doped Nanocrystalline Diamond Thin Films

    PubMed Central

    Grausova, Lubica; Kromka, Alexander; Burdikova, Zuzana; Eckhardt, Adam; Rezek, Bohuslav; Vacik, Jiri; Haenen, Ken; Lisa, Vera; Bacakova, Lucie

    2011-01-01

    Intrinsic nanocrystalline diamond (NCD) films have been proven to be promising substrates for the adhesion, growth and osteogenic differentiation of bone-derived cells. To understand the role of various degrees of doping (semiconducting to metallic-like), the NCD films were deposited on silicon substrates by a microwave plasma-enhanced CVD process and their boron doping was achieved by adding trimethylboron to the CH4:H2 gas mixture, the B∶C ratio was 133, 1000 and 6700 ppm. The room temperature electrical resistivity of the films decreased from >10 MΩ (undoped films) to 55 kΩ, 0.6 kΩ, and 0.3 kΩ (doped films with 133, 1000 and 6700 ppm of B, respectively). The increase in the number of human osteoblast-like MG 63 cells in 7-day-old cultures on NCD films was most apparent on the NCD films doped with 133 and 1000 ppm of B (153,000±14,000 and 152,000±10,000 cells/cm2, respectively, compared to 113,000±10,000 cells/cm2 on undoped NCD films). As measured by ELISA per mg of total protein, the cells on NCD with 133 and 1000 ppm of B also contained the highest concentrations of collagen I and alkaline phosphatase, respectively. On the NCD films with 6700 ppm of B, the cells contained the highest concentration of focal adhesion protein vinculin, and the highest amount of collagen I was adsorbed. The concentration of osteocalcin also increased with increasing level of B doping. The cell viability on all tested NCD films was almost 100%. Measurements of the concentration of ICAM-1, i.e. an immunoglobuline adhesion molecule binding inflammatory cells, suggested that the cells on the NCD films did not undergo significant immune activation. Thus, the potential of NCD films for bone tissue regeneration can be further enhanced and tailored by B doping and that B doping up to metallic-like levels is not detrimental for cells. PMID:21695172

  6. Controlling Osteogenic Stem Cell Differentiation via Soft Bioinspired Hydrogels

    PubMed Central

    Jha, Amit K.; Jackson, Wesley M.; Healy, Kevin E.

    2014-01-01

    Osteogenic differentiation of human mesenchymal stem cells (hMSCs) is guided by various physical and biochemical factors. Among these factors, modulus (i.e., rigidiy) of the ECM has gained significant attention as a physical osteoinductive signal that can contribute to endochondral ossification of a cartilaginous skeletal template. However, MSCs also participate in intramembranous bone formation, which occurs de novo from within or on a more compliant tissue environment. To further understand the role of the matrix interactions in this process, we evaluated osteogenic differentiation of hMSCs cultured on low moduli (102, 390 or 970 Pa) poly(N-isopropylacrylamide) (p(NIPAAm)) based semi-interpenetrating networks (sIPN) modified with the integrin engaging peptide bsp-RGD(15) (0, 105 or 210 µM). Cell adhesion, proliferation, and osteogenic differentiation of hMSCs, as measured by alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), bone sialoprotein-2 (iBSP), and osteocalcien (OCN) protein expression, was highest on substrates with the highest modulus and peptide concentrations. However, within this range of substrate stiffness, many osteogenic cellular functions were enhanced by increasing either the modulus or the peptide density. These findings suggest that within a compliant and low modulus substrate, a high affinity adhesive ligand serves as a substitute for a rigid matrix to foster osteogenic differentiation. PMID:24937602

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

    PubMed

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

    2016-04-01

    Recent in vitro tissue engineering approaches have shown that chondrogenic priming of human bone marrow mesenchymal stem cells (MSCs) can have a positive effect on osteogenesis in vivo. However, whether chondrogenic priming is an effective in vitro bone regeneration strategy is not yet known. In particular, the appropriate timing for chondrogenic priming in vitro is unknown albeit that in vivo cartilage formation persists for a specific period before bone formation. The objective of this study is to determine the optimum time for chondrogenic priming of MSCs to enhance osteogenic differentiation by MSCs in vitro. Pellets derived from murine and human MSCs were cultured in six different media groups: two control groups (chondrogenic and osteogenic) and four chondrogenic priming groups (10, 14, 21 and 28 days priming). Biochemical analyses (Hoechst, sulfate glycosaminoglycan (sGAG), Alkaline Phosphate (ALP), calcium), histology (Alcian Blue, Alizarin Red) and immunohistochemistry (collagen types I, II and X) were performed on the samples at specific times. Our results show that after 49 days the highest amount of sGAG production occurred in MSCs chondrogenically primed for 21 days and 28 days. Moreover we found that chondrogenic priming of MSCs in vitro for specific amounts of time (14 days, 21 days) can have optimum influence on their mineralization capacity and can produce a construct that is mineralized throughout the core. Determining the optimum time for chondrogenic priming to enhance osteogenic differentiation in vitro provides information that might lead to a novel regenerative treatment for large bone defects, as well as addressing the major limitation of core degradation and construct failure. Copyright © 2013 John Wiley & Sons, Ltd. PMID:23922276

  8. Enhancement of growth and osteogenic differentiation of MC3T3-E1 cells via facile surface functionalization of polylactide membrane with chitooligosaccharide based on polydopamine adhesive coating

    NASA Astrophysics Data System (ADS)

    Li, Huihua; Luo, Chuang; Luo, Binghong; Wen, Wei; Wang, Xiaoying; Ding, Shan; Zhou, Changren

    2016-01-01

    To develop a chitooligosaccharide(COS)-functionalized poly(D,L-lactide) (PDLLA) membrane to enhance growth and osteogenic differentiation of MC3T3-E1 cells, firstly a thin polydopamine (PDOPA) layer was adhered to the PDLLA membrane via the self-polymerization and strong adhesion behavior of dopamine. Subsequently, COS was immobilized covalently on the resultant PDLLA/PDOPA composite membrane by coupling with PDOPA active coating. The successful immobilization of the PDOPA and COS was confirmed by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). Scanning electronic microscopy (SEM) and atomic force microscopy (AFM) results indicated that the surface topography and roughness of the membranes were changed, and the root mean square increased from 0.613 nm to 6.96 and 7.12 nm, respectively after coating PDOPA and COS. Water contact angle and surface energy measurements revealed that the membrane hydrophilicity was remarkably improved by surface modification. In vitro cells culture results revealed that the PDOPA- and COS-functionalized surfaces showed a significant increase in MC3T3-E1 cells adhesion, proliferation, osteogenic differentiation and alkaline phosphate activity compared to the pristine PDLLA substrate. Furthermore the COS-functionalized PDLLA membrane was more effectively at enhancing osteoblast activity than the PDOPA-functionalized PDLLA membrane.

  9. p53 Loss Increases the Osteogenic Differentiation of BMSCs

    PubMed Central

    He, Yunlong; de Castro, Luis F; Shin, Min Hwa; Dubois, Wendy; Yang, Howard H.; Jiang, Shunlin; Mishra, Pravin J.; Ren, Ling; Gou, Hongfeng; Lal, Ashish; Khanna, Chand; Merlino, Glenn; Lee, Maxwell; Robey, Pamela G.; Huang, Jing

    2014-01-01

    The tumor suppressor, p53, plays a critical role in suppressing osteosarcoma. Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) have been suggested to give rise to osteosarcomas. However, the role of p53 in BMSCs has not been extensively explored. Here, we report that p53 regulates the lineage choice of mouse BMSCs (mBMSCs). Compared to mBMSCs with wild type p53, mBMSCs deficient in p53 have enhanced osteogenic differentiation, but with similar adipogenic and chondrogenic differentiation. The role of p53 in inhibiting osteogenic lineage differentiation is mainly through the action of Runx2, a master transcription factor required for the osteogenic differentiation of mBMSCs. We find that p53 indirectly represses the expression of Runx2 by activating the microRNA-34 family, which suppresses the translation of Runx2. Since osteosarcoma may derive from BMSCs, we examined whether p53 has a role in the osteogenic differentiation of osteosarcoma cells and found that osteosarcoma cells with p53 deletion have higher levels of Runx2 and faster osteogenic differentiation than those with wild type p53. A systems biology approach reveals that p53-deficient mBMSCs are more closely related to human osteosarcoma while mBMSCs with wild type p53 are similar to normal human BMSCs. In summary, our results indicate that p53 activity can influence cell fate specification of mBMSCs, and provide molecular and cellular insights into the observation that p53 loss is associated with increased osteosarcoma incidence. PMID:25524638

  10. Osteogenic BMPs promote tumor growth of human osteosarcomas that harbor differentiation defects.

    PubMed

    Luo, Xiaoji; Chen, Jin; Song, Wen-Xin; Tang, Ni; Luo, Jinyong; Deng, Zhong-Liang; Sharff, Katie A; He, Gary; Bi, Yang; He, Bai-Cheng; Bennett, Erwin; Huang, Jiayi; Kang, Quan; Jiang, Wei; Su, Yuxi; Zhu, Gao-Hui; Yin, Hong; He, Yun; Wang, Yi; Souris, Jeffrey S; Chen, Liang; Zuo, Guo-Wei; Montag, Anthony G; Reid, Russell R; Haydon, Rex C; Luu, Hue H; He, Tong-Chuan

    2008-12-01

    Osteosarcoma (OS) is the most common primary malignancy of bone. Here, we investigated a possible role of defective osteoblast differentiation in OS tumorigenesis. We found that basal levels of the early osteogenic marker alkaline phosphatase (ALP) activity were low in OS lines. Osteogenic regulators Runx2 and OSX, and the late marker osteopontin (OPN) expressed at low levels in most OS lines, indicating that most OS cells fail to undergo terminal differentiation. Furthermore, OS cells were refractory to osteogenic BMP-induced increases in ALP activity. Osteogenic BMPs were shown to upregulate early target genes, but not late osteogenic markers OPN and osteocalcin (OC). Furthermore, osteogenic BMPs failed to induce bone formation from human OS cells, rather effectively promoted OS tumor growth in an orthotopic OS model. Exogenous expression of early target genes enhanced BMP-stimulated OS tumor growth, whereas osteogenic BMP-promoted OS tumor growth was inhibited by exogenous Runx2 expression. These results suggest that alterations in osteoprogenitors may disrupt osteogenic differentiation pathway. Thus, identifying potential differentiation defects in OS tumors would allow us to reconstruct the tumorigenic events in osteoprogenitors and to develop rational differentiation therapies for clinical OS management. PMID:18838962

  11. Modulating the biochemical and biophysical culture environment to enhance osteogenic differentiation and maturation of human pluripotent stem cell-derived mesenchymal progenitors

    PubMed Central

    2013-01-01

    Advances in the fields of stem cell biology, biomaterials, and tissue engineering over the last decades have brought the possibility of constructing tissue substitutes with a broad range of applications in regenerative medicine, disease modeling, and drug discovery. Different types of human stem cells have been used, each presenting a unique set of advantages and limitations with regard to the desired research goals. Whereas adult stem cells are at the frontier of research for tissue and organ regeneration, pluripotent stem cells represent a more challenging cell source for clinical translation. However, with their unlimited growth and wide differentiation potential, pluripotent stem cells represent an unprecedented resource for the construction of advanced human tissue models for biological studies and drug discovery. At the heart of these applications lies the challenge to reproducibly expand, differentiate, and organize stem cells into mature, stable tissue structures. In this review, we focus on the derivation of mesenchymal tissue progenitors from human pluripotent stem cells and the control of their osteogenic differentiation and maturation by modulation of the biophysical culture environment. Similarly to enhancing bone development, the described principles can be applied to the construction of other mesenchymal tissues for basic and applicative studies. PMID:24004835

  12. Soft matrix supports osteogenic differentiation of human dental follicle cells

    SciTech Connect

    Viale-Bouroncle, Sandra; Voellner, Florian; Moehl, Christoph; Kuepper, Kevin; Brockhoff, Gero; Reichert, Torsten E.; Schmalz, Gottfried; Morsczeck, Christian

    2011-07-08

    Highlights: {yields} Rigid stiffness supports osteogenic differentiation in mesenchymal stem cells (MSCs). {yields} Our study examined stiffness and differentiation of dental follicle cells (DFCs). {yields} Soft ECMs have a superior capacity to support the osteogenic differentiation of DFCs. {yields} DFCs and MSCs react contrarily to soft and rigid surface stiffness. -- Abstract: The differentiation of stem cells can be directed by the grade of stiffness of the developed tissue cells. For example a rigid extracellular matrix supports the osteogenic differentiation in bone marrow derived mesenchymal stem cells (MSCs). However, less is known about the relation of extracellular matrix stiffness and cell differentiation of ectomesenchymal dental precursor cells. Our study examined for the first time the influence of the surface stiffness on the proliferation and osteogenic differentiation of human dental follicle cells (DFCs). Cell proliferation of DFCs was only slightly decreased on cell culture surfaces with a bone-like stiffness. The osteogenic differentiation in DFCs could only be initiated with a dexamethasone based differentiation medium after using varying stiffness. Here, the softest surface improved the induction of osteogenic differentiation in comparison to that with the highest stiffness. In conclusion, different to bone marrow derived MSCs, soft ECMs have a superior capacity to support the osteogenic differentiation of DFCs.

  13. A smart fluorescence nanoprobe for the detection of cellular alkaline phosphatase activity and early osteogenic differentiation.

    PubMed

    Cao, Feng-Yi; Fan, Jin-Xuan; Long, Yue; Zeng, Xuan; Zhang, Xian-Zheng

    2016-07-01

    In the past decades, biomaterials were designed to induce stem cell toward osteogenic differentiation. However, conventional methods for evaluation osteogenic differentiation all required a process of cell fixation or lysis, which induce waste of a large number of cells. In this study, a fluorescence nanoprobe was synthesized by combining phosphorylated fluoresceinamine isomer I (FLA) on the surface of mesoporous silica-coated superparamagnetic iron oxide (Fe3O4@mSiO2) nanoparticles. In the presence of alkaline phosphatase (ALP), the phosphorylated FLA on the nanoprobe would be hydrolyzed, resulting in a fluorescence recovery of FLA. During early osteogenic differentiation, a high-level expression of cellular ALP was induced, which accelerated the hydrolysis of phosphorylated FLA, resulting in an enhancement of cellular fluorescence intensity. This fluorescence nanoprobe provides us a rapid and non-toxic method for the detection of cellular ALP activity and early osteogenic differentiation. PMID:26961462

  14. Calcium phosphate surfaces promote osteogenic differentiation of mesenchymal stem cells

    PubMed Central

    Müller, Petra; Bulnheim, Ulrike; Diener, Annette; Lüthen, Frank; Teller, Marianne; Klinkenberg, Ernst-Dieter; Neumann, Hans-Georg; Nebe, Barbara; Liebold, Andreas; Steinhoff, Gustav; Rychly, Joachim

    2008-01-01

    Abstract Although studies in vivo revealed promising results in bone regeneration after implantation of scaffolds together with osteogenic progenitor cells, basic questions remain how material surfaces control the biology of mesenchymal stem cells (MSC). We used human MSC derived from bone marrow and studied the osteogenic differentiation on calcium phosphate surfaces. In osteogenic differentiation medium MSC differentiated to osteoblasts on hydroxyapatite and BONITmatrix®, a degradable xerogel composite, within 14 days. Cells revealed a higher alkaline phosphatase (ALP) activity and increased RNA expression of collagen I and osteocalcin using real-time RTPCR compared with cells on tissue culture plastic. To test whether material surface characteristics alone are able to stimulate osteogenic differentiation, MSC were cultured on the materials in expansion medium without soluble additives for osteogenic differentiation. Indeed, cells on calcium phosphate without osteogenic differentiation additives developed to osteoblasts as shown by increased ALP activity and expression of osteogenic genes, which was not the case on tissue culture plastic. Because we reasoned that the stimulating effect on osteogenesis by calcium phosphate surfaces depends on an altered cell–extracellular matrix interaction we studied the dynamic behaviour of focal adhesions using cells transfected with GFP labelled vinculin. On BONITmatrix®, an increased mobility of focal adhesions was observed compared with cells on tissue culture plastic. In conclusion, calcium phosphate surfaces are able to drive MSC to osteoblasts in the absence of osteogenic differentiation supplements in the medium. An altered dynamic behaviour of focal adhesions on calcium phosphate surfaces might be involved in the molecular mechanisms which promote osteogenic differentiation. PMID:18366455

  15. Electrospun Gelatin/β-TCP Composite Nanofibers Enhance Osteogenic Differentiation of BMSCs and In Vivo Bone Formation by Activating Ca2+-Sensing Receptor Signaling

    PubMed Central

    Zhang, Xuehui; Meng, Song; Huang, Ying; Xu, Mingming; He, Ying; Lin, Hong; Han, Jianmin; Chai, Yuan; Wei, Yan

    2015-01-01

    Calcium phosphate- (CaP-) based composite scaffolds have been used extensively for the bone regeneration in bone tissue engineering. Previously, we developed a biomimetic composite nanofibrous membrane of gelatin/β-tricalcium phosphate (TCP) and confirmed their biological activity in vitro and bone regeneration in vivo. However, how these composite nanofibers promote the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is unknown. Here, gelatin/β-TCP composite nanofibers were fabricated by incorporating 20 wt% β-TCP nanoparticles into electrospun gelatin nanofibers. Electron microscopy showed that the composite β-TCP nanofibers had a nonwoven structure with a porous network and a rough surface. Spectral analyses confirmed the presence and chemical stability of the β-TCP and gelatin components. Compared with pure gelatin nanofibers, gelatin/β-TCP composite nanofibers caused increased cell attachment, proliferation, alkaline phosphatase activity, and osteogenic gene expression in rat BMSCs. Interestingly, the expression level of the calcium-sensing receptor (CaSR) was significantly higher on the composite nanofibrous scaffolds than on pure gelatin. For rat calvarial critical sized defects, more extensive osteogenesis and neovascularization occurred in the composite scaffolds group compared with the gelatin group. Thus, gelatin/β-TCP composite scaffolds promote osteogenic differentiation of BMSCs in vitro and bone regeneration in vivo by activating Ca2+-sensing receptor signaling. PMID:26124840

  16. MEF2 Transcription Factor Regulates Osteogenic Differentiation of Dental Pulp Stem Cells.

    PubMed

    Shen, Shuling; Huang, Dan; Feng, Guijuan; Zhu, Linhe; Zhang, Ye; Cao, Peipei; Zheng, Ke; Zhang, Dongmei; Feng, Xingmei

    2016-08-01

    The myocyte enhancer factor-2 (MEF2) is a member of the MADS-box family. It controls the expression of genes that are critical for biological processes such as proliferation, cell death, and differentiation. Some studies have shown that MEF2 expression is enhanced in osteogenic progenitor cells established from bone marrow stromal cells with other types of mesenchymal progenitor cells. However, the effect of MEF2 on dental pulp stem cells (DPSCs) is unclear. In this study, we investigate the effect of MEF2 on regulating osteogenic differentiation and proliferation of DPSCs. We find that MEF2 is stably expressed in DPSCs, and the expression is increased time-dependently along with cell osteogenic differentiation. MEF2 expression also increases the alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) activity, and enhances mineralization in DPSCs. SB202190, inhibitor of p38, blocks the p38/MEF2 pathway and osteogenic differentiation. In addition, MEF2 overexpression inhibits DPSC proliferation. In summary, our data indicate that MEF2 not only regulates DPSCs as an inhibitor of cell proliferation but is also a promoter of osteogenic differentiation through the p38/MEF2 signaling pathway. PMID:27459583

  17. The Control of Mesenchymal Stromal Cell Osteogenic Differentiation through Modified Surfaces

    PubMed Central

    2013-01-01

    Stem cells continue to receive widespread attention due to their potential to revolutionise treatments in the fields of both tissue engineering and regenerative medicine. Adult stem cells, specifically mesenchymal stromal cells (MSCs), play a vital role in the natural events surrounding bone healing and osseointegration through being stimulated to differentiate along their osteogenic lineage and in doing so, they form new cortical and trabecular bone tissue. Understanding how to control, manipulate, and enhance the intrinsic healing events modulated through osteogenic differentiation of MSCs by the use of modified surfaces and biomaterials could potentially advance the fields of both orthopaedics and dentistry. This could be by either using surface modification to generate greater implant stability and more rapid healing following implantation or the stimulation of MSCs ex vivo for reimplantation. This review aims to gather publications targeted at promoting, enhancing, and controlling the osteogenic differentiation of MSCs through biomaterials, nanotopographies, and modified surfaces for use in implant procedures. PMID:23766768

  18. microRNA-21 Mediates Stretch-Induced Osteogenic Differentiation in Human Periodontal Ligament Stem Cells

    PubMed Central

    Liu, Dongxu; Feng, Cheng; Zhang, Fan; Yang, Shuangyan; Hu, Yijun; Ding, Gang

    2015-01-01

    microRNAs (miRNAs) are short 20- to 22-nucleotide noncoding RNAs that negatively regulate the expression of target genes at the post-transcriptional level. The expression of specific miRNAs and their roles in the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) exposed to mechanical stretch remain unclear. Here, we found that stretch induced both osteogenic differentiation and the differential expression of miR-21 in PDLSCs. Furthermore, we identified activin receptor type IIB (ACVR2B) as a target gene of miR-21. Luciferase reporter assays showed that miR-21 interacts directly with the 3′-untranslated repeat sequence of ACVR2B mRNA. Mechanical stretch suppressed ACVR2B protein levels in PDLSCs, and this suppressive effect was modulated when endogenous miR-21 levels were either enhanced or inhibited. Both stretch and the expression of miR-21 altered endogenous ACVR2B protein levels and thus the osteogenic differentiation of PDLSCs. In addition, gain- and loss of function of ACVR2B mediated the osteogenic differentiation of PDLSCs. This study demonstrates that miR-21 is a mechanosensitive gene that plays an important role in the osteogenic differentiation of PDLSCs exposed to stretch. PMID:25203845

  19. Defect-Related Luminescent Hydroxyapatite-Enhanced Osteogenic Differentiation of Bone Mesenchymal Stem Cells Via an ATP-Induced cAMP/PKA Pathway.

    PubMed

    Wang, Chao; Liu, Dandan; Zhang, Cuimiao; Sun, Jiadong; Feng, Weipei; Liang, Xing-Jie; Wang, Shuxiang; Zhang, Jinchao

    2016-05-11

    Novel defect-related hydroxyapatite (DHAP), which combines the advantages of HAP and defect-related luminescence, has the potential application in tissue engineering and biomedical area, because of its excellent capability of monitoring the osteogenic differentiation and material biodegradation. Although the extracellular mechanism of DHAP minerals and PO4(3-) functioning in osteogenic differentiation has been widely studied, the intracellular molecular mechanism through which PO4(3-) mediates osteogenesis of bone mesenchymal stem cells (BMSCs) is not clear. We examined a previously unknown molecular mechanism through which PO4(3-) promoted osteogenesis of BMSCs with an emphasis on adenosine-triphosphate (ATP)-induced cAMP/PKA pathway. Our studies showed that DHAP could be uptaken into lysosome, in which PO4(3-) was released from DHAP, because of the acid environment of lysosome. The released PO4(3-) interacted with ADP to form ATP, and then degraded into adenosine, an ATP metabolite, which interacted with A2b adenosine receptor to activate the cAMP/PKA pathway, resulting in the high expression of osteogenesis-related genes, such as Runx2, BMP-2, and OCN. These findings first revealed the function of ATP-metabolism in bone physiological homeostasis, which may be developed to cure bone metabolic diseases. PMID:27088570

  20. Morphology-based prediction of osteogenic differentiation potential of human mesenchymal stem cells.

    PubMed

    Matsuoka, Fumiko; Takeuchi, Ichiro; Agata, Hideki; Kagami, Hideaki; Shiono, Hirofumi; Kiyota, Yasujiro; Honda, Hiroyuki; Kato, Ryuji

    2013-01-01

    Human bone marrow mesenchymal stem cells (hBMSCs) are widely used cell source for clinical bone regeneration. Achieving the greatest therapeutic effect is dependent on the osteogenic differentiation potential of the stem cells to be implanted. However, there are still no practical methods to characterize such potential non-invasively or previously. Monitoring cellular morphology is a practical and non-invasive approach for evaluating osteogenic potential. Unfortunately, such image-based approaches had been historically qualitative and requiring experienced interpretation. By combining the non-invasive attributes of microscopy with the latest technology allowing higher throughput and quantitative imaging metrics, we studied the applicability of morphometric features to quantitatively predict cellular osteogenic potential. We applied computational machine learning, combining cell morphology features with their corresponding biochemical osteogenic assay results, to develop prediction model of osteogenic differentiation. Using a dataset of 9,990 images automatically acquired by BioStation CT during osteogenic differentiation culture of hBMSCs, 666 morphometric features were extracted as parameters. Two commonly used osteogenic markers, alkaline phosphatase (ALP) activity and calcium deposition were measured experimentally, and used as the true biological differentiation status to validate the prediction accuracy. Using time-course morphological features throughout differentiation culture, the prediction results highly correlated with the experimentally defined differentiation marker values (R>0.89 for both marker predictions). The clinical applicability of our morphology-based prediction was further examined with two scenarios: one using only historical cell images and the other using both historical images together with the patient's own cell images to predict a new patient's cellular potential. The prediction accuracy was found to be greatly enhanced by incorporation

  1. 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. PMID:26724678

  2. 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

  3. Pulsed electromagnetic fields stimulate osteogenic differentiation in human bone marrow and adipose tissue derived mesenchymal stem cells.

    PubMed

    Ongaro, Alessia; Pellati, Agnese; Bagheri, Leila; Fortini, Cinzia; Setti, Stefania; De Mattei, Monica

    2014-09-01

    Pulsed electromagnetic fields (PEMFs) play a regulatory role on osteoblast activity and are clinically beneficial during fracture healing. Human mesenchymal stem cells (MSCs) derived from different sources have been extensively used in bone tissue engineering. Compared with MSCs isolated from bone marrow (BMSCs), those derived from adipose tissue (ASCs) are easier to obtain and available in larger amounts, although they show a less osteogenic differentiation potential than BMSCs. The hypothesis tested in this study was to evaluate whether PEMFs favor osteogenic differentiation both in BMSCs and in ASCs and to compare the role of PEMFs alone and in combination with the biochemical osteogenic stimulus bone morphogenetic protein (BMP)-2. Early and later osteogenic markers, such as alkaline phosphatase (ALP) activity, osteocalcin levels, and matrix mineralization, were analyzed at different times during osteogenic differentiation. Results showed that PEMFs induced osteogenic differentiation by increasing ALP activity, osteocalcin, and matrix mineralization in both BMSCs and ASCs, suggesting that PEMF activity is maintained during the whole differentiation period. The addition of BMP-2 in PEMF exposed cultures further increased all the osteogenic markers in BMSCs, while in ASCs, the stimulatory role of PEMFs was independent of BMP-2. Our results indicate that PEMFs may stimulate an early osteogenic induction in both BMSCs and ASCs and they suggest PEMFs as a bioactive factor to enhance the osteogenesis of ASCs, which are an attractive cell source for clinical applications. In conclusion, PEMFs may be considered a possible tool to improve autologous cell-based regeneration of bone defects in orthopedics. PMID:25099126

  4. Plasma Surface Modification of Polyhedral Oligomeric Silsequioxane-Poly(carbonate-urea) Urethane with Allylamine Enhances the Response and Osteogenic Differentiation of Adipose-Derived Stem Cells.

    PubMed

    Chaves, Camilo; Alshomer, Feras; Palgrave, Robert G; Kalaskar, Deepak M

    2016-07-27

    This study present amino functionalization of biocompatible polymer polyhedral oligomeric silsequioxane-poly(carbonate-urea) urethane (POSS-PCU) using plasma polymerization process to induce osteogenic differentiation of adipose derived stem cells (ADSCs). Optimization of plasma polymerization process was carried out keeping cell culture application in mind. Thus, samples were rigorously tested for retention of amino groups under both dry and wet conditions. Physio-chemical characterization was carried out using ninhydrin test, X-ray photon spectroscopy, scanning electron microscopy, and static water contact analysis. Results from physio chemical characterization shows that functionalization of the amino group is not stable under wet conditions and optimization of plasma process is required for stable bonding of amino groups to the POSS-PCU polymer. Optimized samples were later tested in vitro in short and long-term culture to study differentiation of ADSCs on amino modified samples. Short-term cell culture shows that initial cell attachment was significantly (p < 0.001) improved on amine modified samples (NH2-POSS-PCU) compared to unmodified POSS-PCU. NH2-POSS-PCU samples also facilitates osteogenic differentiation of ADSCs as confirmed by immunological staining of cells for extracellular markers such as collagen Type I and osteopontin. Quantification of total collagen and ALP activity also shows significant (p < 0.001) increase on NH2-POSS-PCU samples compared to unmodified POSS-PCU. A pilot study also confirms that these optimized amino modified POSS-PCU samples can further be functionalized using bone inducing peptide such as KRSR using conventional wet chemistry. This further provides an opportunity for biofunctionalization of the polymer for various tissue specific applications. PMID:27384590

  5. Enhancement of osteogenic differentiation and proliferation in human mesenchymal stem cells by a modified low intensity ultrasound stimulation under simulated microgravity.

    PubMed

    Uddin, Sardar M Z; Qin, Yi-Xian

    2013-01-01

    Adult stem cells can differentiate into multiple lineages depending on their exposure to differing biochemical and biomechanical inductive factors. Lack of mechanical signals due to disuse can inhibit osteogenesis and induce adipogenesis of mesenchymal stem cells (MSCs). Long-term bed rest due to both brain/spinal cord injury and space travel can lead to disuse osteoporosis that is in part caused by a reduced number of osteoblasts. Thus, it is essential to provide proper mechanical stimulation for cellular viability and osteogenesis, particularly under disuse conditions. The objective of this study was to examine the effects of low intensity pulsed ultrasound (LIPUS) on the osteogenic differentiation of adipose-derived human stem cells (Ad-hMSC) in simulated microgravity conditions. Cells were cultured in a 1D clinostat to simulate microgravity (SMG) and treated with LIPUS at 30mW/cm(2) for 20 min/day. It was hypothesized that the application of LIPUS to SMG cultures would restore osteogenesis in Ad-hMSCs. The results showed significant increases in ALP, OSX, RANKL, RUNX2, and decreases in OPG in LIPUS treated SMG cultures of Ad-MSC compared to non-treated cultures. LIPUS also restored OSX, RUNX2 and RANKL expression in osteoblast cells. SMG significantly reduced ALP positive cells by 70% (p<0.01) and ALP activity by 22% (p<0.01), while LIPUS treatment restored ALP positive cell number and activity to equivalence with normal gravity controls. Extracellular matrix collagen and mineralization was assessed by Sirius red and Alizarin red staining, respectively. SMG cultures showed little or no collagen or mineralization, but LIPUS treatment restored collagen content to 50% (p<0.001) and mineralization by 45% (p<0.001) in LIPUS treated-SMG cultures relative to SMG-only cultures. The data suggest that LIPUS treatment can restore normal osteogenic differentiation of MSCs from disuse by daily short duration stimulation. PMID:24069248

  6. Three-dimensional spherical spatial boundary conditions differentially regulate osteogenic differentiation of mesenchymal stromal cells

    PubMed Central

    Lo, Yin-Ping; Liu, Yi-Shiuan; Rimando, Marilyn G.; Ho, Jennifer Hui-Chun; Lin, Keng-hui; Lee, Oscar K.

    2016-01-01

    The spatial boundary condition (SBC) arising from the surrounding microenvironment imposes specific geometry and spatial constraints that affect organogenesis and tissue homeostasis. Mesenchymal stromal cells (MSCs) sensitively respond to alterations of mechanical cues generated from the SBC. However, mechanical cues provided by a three-dimensional (3D) environment are deprived in a reductionist 2D culture system. This study investigates how SBC affects osteogenic differentiation of MSCs using 3D scaffolds with monodispersed pores and homogenous spherical geometries. MSCs cultured under SBCs with diameters of 100 and 150 μm possessed the greatest capability of osteogenic differentiation. This phenomenon was strongly correlated with MSC morphology, organization of actin cytoskeleton, and distribution of focal adhesion involving α2 and α5 integrins. Further silencing either α2 or α5 integrin significantly reduced the above mentioned mechanosensitivity, indicating that the α2 and α5 integrins as mechano-sensitive molecules mediate MSCs’ ability to provide enhanced osteogenic differentiation in response to different spherical SBCs. Taken together, the findings provide new insights regarding how MSCs respond to mechanical cues from the surrounding microenvironment in a spherical SBC, and such biophysical stimuli should be taken into consideration in tissue engineering and regenerative medicine in conjunction with biochemical cues. PMID:26884253

  7. 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

  8. 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.

  9. Hyaluronan scaffold supports osteogenic differentiation of bone marrow concentrate cells.

    PubMed

    Cavallo, C; Desando, G; Ferrari, A; Zini, N; Mariani, E; Grigolo, B

    2016-01-01

    Osteochondral lesions are considered a challenge for orthopedic surgeons. Currently, the treatments available are often unsatisfactory and unable to stimulate tissue regeneration. Tissue engineering offers a new therapeutic strategy, taking into account the role exerted by cells, biomaterial and growth factors in restoring tissue damage. In this light, Mesenchymal Stem Cells (MSCs) have been indicated as a fascinating tool for regenerative medicine thanks to their ability to differentiate into bone, cartilage and adipose tissue. However, in vitro-cultivation of MSCs could be associated with some risks such as de-differentiation/reprogramming, infection and contaminations of the cells. To overcome these shortcomings, a new approach is represented by the use of Bone Marrow Concentrate (BMC), that could allow the delivery of cells surrounded by their microenvironment in injured tissue. For this purpose, cells require a tridimensional scaffold that can support their adhesion, proliferation and differentiation. This study is focused on the potentiality of BMC seeded onto a hyaluronan-based scaffold (Hyaff-11) to differentiate into osteogenic lineage. This process depends on the specific interaction between cells derived from bone marrow (surrounded by their niche) and scaffold, that create an environment able to support the regeneration of damaged tissue. The data obtained from the present study demonstrate that BMC grown onto Hyaff-11 are able to differentiate toward osteogenic sense, producing specific osteogenic genes and matrix proteins. PMID:27358127

  10. Autocrine fibroblast growth factor 18 mediates dexamethasone-induced osteogenic differentiation of murine mesenchymal stem cells.

    PubMed

    Hamidouche, Zahia; Fromigué, Olivia; Nuber, Ulrike; Vaudin, Pascal; Pages, Jean-Christophe; Ebert, Regina; Jakob, Franz; Miraoui, Hichem; Marie, Pierre J

    2010-08-01

    The potential of mesenchymal stem cells (MSC) to differentiate into functional bone forming cells provides an important tool for bone regeneration. The identification of factors capable of promoting osteoblast differentiation in MSCs is therefore critical to enhance the osteogenic potential of MSCs. Using microarray analysis combined with biochemical and molecular approach, we found that FGF18, a member of the FGF family, is upregulated during osteoblast differentiation induced by dexamethasone in murine MSCs. We showed that overexpression of FGF18 by lentiviral (LV) infection, or treatment of MSCs with recombinant human (rh)FGF18 increased the expression of the osteoblast specific transcription factor Runx2, and enhanced osteoblast phenotypic marker gene expression and in vitro osteogenesis. Molecular silencing using lentiviral shRNA demonstrated that downregulation of FGFR1 or FGFR2 abrogated osteoblast gene expression induced by either LV-FGF18 or rhFGF18, indicating that FGF18 enhances osteoblast differentiation in MSCs via activation of FGFR1 or FGFR2 signaling. Biochemical and pharmacological analyses showed that the induction of phenotypic osteoblast markers by LV-FGF18 is mediated by activation of ERK1/2-MAPKs and PI3K signaling in MSCs. These results reveal that FGF18 is an essential autocrine positive regulator of the osteogenic differentiation program in murine MSCs and indicate that osteogenic differentiation induced by FGF18 in MSCs is triggered by FGFR1/FGFR2-mediated ERK1/2-MAPKs and PI3K signaling. PMID:20432451

  11. Investigating the Role of FGF18 in the Cultivation and Osteogenic Differentiation of Mesenchymal Stem Cells

    PubMed Central

    Lee, Sujin; Koh, Young-Hyag; Kim, Hae-Won; Suh, Chang Kook; Jang, Jun-Hyeog

    2012-01-01

    Fibroblast growth factor18 (FGF18) belongs to the FGF family and is a pleiotropic protein that stimulates proliferation in several tissues. Bone marrow mesenchymal stem cells (BMSCs) participate in the normal replacement of damaged cells and in disease healing processes within bone and the haematopoietic system. In this study, we constructed FGF18 and investigated its effects on rat BMSCs (rBMSCs). The proliferative effects of FGF18 on rBMSCs were examined using an MTS assay. To validate the osteogenic differentiation effects of FGF18, ALP and mineralization activity were examined as well as osteogenic differentiation-related gene levels. FGF18 significantly enhanced rBMSCs proliferation (p<0.001) and induced the osteogenic differentiation by elevating ALP and mineralization activity of rBMSCs (p<0.001). Furthermore, these osteogenic differentiation effects of FGF18 were confirmed via increasing the mRNA levels of collagen type I (Col I), bone morphogenetic protein 4 (BMP4), and Runt-related transcription factor 2 (Runx2) at 3 and 7 days. These results suggest that FGF18 could be used to improve bone repair and regeneration. PMID:22937141

  12. Stereolithographic Bone Scaffold Design Parameters: Osteogenic Differentiation and Signal Expression

    PubMed Central

    Kim, Kyobum; Yeatts, Andrew; Dean, David

    2010-01-01

    Scaffold design parameters including porosity, pore size, interconnectivity, and mechanical properties have a significant influence on osteogenic signal expression and differentiation. This review evaluates the influence of each of these parameters and then discusses the ability of stereolithography (SLA) to be used to tailor scaffold design to optimize these parameters. Scaffold porosity and pore size affect osteogenic cell signaling and ultimately in vivo bone tissue growth. Alternatively, scaffold interconnectivity has a great influence on in vivo bone growth but little work has been done to determine if interconnectivity causes changes in signaling levels. Osteogenic cell signaling could be also influenced by scaffold mechanical properties such as scaffold rigidity and dynamic relationships between the cells and their extracellular matrix. With knowledge of the effects of these parameters on cellular functions, an optimal tissue engineering scaffold can be designed, but a proper technology must exist to produce this design to specification in a repeatable manner. SLA has been shown to be capable of fabricating scaffolds with controlled architecture and micrometer-level resolution. Surgical implantation of these scaffolds is a promising clinical treatment for successful bone regeneration. By applying knowledge of how scaffold parameters influence osteogenic cell signaling to scaffold manufacturing using SLA, tissue engineers may move closer to creating the optimal tissue engineering scaffold. PMID:20504065

  13. Bioreducible-Cationic Poly(amido amine)s for Enhanced Gene Delivery and Osteogenic Differentiation of Tonsil-Derived Mesenchymal Stem Cells.

    PubMed

    Jeong, Hansaem; Lee, Eun-Seo; Jung, Giyoung; Park, Jungha; Jeong, Byeongmoon; Ryu, Kyung Ha; Hwang, Nathaniel S; Lee, Hyukjin

    2016-05-01

    The development of efficient and safe gene delivery carriers has been a major challenge in the clinical application of non-viral gene therapy. Herein, we report novel bioreducible poly(amido amine)s for the efficient delivery of genetic material such as plasmid DNA. A library of 34 different bioreducible polymer compounds was synthesized and screened to find lead materials for in vitro gene transfection. Our lead material (CBA-106) allows effortless polyplex formation with genetic materials by electrostatic interactions at the weight ratio of 1:5 (DNA/polymer). Polyplexes were further characterized by DLS and AFM analysis. Enhanced serum stability and bioreducibility under physiological conditions were confirmed, in addition to low cellular cytotoxicity. When compared with a commercially available gene delivery carrier (Lipofectamine 2000), CBA-1 06 shows comparable or even surpassing gene transfection efficiency. Furthermore, BMP-2 plasmids were efficiently delivered to tonsil-derived mesenchymal stem cells (TMSCs) for osteogenic commitment in vitro and in vivo. Taken together, our results clearly demonstrate the potential of novel bioreducible polymeric systems for gene delivery applications. We suggest that our system can provide a valuable platform for the broad application of gene regulation in cell therapy and regenerative medicine. PMID:27305823

  14. Mechanical stimulation orchestrates the osteogenic differentiation of human bone marrow stromal cells by regulating HDAC1.

    PubMed

    Wang, J; Wang, C D; Zhang, N; Tong, W X; Zhang, Y F; Shan, S Z; Zhang, X L; Li, Q F

    2016-01-01

    Mechanical stimulation and histone deacetylases (HDACs) have essential roles in regulating the osteogenic differentiation of bone marrow stromal cells (BMSCs) and bone formation. However, little is known regarding what regulates HDAC expression and therefore the osteogenic differentiation of BMSCs during osteogenesis. In this study, we investigated whether mechanical loading regulates HDAC expression directly and examined the role of HDACs in mechanical loading-triggered osteogenic differentiation and bone formation. We first studied the microarrays of samples from patients with osteoporosis and found that the NOTCH pathway and skeletal development gene sets were downregulated in the BMSCs of patients with osteoporosis. Then we demonstrated that mechanical stimuli can regulate osteogenesis and bone formation both in vivo and in vitro. NOTCH signaling was upregulated during cyclic mechanical stretch (CMS)-induced osteogenic differentiation, whereas HDAC1 protein expression was downregulated. The perturbation of HDAC1 expression also had a significant effect on matrix mineralization and JAG1-mediated Notch signaling, suggesting that HDAC1 acts as an endogenous attenuator of Notch signaling in the mechanotransduction of BMSCs. Chromatin immunoprecipitation (ChIP) assay results suggest that HDAC1 modulates the CMS-induced histone H3 acetylation level at the JAG1 promoter. More importantly, we found an inhibitory role of Hdac1 in regulating bone formation in response to hindlimb unloading in mice, and pretreatment with an HDAC1 inhibitor partly rescued the osteoporosis caused by mechanical unloading. Our results demonstrate, for the first time, that mechanical stimulation orchestrates genes expression involved in the osteogenic differentiation of BMSCs via the direct regulation of HDAC1, and the therapeutic inhibition of HDAC1 may be an efficient strategy for enhancing bone formation under mechanical stimulation. PMID:27171263

  15. Mechanical stimulation orchestrates the osteogenic differentiation of human bone marrow stromal cells by regulating HDAC1

    PubMed Central

    Wang, J; Wang, C D; Zhang, N; Tong, W X; Zhang, Y F; Shan, S Z; Zhang, X L; Li, Q F

    2016-01-01

    Mechanical stimulation and histone deacetylases (HDACs) have essential roles in regulating the osteogenic differentiation of bone marrow stromal cells (BMSCs) and bone formation. However, little is known regarding what regulates HDAC expression and therefore the osteogenic differentiation of BMSCs during osteogenesis. In this study, we investigated whether mechanical loading regulates HDAC expression directly and examined the role of HDACs in mechanical loading-triggered osteogenic differentiation and bone formation. We first studied the microarrays of samples from patients with osteoporosis and found that the NOTCH pathway and skeletal development gene sets were downregulated in the BMSCs of patients with osteoporosis. Then we demonstrated that mechanical stimuli can regulate osteogenesis and bone formation both in vivo and in vitro. NOTCH signaling was upregulated during cyclic mechanical stretch (CMS)-induced osteogenic differentiation, whereas HDAC1 protein expression was downregulated. The perturbation of HDAC1 expression also had a significant effect on matrix mineralization and JAG1-mediated Notch signaling, suggesting that HDAC1 acts as an endogenous attenuator of Notch signaling in the mechanotransduction of BMSCs. Chromatin immunoprecipitation (ChIP) assay results suggest that HDAC1 modulates the CMS-induced histone H3 acetylation level at the JAG1 promoter. More importantly, we found an inhibitory role of Hdac1 in regulating bone formation in response to hindlimb unloading in mice, and pretreatment with an HDAC1 inhibitor partly rescued the osteoporosis caused by mechanical unloading. Our results demonstrate, for the first time, that mechanical stimulation orchestrates genes expression involved in the osteogenic differentiation of BMSCs via the direct regulation of HDAC1, and the therapeutic inhibition of HDAC1 may be an efficient strategy for enhancing bone formation under mechanical stimulation. PMID:27171263

  16. The Casitas B lineage lymphoma (Cbl) mutant G306E enhances osteogenic differentiation in human mesenchymal stromal cells in part by decreased Cbl-mediated platelet-derived growth factor receptor alpha and fibroblast growth factor receptor 2 ubiquitination.

    PubMed

    Sévère, Nicolas; Miraoui, Hichem; Marie, Pierre J

    2011-07-01

    Human bone marrow-derived mesenchymal stromal cells (hMSCs) have the capacity to differentiate into several cell types including osteoblasts and are therefore an important cell source for bone tissue regeneration. A crucial issue is to identify mechanisms that trigger hMSC osteoblast differentiation to promote osteogenic potential. Casitas B lineage lymphoma (Cbl) is an E3 ubiquitin ligase that ubiquitinates and targets several molecules for degradation. We hypothesized that attenuation of Cbl-mediated degradation of receptor tyrosine kinases (RTKs) may promote osteogenic differentiation in hMSCs. We show here that specific inhibition of Cbl interaction with RTKs using a Cbl mutant (G306E) promotes expression of osteoblast markers (Runx2, alkaline phosphatase, type 1 collagen, osteocalcin) and increases osteogenic differentiation in clonal bone marrow-derived hMSCs and primary hMSCs. Analysis of molecular mechanisms revealed that the Cbl mutant increased PDGF receptor α and FGF receptor 2 but not EGF receptor expression in hMSCs, resulting in increased ERK1/2 and PI3K signaling. Pharmacological inhibition of FGFR or PDGFR abrogated in vitro osteogenesis induced by the Cbl mutant. The data reveal that specific inhibition of Cbl interaction with RTKs promotes the osteogenic differentiation program in hMSCs in part by decreased Cbl-mediated PDGFRα and FGFR2 ubiquitination, providing a novel mechanistic approach targeting Cbl to promote the osteogenic capacity of hMSCs. PMID:21596750

  17. Epigenetic modifications in osteogenic differentiation and transformation.

    PubMed

    Thomas, David; Kansara, Maya

    2006-07-01

    Almost all tumors are characterized by both architectural and cellular abnormalities in differentiation. Osteoblast development is relatively well understood, making osteosarcoma a good model for understanding how tumorigenesis perturbs normal differentiation. We argue that there are two key transition points in normal cellular differentiation that are the focus of oncogenic events, in both of which epigenetic processes are critical. The first is the transition from an uncommitted pluripotent precursor (mesenchymal stem cell) to the 'transit-amplifying compartment' of the osteoblast lineage. This transition, normally exquisitely regulated in space and time, is abnormal in cancer. The second involves termination of lineage expansion, equally tightly regulated under normal circumstances. In cancer, the mechanisms that mandate eventual cessation of cell division are almost universally disrupted. This model predicts that key differentiation genes in bone, such as RUNX2, act in an oncogenic fashion to initiate entry into a proliferative phase of cell differentiation, and anti-oncogenically into the post-mitotic state, resulting in ambivalent roles in tumorigenesis. Polycomb genes exemplify epigenetic processes in the stem cell compartment and tumorigenesis, and are implicated in skeletal development in vivo. The epigenetic functions of the retinoblastoma protein, which plays a key role in tumorigenesis in bone, is discussed in the context of terminal cell cycle exit. PMID:16598744

  18. Osteogenic Differentiation of MSC through Calcium Signaling Activation: Transcriptomics and Functional Analysis

    PubMed Central

    Viti, Federica; Landini, Martina; Mezzelani, Alessandra; Petecchia, Loredana; Milanesi, Luciano; Scaglione, Silvia

    2016-01-01

    The culture of progenitor mesenchymal stem cells (MSC) onto osteoconductive materials to induce a proper osteogenic differentiation and mineralized matrix regeneration represents a promising and widely diffused experimental approach for tissue-engineering (TE) applications in orthopaedics. Among modern biomaterials, calcium phosphates represent the best bone substitutes, due to their chemical features emulating the mineral phase of bone tissue. Although many studies on stem cells differentiation mechanisms have been performed involving calcium-based scaffolds, results often focus on highlighting production of in vitro bone matrix markers and in vivo tissue ingrowth, while information related to the biomolecular mechanisms involved in the early cellular calcium-mediated differentiation is not well elucidated yet. Genetic programs for osteogenesis have been just partially deciphered, and the description of the different molecules and pathways operative in these differentiations is far from complete, as well as the activity of calcium in this process. The present work aims to shed light on the involvement of extracellular calcium in MSC differentiation: a better understanding of the early stage osteogenic differentiation program of MSC seeded on calcium-based biomaterials is required in order to develop optimal strategies to promote osteogenesis through the use of new generation osteoconductive scaffolds. A wide spectrum of analysis has been performed on time-dependent series: gene expression profiles are obtained from samples (MSC seeded on calcium-based scaffolds), together with related microRNAs expression and in vivo functional validation. On this basis, and relying on literature knowledge, hypotheses are made on the biomolecular players activated by the biomaterial calcium-phosphate component. Interestingly, a key role of miR-138 was highlighted, whose inhibition markedly increases osteogenic differentiation in vitro and enhance ectopic bone formation in vivo

  19. Ethanol alters the osteogenic differentiation of amniotic fluid-derived stem cells

    PubMed Central

    Hipp, Jennifer A; Hipp, Jason D; Atala, Anthony; Soker, Shay

    2010-01-01

    Background Fetal Alcohol Spectrum Disorder (FASD) is a set of developmental defects caused by prenatal alcohol exposure. Clinical manifestations of FASD are highly variable and include mental retardation and developmental defects of the heart, kidney, muscle, skeleton, and craniofacial structures. Specific effects of ethanol on fetal cells include induction of apoptosis as well as inhibition of proliferation, differentiation, and migration. This complex set of responses suggests that a bioinformatics approach could clarify some of the pathways involved in these responses. Methods In this study, the responses of fetal stem cells derived from the amniotic fluid (AFSCs) to treatment with ethanol have been examined. Large-scale transcriptome analysis of ethanol-treated AFSCs indicates that genes involved in skeletal development and ossification are up-regulated in these cells. Therefore, the effect of ethanol on osteogenic differentiation of AFSCs was studied. Results Exposure to ethanol during the first 48 hours of an osteogenic differentiation protocol increased in vitro calcium deposition by AFSCs and increased alkaline phosphatase activity. In contrast, ethanol treatment later in the differentiation protocol (day 8) had no significant effect on the activity of alkaline phosphatase. Conclusions These results suggest that transient exposure of AFSCs to ethanol during early differentiation enhances osteogenic differentiation of the cells. PMID:20608908

  20. MicroRNA functionalized microporous titanium oxide surface by lyophilization with enhanced osteogenic activity.

    PubMed

    Wu, Kaimin; Song, Wen; Zhao, Lingzhou; Liu, Mengyuan; Yan, Jun; Andersen, Morten Østergaard; Kjems, Jørgen; Gao, Shan; Zhang, Yumei

    2013-04-10

    Developing biomedical titanium (Ti) implants with high osteogenic ability and consequent rigid osseointegration is a constant requirement from the clinic. In this study, we fabricate novel miRNA functionalized microporous Ti implants by lyophilizing miRNA lipoplexes onto a microporous titanium oxide surface formed by microarc oxidation (MAO). The microporous titanium oxide surface provides a larger surface area for miRNA loading and enables spatial retention of the miRNAs within the pores until cellular delivery. The loading of lipoplexes into the micropores on the MAO Ti surface is facilitated by the superhydrophilicity and Ti-OH groups gathering of the MAO surface after UV irradiation followed by lyophilization. A high miRNA transfection efficiency was observed in mesenchymal stem cells (MSCs) seeded onto the miRNA functionalized surface with no apparent cytotoxicity. When functionalizing the Ti surface with miR-29b that enhances osteogenic activity and antimiR-138 that inhibits miR-138 inhibition of endogenous osteogenesis, clear stimulation of MSC osteogenic differentiation was observed, in terms of up-regulating osteogenic expression and enhancing alkaline phosphatase production, collagen secretion and ECM mineralization. The novel miRNA functionalized Ti implants with enhanced osteogenic activity promisingly lead to more rapid and robust osseointegration of a clinical bone implant interface. Our study implies that lyophilization may constitute a versatile method for miRNA loading to other biomaterials with the aim of controlling cellular function. PMID:23459382

  1. Dissection of the Osteogenic Effects of Laminin-332 Utilizing Specific LG Domains: LG3 Induces Osteogenic Differentiation, but not Mineralization

    PubMed Central

    Klees, Robert F.; Salasznyk, Roman M.; Ward, Donald F.; Crone, Donna E.; Williams, William A.; Harris, Mark P.; Boskey, Adele; Quaranta, Vito; Plopper, George E.

    2008-01-01

    The overall mechanisms governing the role of laminins during osteogenic differentiation of human mesenchymal stem cells (hMSC) are poorly understood. We previously reported that laminin-332 induces an osteogenic phenotype in hMSC and does so through a focal adhesion kinase (FAK) and extracellular signal-related kinase (ERK) dependent pathway. We hypothesized that this is a result of integrin-ECM binding, and that it occurs via the known α3 LG3 integrin binding domain of laminin-332. To test this hypothesis we cultured hMSC on several different globular domains of laminin-332. hMSC adhered best to the LG3 domain, and this adhesion maximally activated FAK and ERK within 120 minutes. Prolonged culturing (8 or 16 days) of hMSC on LG3 led to activation of the osteogenic transcription factor Runx2 and expression of key osteogenic markers (osterix, bone sialoprotein 2, osteocalcin, alkaline phosphatase, extracellular calcium) in hMSC. LG3 domain binding did not increase matrix mineralization, demonstrating that the LG3 domain alone is not sufficient to induce complete osteogenic differentiation in vitro. We conclude that the LG3 domain mediates attachment of hMSC to laminin-332 and that this adhesion recapitulates most, but not all, of the osteogenic differentiation associated with laminin-5 binding to hMSC. PMID:18206871

  2. Epigenetic Plasticity Drives Adipogenic and Osteogenic Differentiation of Marrow-derived Mesenchymal Stem Cells.

    PubMed

    Meyer, Mark B; Benkusky, Nancy A; Sen, Buer; Rubin, Janet; Pike, J Wesley

    2016-08-19

    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

  3. Effect of basic fibroblast growth factor in mouse embryonic stem cell culture and osteogenic differentiation.

    PubMed

    Rose, Laura C; Fitzsimmons, Ross; Lee, Poh; Krawetz, Roman; Rancourt, Derrick E; Uludağ, Hasan

    2013-05-01

    Embryonic stem cells are actively explored as a cell source in tissue engineering and regenerative medicine involving bone repair. Basic fibroblast growth factor (bFGF) has been a valuable growth factor to support the culture of human stem cells as well as their osteogenic differentiation, but the influence of bFGF on mouse embryonic stem (mES) cells is not known. Towards this goal, D3 cells were treated with bFGF during maintenance conditions and during spontaneous and osteogenic differentiation. In feeder-free monolayers, up to 40 ng/ml of exogenous bFGF did not support self-renewal of mES without LIF during cell expansion. During spontaneous differentiation in high-density cultures, bFGF stimulated cell proliferation under certain conditions but did not influence differentiation, as judged by stage-specific embryonic antigen-1 expression. The addition of bFGF reduced the alkaline phosphatase (ALP) activity associated with osteoblast activity during differentiation induced by osteogenic supplements, although the extent of mineralization was unaffected by bFGF. The bFGF increased the mesenchymal stem cell marker Sca-1 in an mES cell population and led to an enhanced increase in osteocalcin and runx2 expression in combination with BMP-2. These results suggest that bFGF could be utilized to expand the cell population in high-density cultures in addition to enriching the BMP-2 responsiveness of mES cells. PMID:22674886

  4. 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. PMID:21555842

  5. Mechanism of osteogenic and adipogenic differentiation of tendon stem cells induced by sirtuin 1.

    PubMed

    Liu, Junpeng; Han, Weifeng; Chen, Lei; Tang, Kanglai

    2016-08-01

    The aim of the present study was to assess the expression of sirtuin (Sirt)1 in tendon stem cells (TSCs) and to elucidate its association with osteogenic and adipogenic differentiation of TSCs. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analyses were performed to detect Sirt1 mRNA and protein levels in TSCs, respectively. TSCs were positive for Sirt1 expression, which was elevated by Sirt1 activator SRT1720 in a time- and concentration- dependent manner, and decreased by Sirt1 inhibitor EX527. TSCs were treated with SRT1720 and EX527 for various time periods and resulting changes in osteogenic and adipogenic protein markers were analyzed using alizarin red and oil red O staining. According to RT-qPCR and western blot analyses, the associated factors β‑catenin, Runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 2 were elevated following increases of Sirt1 levels, while CCAAT/enhancer binding protein (CEBP)α and peroxisome proliferator-activated receptor (PPAR)γ were decreased. These results suggested that osteogenic differentiation capacity was enhanced, while adipogenic differentiation capacity declined. Further mechanistic study revealed that phosphoinositide‑3 kinase (PI3K) and AKT were decreased following activation of Sirt1. In conclusion, the present study suggested that Sirt1 promotes the osteogenic differentiation of TSCs through upregulating β‑catenin and Runx2 and inhibits the adipogenic differentiation of TSCs through the PI3K/AKT pathway with downregulation of CEBPα and PPARγ. PMID:27357961

  6. Differentiation of skeletal osteogenic progenitor cells to osteoblasts with 3,4-diarylbenzopyran based amide derivatives: Novel osteogenic agents.

    PubMed

    Gupta, Atul; Ahmad, Imran; Kureel, Jyoti; John, Aijaz A; Sultan, Eram; Chanda, Debabrata; Agarwal, Naresh Kumar; Alauddin; Wahajuddin; Prabhaker, S; Verma, Amita; Singh, Divya

    2016-10-01

    A series of 3,4-diarylbenzopyran based amide derivatives was synthesized and evaluated for osteogenic activity in in vitro and in vivo models of osteoporosis. Compounds 17a, 21b-c and 22a-b showed significant osteogenic activity in osteoblast differentiation assay. Among the synthesized compounds, 22b was identified as lead molecule which showed significant osteogenic activity at 1 pM concentration in osteoblast differentiation assay and at 1 mg kg(-1) body weight dose in estrogen deficient balb/c mice model. In vitro bone mineralization and expression of osteogenic marker genes viz BMP-2, RUNX-2, OCN, and collagen type 1 further confirmed the osteogenic potential of 22b. Gene expression study for estrogen receptor α and β (ER-α and ER-β) in mouse calvarial osteoblasts (MCOs) unveiled that possibly 22b exerted osteogenic efficacy via activation of Estrogen receptor-β preferentially. In vivo pharmacokinetic, estrogenicity and acute toxicity studies of 22b showed that it had good bioavailability and was devoid of uterine estrogenicity at 1 mg kg(-1) and inherent toxicity up to 1000 mg kg(-1) body weight dose respectively. PMID:27236065

  7. Suppression of ornithine decarboxylase promotes osteogenic differentiation of human bone marrow-derived mesenchymal stem cells.

    PubMed

    Tsai, Yo-Hsian; Lin, Kuan-Lian; Huang, Yuan-Pin; Hsu, Yi-Chiang; Chen, Chung-Hwan; Chen, Yuhsin; Sie, Min-Hua; Wang, Gwo-Jaw; Lee, Mon-Juan

    2015-07-22

    Ornithine decarboxylase (ODC) is the rate-limiting enzyme for polyamine biosynthesis. Suppression of ODC by its irreversible inhibitor, α-difluoromethylornithine (DFMO), or by RNA interference through siRNA, enhanced osteogenic gene expression and alkaline phosphatase activity, and accelerated matrix mineralization of human bone marrow-derived mesenchymal stem cells (hBMSCs). Besides, adipogenic gene expression and lipid accumulation was attenuated, indicating that the enhanced osteogenesis was accompanied by down-regulation of adipogenesis when ODC was suppressed. A decrease in the intracellular polyamine content of hBMSCs during osteogenic induction was observed, suggesting that the level of endogenous polyamines is regulated during differentiation of hBMSCs. This study elucidates the role of polyamine metabolism in the lineage commitment of stem cells and provides a potential new indication for DFMO as bone-stimulating drug. PMID:26140984

  8. Simvastatin induces osteogenic differentiation of murine embryonic stem cells.

    PubMed

    Pagkalos, Joseph; Cha, Jae Min; Kang, Yunyi; Heliotis, Manolis; Tsiridis, Eleftherios; Mantalaris, Athanasios

    2010-11-01

    Statins are potent inhibitors of cholesterol synthesis. Several statins are available with different molecular and pharmacokinetic properties. Simvastatin is more lipophilic than pravastatin and has a higher affinity to phospholipid membranes than atorvastatin, allowing its passive diffusion through the cell membrane. In vitro studies on bone marrow stromal cells, osteoblast-like cells, and embryonic stem cells have shown statins to have cholesterol-independent anabolic effects on bone metabolism; alas, statins were supplemented in osteogenic medium, which does not facilitate elucidation of their potential osteoinductive properties. Embryonic stem cells (ESCs), derived from the inner cell mass of the blastocyst, are unique in that they enjoy perpetual self-proliferation, are pluripotent, and are able to differentiate toward all the cellular lineages composing the body, including the osteogenic lineage. Consequently, ESCs represent a potentially potent cell source for future clinical cellular therapies of various bone diseases, even though there are several hurdles that still need to be overcome. Herein we demonstrate, for the first time to our knowledge, that simvastatin induces murine ESC (mESC) differentiation toward the osteogenic lineage in the absence of osteoinductive supplements. Specifically, we found that a simvastatin concentration in the micromolar range and higher was toxic to the cells and that an effective concentration for osteoinduction is 0.1 nM, as shown by increased alizarin red staining as well as increased osteocalcin and osetrix gene expression. These results suggest that in the future, lipophilic simvastatin may provide a novel pharmacologic agent for bone tissue engineering applications. PMID:20564244

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

    PubMed

    Brauer, A; Pohlemann, T; Metzger, W

    2016-03-01

    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. PMID:26795823

  10. Osteogenic differentiation of preosteoblasts on a hemostatic gelatin sponge.

    PubMed

    Kuo, Zong-Keng; Lai, Po-Liang; Toh, Elsie Khai-Woon; Weng, Cheng-Hsi; Tseng, Hsiang-Wen; Chang, Pei-Zen; Chen, Chih-Chen; Cheng, Chao-Min

    2016-01-01

    Bone tissue engineering provides many advantages for repairing skeletal defects. Although many different kinds of biomaterials have been used for bone tissue engineering, safety issues must be considered when using them in a clinical setting. In this study, we examined the effects of using a common clinical item, a hemostatic gelatin sponge, as a scaffold for bone tissue engineering. The use of such a clinically acceptable item may hasten the translational lag from laboratory to clinical studies. We performed both degradation and biocompatibility studies on the hemostatic gelatin sponge, and cultured preosteoblasts within the sponge scaffold to demonstrate its osteogenic differentiation potential. In degradation assays, the gelatin sponge demonstrated good stability after being immersed in PBS for 8 weeks (losing only about 10% of its net weight and about 54% decrease of mechanical strength), but pepsin and collagenases readily biodegraded it. The gelatin sponge demonstrated good biocompatibility to preosteoblasts as demonstrated by MTT assay, confocal microscopy, and scanning electron microscopy. Furthermore, osteogenic differentiation and the migration of preosteoblasts, elevated alkaline phosphatase activity, and in vitro mineralization were observed within the scaffold structure. Each of these results indicates that the hemostatic gelatin sponge is a suitable scaffold for bone tissue engineering. PMID:27616161

  11. Lifetime fluorescence spectroscopy for in situ investigation of osteogenic differentiation

    NASA Astrophysics Data System (ADS)

    Marcu, Laura; Elbarbary, Amir; Zuk, Patricia; De Ugarte, Daniel A.; Benhaim, Prosper; Kurt, Hamza; Hedrick, Marc H.; Ashjian, Peter

    2003-07-01

    Time-Resolved Laser-Induced Fluorescence Spectroscopy (TR-LIFS) represents a potential tool for the in-situ characterization of bioengineered tissues. In this study, we evaluate the application of TR-LIFS to non-intrusive monitoring of matrix composition during osteogenetic differentiation. Human adipose-derived stem cells, harvested from 3 patients, were induced in osteogenic media for 3, 5, and 7 weeks. Samples were subsequently collected and probed for time-resolved fluorescence emission with a pulsed nitrogen laser. Fluorescence parameters, derived from both spectral- and time-domain, were used for sample characterization. The samples were further analyzed using Western blot analysis and computer-based densitometry. A significant change in the fluorescence parameters was detected for samples beyond 3 weeks of osteogenic differentiation. The spectroscopic observations: 1) show increase of collagen I when contrasted against the time-resolved fluorescence spectra of commercially available collagens; and 2) are in agreement with Western blot analysis that demonstrated significant increase in collagen I content between 3- vs. 5-weeks and 3- vs. 7-weeks and no changes for collagens III, IV, and V. Our results suggest that TR-LIFS can be used as a non-invasive means for the detection of specific collagens in maturing connective tissues.

  12. Osteogenic differentiation of human dental papilla mesenchymal cells

    SciTech Connect

    Ikeda, Etsuko; Hirose, Motohiro . E-mail: motohiro-hirose@aist.go.jp; Kotobuki, Noriko; Shimaoka, Hideki; Tadokoro, Mika; Maeda, Masahiko; Hayashi, Yoshiko; Kirita, Tadaaki; Ohgushi, Hajime

    2006-04-21

    We isolated dental papilla from impacted human molar and proliferated adherent fibroblastic cells after collagenase treatment of the papilla. The cells were negative for hematopoietic markers but positive for CD29, CD44, CD90, CD105, and CD166. When the cells were further cultured in the presence of {beta}-glycerophosphate, ascorbic acid, and dexamethasone for 14 days, mineralized areas together with osteogenic differentiation evidenced by high alkaline phosphatase activity and osteocalcin contents were observed. The differentiation was confirmed at both protein and gene expression levels. The cells can also be cryopreserved and, after thawing, could show in vivo bone-forming capability. These results indicate that mesenchymal type cells localize in dental papilla and that the cells can be culture expanded/utilized for bone tissue engineering.

  13. 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

  14. Single-pulsed electromagnetic field therapy increases osteogenic differentiation through Wnt signaling pathway and sclerostin downregulation.

    PubMed

    Lin, Chih-Chun; Lin, Ru-Wei; Chang, Chih-Wei; Wang, Gwo-Jaw; Lai, Kuo-An

    2015-10-01

    Pulsed electromagnetic field (PEMF) therapy has been used for more than three decades to treat bone diseases. The main complaint about using PEMF is that it is time-consuming. Previously, we showed single-pulsed electromagnetic field (SPEMF) applied for 3 min daily increased osteogenic differentiation of mesenchymal stem cells and accelerated bone growth in a long bone defect model. In the current study, we investigated the mechanism of SPEMF to increase osteogenic differentiation in osteoblastic cells. We found that both short-term (SS) and long-term (SL) SPEMF treatment increased mineralization, while alkaline phosphatase (ALP) activity increased during the first 5 days of SPEMF treatment. SS treatment increased gene expression of Wnt1, Wnt3a, Wnt10b, Fzd9, ALP, and Bmp2. Also, SPEMF inhibited sclerostin after 5 days of treatment, and that inhibition was more significant with SL treatment. SL SPEMF increased expression of parathyroid hormone-related protein (PTHrP) but decreased expression of Sost gene, which encodes sclerostin. Together, the early osteogenic effect of SPEMF utilizes the canonical Wnt signaling pathway while the inhibitory effect of long-term SPEMF on sclerostin may be attributable to PTHrP upregulation. This study enhances our understanding of cellular mechanisms to support the previous finding and may provide new insight for clinical applications. PMID:26364557

  15. Collagen nanofibres are a biomimetic substrate for the serum-free osteogenic differentiation of human adipose stem cells

    PubMed Central

    Sefcik, Lauren S.; Neal, Rebekah A.; Kaszuba, Stephanie N.; Parker, Anna M.; Katz, Adam J.; Ogle, Roy C.; Botchwey, Edward A.

    2011-01-01

    Electrospinning has recently gained widespread attention as a process capable of producing nanoscale fibres that mimic native extracellular matrix. In this study, we compared the osteogenic differentiation behaviour of human adipose stem cells (ASCs) on a 3D nanofibre matrix of type I rat tail collagen (RTC) and a 2D RTC collagen-coated substrate, using a novel serum-free osteogenic medium. The serum-free medium significantly enhanced the numbers of proliferating cells in culture, compared to ASCs in traditional basal medium containing 10% animal serum, highlighting a potential clinical role for in vitro stem cell expansion. Osteogenic differentiation behaviour was assessed at days 7, 14 and 21 using quantitative real-time RT–PCR analysis of the osteogenic genes collagen I (Coll I), alkaline phosphatase (ALP), osteopontin (OP), osteonectin (ON), osteocalcin (OC) and core-binding factor-α (cbfa1). All genes were upregulated (>one-fold) in ASCs cultured on nanofibre scaffolds over 2D collagen coatings by day 21. Synthesis of mineralized extracellular matrix on the scaffolds was assessed on day 21 with Alizarin red staining. These studies demonstrate that 3D nanoscale morphology plays a critical role in regulating cell fate processes and in vitro osteogenic differentiation of ASCs under serum-free conditions. PMID:18493910

  16. Inhibiting PPARγ by erythropoietin while upregulating TAZ by IGF1 synergistically promote osteogenic differentiation of mesenchymal stem cells.

    PubMed

    Zhou, Jianwei; Wei, Fangyuan; Ma, Yuquan

    2016-09-01

    Erythropoietin (EPO) is reported to promote osteogenesis and inhibit adipogenesis of mesenchymal stem cells (MSC) through inhibiting PPARγ, while insulin-like growth factor 1 (IGF1) is able to enhance osteogenesis via upregulating transcriptional coactivator with PDZ-binding motif (TAZ). The different targets of EPO and IGF1 suggested their potential synergism to enhance osteogenesis. In this study, we aimed to determine the potential synergism of EPO and IGF1 and its efficacy on MSC differentiation. Rat adipose-derived mesenchymal stem cells (ADSCs) were separately treated with EPO, IGF1 and EPO/IGF1. It was observed that the co-treatment using EPO and IGF1 was able to potently promote the osteogenic differentiation of rat ADSCs compared with EPO or IGF1 alone, which offered a promising effective option to strengthen bone tissue regeneration for bone defects. Further, we demonstrated that the enhanced osteogenic differentiation by EPO and IGF1 co-treatment was almost counteracted by activating PPARγ through PPARγ agonist, RSG, and blocking TAZ through TAZ silencing RNA, siTAZ. Thus, it could be concluded that EPO and IGF1 possessed a potent synergism in promoting osteogenic differentiation, and the synergism was mainly attributed to co-regulation of different osteogenic regulators PPARγ and TAZ, which were targeted genes of EPO and IGF1 respectively. PMID:27422606

  17. Microbioreactor Array Screening of Wnt Modulators and Microenvironmental Factors in Osteogenic Differentiation of Mesenchymal Progenitor Cells

    PubMed Central

    Padmanabhan, Harish; Cooper-White, Justin J.

    2013-01-01

    Cellular microenvironmental conditions coordinate to regulate stem cell populations and their differentiation. Mesenchymal precursor cells (MPCs), which have significant potential for a wide range of therapeutic applications, can be expanded or differentiated into osteo- chondro- and adipogenic lineages. The ability to establish, screen, and control aspects of the microenvironment is paramount if we are to elucidate the complex interplay of signaling events that direct cell fate. Whilst modulation of Wnt signaling may be useful to direct osteogenesis in MPCs, there is still significant controversy over how the Wnt signaling pathway influences osteogenesis. In this study, we utilised a full-factorial microbioreactor array (MBA) to rapidly, combinatorially screen several Wnt modulatory compounds (CHIR99021, IWP-4 and IWR-1) and characterise their effects upon osteogenesis. The MBA screening system showed excellent consistency between donors and experimental runs. CHIR99021 (a Wnt agonist) had a profoundly inhibitory effect upon osteogenesis, contrary to expectations, whilst the effects of the IWP-4 and IWR-1 (Wnt antagonists) were confirmed to be inhibitory to osteogenesis, but to a lesser extent than observed for CHIR99021. Importantly, we demonstrated that these results were translatable to standard culture conditions. Using RT-qPCR of osteogenic and Wnt pathway markers, we showed that CHIR exerted its effects via inhibition of ALP and SPP1 expression, even though other osteogenic markers (RUNX2, MSX2, DLX, COL1A1) were upregulated. Lastly, this MBA platform, due to the continuous provision of medium from the first to the last of ten serially connected culture chambers, permitted new insight into the impacts of paracrine signaling on osteogenic differentiation in MPCs, with factors secreted by the MPCs in upstream chambers enhancing the differentiation of cells in downstream chambers. Insights provided by this cell-based assay system will be key to better

  18. Surface thermal oxidation on titanium implants to enhance osteogenic activity and in vivo osseointegration.

    PubMed

    Wang, Guifang; Li, Jinhua; Lv, Kaige; Zhang, Wenjie; Ding, Xun; Yang, Guangzheng; Liu, Xuanyong; Jiang, Xinquan

    2016-01-01

    Thermal oxidation, which serves as a low-cost, effective and relatively simple/facile method, was used to modify a micro-structured titanium surface in ambient atmosphere at 450 °C for different time periods to improve in vitro and in vivo bioactivity. The surface morphology, crystallinity of the surface layers, chemical composition and chemical states were evaluated by field-emission scanning electron microscopy (FESEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). Cell behaviours including cell adhesion, attachment, proliferation, and osteogenic differentiation were observed in vitro study. The ability of the titanium surface to promote osseointegration was evaluated in an in vivo animal model. Surface thermal oxidation on titanium implants maintained the microstructure and, thus, both slightly changed the nanoscale structure of titanium and enhanced the crystallinity of the titanium surface layer. Cells cultured on the three oxidized titanium surfaces grew well and exhibited better osteogenic activity than did the control samples. The in vivo bone-implant contact also showed enhanced osseointegration after several hours of oxidization. This heat-treated titanium enhanced the osteogenic differentiation activity of rBMMSCs and improved osseointegration in vivo, suggesting that surface thermal oxidation could potentially be used in clinical applications to improve bone-implant integration. PMID:27546196

  19. Surface thermal oxidation on titanium implants to enhance osteogenic activity and in vivo osseointegration

    PubMed Central

    Wang, Guifang; Li, Jinhua; Lv, Kaige; Zhang, Wenjie; Ding, Xun; Yang, Guangzheng; Liu, Xuanyong; Jiang, Xinquan

    2016-01-01

    Thermal oxidation, which serves as a low-cost, effective and relatively simple/facile method, was used to modify a micro-structured titanium surface in ambient atmosphere at 450 °C for different time periods to improve in vitro and in vivo bioactivity. The surface morphology, crystallinity of the surface layers, chemical composition and chemical states were evaluated by field-emission scanning electron microscopy (FESEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). Cell behaviours including cell adhesion, attachment, proliferation, and osteogenic differentiation were observed in vitro study. The ability of the titanium surface to promote osseointegration was evaluated in an in vivo animal model. Surface thermal oxidation on titanium implants maintained the microstructure and, thus, both slightly changed the nanoscale structure of titanium and enhanced the crystallinity of the titanium surface layer. Cells cultured on the three oxidized titanium surfaces grew well and exhibited better osteogenic activity than did the control samples. The in vivo bone-implant contact also showed enhanced osseointegration after several hours of oxidization. This heat-treated titanium enhanced the osteogenic differentiation activity of rBMMSCs and improved osseointegration in vivo, suggesting that surface thermal oxidation could potentially be used in clinical applications to improve bone-implant integration. PMID:27546196

  20. Voriconazole Enhances the Osteogenic Activity of Human Osteoblasts In Vitro through a Fluoride-Independent Mechanism

    PubMed Central

    Allen, Kahtonna C.; Sanchez, Carlos J.; Niece, Krista L.; Wenke, Joseph C.

    2015-01-01

    Periostitis, which is characterized by bony pain and diffuse periosteal ossification, has been increasingly reported with prolonged clinical use of voriconazole. While resolution of clinical symptoms following discontinuation of therapy suggests a causative role for voriconazole, the biological mechanisms contributing to voriconazole-induced periostitis are unknown. To elucidate potential mechanisms, we exposed human osteoblasts in vitro to voriconazole or fluconazole at 15 or 200 μg/ml (reflecting systemic or local administration, respectively), under nonosteogenic or osteogenic conditions, for 1, 3, or 7 days and evaluated the effects on cell proliferation (reflected by total cellular DNA) and osteogenic differentiation (reflected by alkaline phosphatase activity, calcium accumulation, and expression of genes involved in osteogenic differentiation). Release of free fluoride, vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) was also measured in cell supernatants of osteoblasts exposed to triazoles, with an ion-selective electrode (for free fluoride) and enzyme-linked immunosorbent assays (ELISAs) (for VEGF and PDGF). Voriconazole but not fluconazole significantly enhanced the proliferation and differentiation of osteoblasts. In contrast to clinical observations, no increases in free fluoride levels were detected following exposure to either voriconazole or fluconazole; however, significant increases in the expression of VEGF and PDGF by osteoblasts were observed following exposure to voriconazole. Our results demonstrate that voriconazole can induce osteoblast proliferation and enhance osteogenic activity in vitro. Importantly, and in contrast to the previously proposed mechanism of fluoride-stimulated osteogenesis, our findings suggest that voriconazole-induced periostitis may also occur through fluoride-independent mechanisms that enhance the expression of cytokines that can augment osteoblastic activity. PMID:26324277

  1. Voriconazole Enhances the Osteogenic Activity of Human Osteoblasts In Vitro through a Fluoride-Independent Mechanism.

    PubMed

    Allen, Kahtonna C; Sanchez, Carlos J; Niece, Krista L; Wenke, Joseph C; Akers, Kevin S

    2015-12-01

    Periostitis, which is characterized by bony pain and diffuse periosteal ossification, has been increasingly reported with prolonged clinical use of voriconazole. While resolution of clinical symptoms following discontinuation of therapy suggests a causative role for voriconazole, the biological mechanisms contributing to voriconazole-induced periostitis are unknown. To elucidate potential mechanisms, we exposed human osteoblasts in vitro to voriconazole or fluconazole at 15 or 200 μg/ml (reflecting systemic or local administration, respectively), under nonosteogenic or osteogenic conditions, for 1, 3, or 7 days and evaluated the effects on cell proliferation (reflected by total cellular DNA) and osteogenic differentiation (reflected by alkaline phosphatase activity, calcium accumulation, and expression of genes involved in osteogenic differentiation). Release of free fluoride, vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) was also measured in cell supernatants of osteoblasts exposed to triazoles, with an ion-selective electrode (for free fluoride) and enzyme-linked immunosorbent assays (ELISAs) (for VEGF and PDGF). Voriconazole but not fluconazole significantly enhanced the proliferation and differentiation of osteoblasts. In contrast to clinical observations, no increases in free fluoride levels were detected following exposure to either voriconazole or fluconazole; however, significant increases in the expression of VEGF and PDGF by osteoblasts were observed following exposure to voriconazole. Our results demonstrate that voriconazole can induce osteoblast proliferation and enhance osteogenic activity in vitro. Importantly, and in contrast to the previously proposed mechanism of fluoride-stimulated osteogenesis, our findings suggest that voriconazole-induced periostitis may also occur through fluoride-independent mechanisms that enhance the expression of cytokines that can augment osteoblastic activity. PMID:26324277

  2. Tissue transglutaminase is involved in mechanical load-induced osteogenic differentiation of human ligamentum flavum cells.

    PubMed

    Chao, Yuan-Hung; Huang, Shih-Yung; Yang, Ruei-Cheng; Sun, Jui-Sheng

    2016-07-01

    Mechanical load-induced osteogenic differentiation might be the key cellular event in the calcification and ossification of ligamentum flavum. The aim of this study was to investigate the influence of tissue transglutaminase (TGM2) on mechanical load-induced osteogenesis of ligamentum flavum cells. Human ligamentum flavum cells were obtained from 12 patients undergoing lumbar spine surgery. Osteogenic phenotypes of ligamentum flavum cells, such as alkaline phosphatase (ALP), Alizarin red-S stain, and gene expression of osteogenic makers were evaluated following the administration of mechanical load and BMP-2 treatment. The expression of TGM2 was evaluated by real-time PCR, Western blotting, and enzyme-linked immunosorbent assay (ELISA) analysis. Our results showed that mechanical load in combination with BMP-2 enhanced calcium deposition and ALP activity. Mechanical load significantly increased ALP and OC gene expression on day 3, whereas BMP-2 significantly increased ALP, OPN, and Runx2 on day 7. Mechanical load significantly induced TGM2 gene expression and enzyme activity in human ligamentum flavum cells. Exogenous TGM2 increased ALP and OC gene expression; while, inhibited TG activity significantly attenuated mechanical load-induced and TGM2-induced ALP activity. In summary, mechanical load-induced TGM2 expression and enzyme activity is involved in the progression of the calcification of ligamentum flavum. PMID:27115725

  3. Adhesion, Vitality and Osteogenic Differentiation Capacity of Adipose Derived Stem Cells Seeded on Nitinol Nanoparticle Coatings

    PubMed Central

    Strauß, Sarah; Neumeister, Anne; Barcikowski, Stephan; Kracht, Dietmar; Kuhbier, Jörn W.; Radtke, Christine; Reimers, Kerstin; Vogt, Peter M.

    2013-01-01

    Autologous cells can be used for a bioactivation of osteoimplants to enhance osseointegration. In this regard, adipose derived stem cells (ASCs) offer interesting perspectives in implantology because they are fast and easy to isolate. However, not all materials licensed for bone implants are equally suited for cell adhesion. Surface modifications are under investigation to promote cytocompatibility and cell growth. The presented study focused on influences of a Nitinol-nanoparticle coating on ASCs. Possible toxic effects as well as influences on the osteogenic differentiation potential of ASCs were evaluated by viability assays, scanning electron microscopy, immunofluorescence and alizarin red staining. It was previously shown that Nitinol-nanoparticles exert no cell toxic effects to ASCs either in soluble form or as surface coating. Here we could demonstrate that a Nitinol-nanoparticle surface coating enhances cell adherence and growth on Nitinol-surfaces. No negative influence on the osteogenic differentiation was observed. Nitinol-nanoparticle coatings offer new possibilities in implantology research regarding bioactivation by autologous ASCs, respectively enhancement of surface attraction to cells. PMID:23308190

  4. Adenosine Signaling Mediates Osteogenic Differentiation of Human Embryonic Stem Cells on Mineralized Matrices

    PubMed Central

    Rao, Vikram; Shih, Yu-Ru V.; Kang, Heemin; Kabra, Harsha; Varghese, Shyni

    2015-01-01

    Human embryonic stem cells (hESCs) are attractive cell sources for tissue engineering and regenerative medicine due to their self-renewal and differentiation ability. Design of biomaterials with an intrinsic ability that promotes hESC differentiation to the targeted cell type boasts significant advantages for tissue regeneration. We have previously developed biomineralized calcium phosphate (CaP) matrices that inherently direct osteogenic differentiation of hESCs without the need of osteogenic-inducing chemicals or growth factors. Here, we show that CaP matrix-driven osteogenic differentiation of hESCs occurs through A2b adenosine receptor (A2bR). The inhibition of the receptor with an A2bR-specific antagonist attenuated mineralized matrix-mediated osteogenic differentiation of hESCs. In addition, when cultured on matrices in an environment deficient of CaP minerals, exogenous adenosine promoted osteogenic differentiation of hESCs, but was attenuated by the inhibition of A2bR. Such synthetic matrices that intrinsically support osteogenic commitment of hESCs are not only beneficial for bone tissue engineering but can also be used as a platform to study the effect of the physical and chemical cues to the extracellular milieu on stem cell commitment. Insights into the cell signaling during matrix-induced differentiation of stem cells will also help define the key processes and enable discovery of new targets that promote differentiation of pluripotent stem cells for bone tissue engineering. PMID:26618155

  5. Preparation and characterization of composite nanofibers of polycaprolactone and nanohydroxyapatite for osteogenic differentiation of mesenchymal stem cells.

    PubMed

    Chen, Jyh-Ping; Chang, Yin-Shin

    2011-08-01

    Nanocomposites of nanohydroxyapatite (nHAP) dispersed in poly(ɛ-caprolactone) (PCL) were prepared by electrospinning (ES) to obtain PCL/nHAP nanofibers. Nanofibers with similar diameters (340 ± 30 nm) but different nHAP concentrations (0-50%) were fabricated and studied for growth and osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs). The nanofibrous membranes were subjected to detailed analysis for its physicochemical properties by scanning electron microscopy (SEM), thermogravimetric analysis, X-ray diffraction, Fourier-transform infrared spectroscopy, and mechanical tensile testing. nHAP particles (~30 nm diameter) embedded in nanofibers increased the nanofibrous membrane's ultimate stress and the elastic modulus, while decreased the strain at failure. When cultured under an osteogenic stimulation condition on nanofibers, MSCs showed normal phenotypic cell morphology, and time-dependent mineralization and osteogenic differentiation from SEM observations and alkaline phosphatase activity assays. The nanofibers could support the growth of mesenchymal stem cells without compromising their osteogenic differentiation capability up to 21 days and the enhancement of cell differentiation by nHAP is positively correlated with its concentration in the nanofibers. Energy dispersive X-ray analysis of Ca and P elements indicated mineral deposits on the cell surface. The mineralization extent was significantly raised in nanofibers with 50% nHAP where a Ca/P ratio similar to that of bone was found. The present study indicated that electrospun composite PCL/nHAP nanofibrous membranes are suitable for mineralization of MSCs intended for bone tissue engineering. PMID:21514800

  6. 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. PMID:25894066

  7. Enhanced osteogenic activity of poly ether ether ketone using calcium plasma immersion ion implantation.

    PubMed

    Lu, Tao; Qian, Shi; Meng, Fanhao; Ning, Congqin; Liu, Xuanyong

    2016-06-01

    As a promising implantable material, poly ether ether ketone (PEEK) possesses similar elastic modulus to that of cortical bones yet suffers from bio-inertness and poor osteogenic properties, which limits its application as orthopedic implants. In this work, calcium is introduced onto PEEK surface using calcium plasma immersion ion implantation (Ca-PIII). The results obtained from scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) confirm the modified layer with varying contents of calcium are formed on PEEK surfaces. Water contact angle measurements reveal the increasing hydrophobicity of both Ca-PIII treated surfaces. In vitro cell adhesion, viability assay, alkaline phosphatase activity and collagen secretion analyses disclose improved the adhesion, proliferation, and osteo-differentiation of rat bone mesenchymal stem cells (bMSCs) on Ca-PIII treated surfaces. The obtained results indicate that PEEK surface with enhanced osteogenic activity can be produced by calcium incorporation. PMID:26954085

  8. Controlling Growth and Osteogenic Differentiation of Osteoblasts on Microgrooved Polystyrene Surfaces

    PubMed Central

    Sun, Lanying; Pereira, Daniel; Wang, Qibao; Barata, David Baião; Truckenmüller, Roman; Li, Zhaoyuan; Xu, Xin; Habibovic, Pamela

    2016-01-01

    Surface topography is increasingly being recognized as an important factor to control the response of cells and tissues to biomaterials. In the current study, the aim was to obtain deeper understanding of the effect of microgrooves on shape and orientation of osteoblast-like cells and to relate this effect to their proliferation and osteogenic differentiation. To this end, two microgrooved polystyrene (PS) substrates, differing in the width of the grooves (about 2 μm and 4 μm) and distance between individual grooves (about 6 μm and 11 μm, respectively) were fabricated using a combination of photolithography and hot embossing. MG-63 human osteosarcoma cells were cultured on these microgrooved surfaces, with unpatterned hot-embossed PS substrate as a control. Scanning electron- and fluorescence microscopy analyses showed that on patterned surfaces, the cells aligned along the microgrooves. The cells cultured on 4 μm-grooves / 11 μm-ridges surface showed a more pronounced alignment and a somewhat smaller cell area and cell perimeter as compared to cells cultured on surface with 2 μm-grooves / 6 μm-ridges or unpatterned PS. PrestoBlue analysis and quantification of DNA amounts suggested that microgrooves used in this experiment did not have a strong effect on cell metabolic activity or proliferation. However, cell differentiation towards the osteogenic lineage was significantly enhanced when MG-63 cells were cultured on the 2/6 substrate, as compared to the 4/11 substrate or unpatterned PS. This effect on osteogenic differentiation may be related to differences in cell spreading between the substrates. PMID:27571520

  9. Controlling Growth and Osteogenic Differentiation of Osteoblasts on Microgrooved Polystyrene Surfaces.

    PubMed

    Sun, Lanying; Pereira, Daniel; Wang, Qibao; Barata, David Baião; Truckenmüller, Roman; Li, Zhaoyuan; Xu, Xin; Habibovic, Pamela

    2016-01-01

    Surface topography is increasingly being recognized as an important factor to control the response of cells and tissues to biomaterials. In the current study, the aim was to obtain deeper understanding of the effect of microgrooves on shape and orientation of osteoblast-like cells and to relate this effect to their proliferation and osteogenic differentiation. To this end, two microgrooved polystyrene (PS) substrates, differing in the width of the grooves (about 2 μm and 4 μm) and distance between individual grooves (about 6 μm and 11 μm, respectively) were fabricated using a combination of photolithography and hot embossing. MG-63 human osteosarcoma cells were cultured on these microgrooved surfaces, with unpatterned hot-embossed PS substrate as a control. Scanning electron- and fluorescence microscopy analyses showed that on patterned surfaces, the cells aligned along the microgrooves. The cells cultured on 4 μm-grooves / 11 μm-ridges surface showed a more pronounced alignment and a somewhat smaller cell area and cell perimeter as compared to cells cultured on surface with 2 μm-grooves / 6 μm-ridges or unpatterned PS. PrestoBlue analysis and quantification of DNA amounts suggested that microgrooves used in this experiment did not have a strong effect on cell metabolic activity or proliferation. However, cell differentiation towards the osteogenic lineage was significantly enhanced when MG-63 cells were cultured on the 2/6 substrate, as compared to the 4/11 substrate or unpatterned PS. This effect on osteogenic differentiation may be related to differences in cell spreading between the substrates. PMID:27571520

  10. Controlled Osteogenic Differentiation of Mouse Mesenchymal Stem Cells by Tetracycline-Controlled Transcriptional Activation of Amelogenin

    PubMed Central

    Wang, Fangfang; Okawa, Hiroko; Kamano, Yuya; Niibe, Kunimichi; Kayashima, Hiroki; Osathanon, Thanaphum; Pavasant, Prasit; Saeki, Makio; Yatani, Hirofumi; Egusa, Hiroshi

    2015-01-01

    Regenerative dental therapies for bone tissues rely on efficient targeting of endogenous and transplanted mesenchymal stem cells (MSCs) to guide bone formation. Amelogenin is the primary component of Emdogain, which is used to regenerate periodontal defects; however, the mechanisms underlying the therapeutic effects on alveolar bone remain unclear. The tetracycline (Tet)-dependent transcriptional regulatory system is a good candidate to investigate distinct roles of genes of interest during stem cell differentiation. Here, we investigated amelogenin-dependent regulation of osteogenesis in MSCs by establishing a Tet-controlled transcriptional activation system. Clonal mouse bone marrow-derived MSCs were lentivirally transduced with the Tet repressor (TetR) expression vector followed by drug selection to obtain MSCs constitutively expressing TetR (MSCs-TetR). Expression vectors that contained the Tet operator and amelogenin-coding (Amelx) cDNA fragments were constructed using the Gateway system and lentivirally introduced into MSCs-TetR to generate a Tet regulation system in MSCs (MSCs-TetR/Amelx). MSCs-TetR/Amelx significantly overexpressed the Amelx gene and protein in the presence of the tetracycline derivative doxycycline. Concomitant expression of osterix, bone sialoprotein (BSP), osteopontin, and osteocalcin was modulated by addition or removal of doxycycline under osteogenic guidance. During osteogenic induction, MSCs-TetR/Amelx treated with doxycycline showed significantly increased gene expression of osterix, type I collagen, BSP, and osteocalcin in addition to increased alkaline phosphatase activity and mineralized nodule formation. Enhanced extracellular matrix calcification was observed when forced Amelx expression commenced at the early stage but not at the intermediate or late stages of osteogenesis. These results suggest that a Tet-controlled Amelx gene regulation system for mouse MSCs was successfully established, in which transcriptional activation

  11. Controlled Osteogenic Differentiation of Mouse Mesenchymal Stem Cells by Tetracycline-Controlled Transcriptional Activation of Amelogenin.

    PubMed

    Wang, Fangfang; Okawa, Hiroko; Kamano, Yuya; Niibe, Kunimichi; Kayashima, Hiroki; Osathanon, Thanaphum; Pavasant, Prasit; Saeki, Makio; Yatani, Hirofumi; Egusa, Hiroshi

    2015-01-01

    Regenerative dental therapies for bone tissues rely on efficient targeting of endogenous and transplanted mesenchymal stem cells (MSCs) to guide bone formation. Amelogenin is the primary component of Emdogain, which is used to regenerate periodontal defects; however, the mechanisms underlying the therapeutic effects on alveolar bone remain unclear. The tetracycline (Tet)-dependent transcriptional regulatory system is a good candidate to investigate distinct roles of genes of interest during stem cell differentiation. Here, we investigated amelogenin-dependent regulation of osteogenesis in MSCs by establishing a Tet-controlled transcriptional activation system. Clonal mouse bone marrow-derived MSCs were lentivirally transduced with the Tet repressor (TetR) expression vector followed by drug selection to obtain MSCs constitutively expressing TetR (MSCs-TetR). Expression vectors that contained the Tet operator and amelogenin-coding (Amelx) cDNA fragments were constructed using the Gateway system and lentivirally introduced into MSCs-TetR to generate a Tet regulation system in MSCs (MSCs-TetR/Amelx). MSCs-TetR/Amelx significantly overexpressed the Amelx gene and protein in the presence of the tetracycline derivative doxycycline. Concomitant expression of osterix, bone sialoprotein (BSP), osteopontin, and osteocalcin was modulated by addition or removal of doxycycline under osteogenic guidance. During osteogenic induction, MSCs-TetR/Amelx treated with doxycycline showed significantly increased gene expression of osterix, type I collagen, BSP, and osteocalcin in addition to increased alkaline phosphatase activity and mineralized nodule formation. Enhanced extracellular matrix calcification was observed when forced Amelx expression commenced at the early stage but not at the intermediate or late stages of osteogenesis. These results suggest that a Tet-controlled Amelx gene regulation system for mouse MSCs was successfully established, in which transcriptional activation

  12. miR-124 negatively regulates osteogenic differentiation and in vivo bone formation of mesenchymal stem cells.

    PubMed

    Qadir, Abdul S; Um, Soyoun; Lee, Heesu; Baek, Kyunghwa; Seo, Byoung Moo; Lee, Gene; Kim, Gwan-Shik; Woo, Kyung Mi; Ryoo, Hyun-Mo; Baek, Jeong-Hwa

    2015-05-01

    MicroRNAs are novel key regulators of cellular differentiation. Dlx transcription factors play an important role in osteoblast differentiation, and Dlx5 and Dlx2 are known targets of miR-124. Therefore, in the present study, we investigated the regulatory effects of miR-124 on the osteogenic differentiation and in vivo bone formation of mesenchymal stem cells (MSCs). During osteogenic induction by BMP2, the expression levels of miR-124 were inversely correlated with those of osteogenic differentiation marker genes in human and mouse bone marrow-derived MSCs, MC3T3-E1 cells and C2C12 cells. The overexpression of a miR-124 mimic significantly decreased the expression levels of Dlx5, Dlx3, and Dlx2, whereas the silencing of miR-124 with hairpin inhibitors significantly increased the expression of these Dlx genes. Luciferase reporter assays demonstrated that miR-124 directly targets the 3'UTRs of Dlx3, Dlx5, and Dlx2. The overexpression of a miR-124 mimic suppressed the osteogenic marker gene expression levels, alkaline phosphatase activity and matrix mineralization, which were all significantly increased by the overexpression of a miR-124 inhibitor. When ectopic bone formation was induced by the subcutaneous transplantation of human bone marrow-derived MSCs in nude mice, MSCs overexpressing a miR-124 inhibitor significantly enhanced woven bone formation compared with control MSCs. However, MSCs overexpressing a miR-124 mimic exhibited increased adipocyte differentiation at the expense of ectopic bone formation. These results suggest that miR-124 is a negative regulator of osteogenic differentiation and in vivo bone formation and that the targeting of Dlx5, Dlx3, and Dlx2 genes partly contributes to this inhibitory effect exerted by miR-124. PMID:25424317

  13. Evaluating the effects of charged oligopeptide motifs coupled with RGD on osteogenic differentiation of mesenchymal stem cells.

    PubMed

    Cao, Feng-Yi; Yin, Wei-Na; Fan, Jin-Xuan; Tao, Li; Qin, Si-Yong; Zhuo, Ren-Xi; Zhang, Xian-Zheng

    2015-04-01

    Mesenchymal stem cells, due to their multilineage differentiation potential, have emerged as a promising cell candidate for cell-based therapy. In recent years, biomaterials were artificially synthesized to control the differentiation of mesenchymal stem cells. In this study, a series of charged or neutral oligopeptide motifs coupled with RGD were synthesized and used for surface modification using quartz substrates as model. Cell behaviors on the modified surfaces with different charged oligopeptide motifs were studied. It was found that these different charged oligopeptide motifs coupled with RGD were biocompatible for cell proliferation and adhesion. Moreover, it was demonstrated that the positively charged oligopeptide motif could inhibit osteogenic differentiation, while the negatively charged and neutral oligopeptide motifs could enhance osteogenic differentiation in the presence of RGD. This work may bring us enlightenment that different charged oligopeptide motifs coupled with RGD may be used for biomaterial surface modification for different stem cell-based therapies. PMID:25748883

  14. Biochanin A Promotes Osteogenic but Inhibits Adipogenic Differentiation: Evidence with Primary Adipose-Derived Stem Cells

    PubMed Central

    Su, Shu-Jem; Su, Shu-Hui; Shyu, Huey-Wen; Chen, Kuan-Ming; Yeh, Hua

    2013-01-01

    Biochanin A has promising effects on bone formation in vivo, although the underlying mechanism remains unclear yet. This study therefore aimed to investigate whether biochanin A regulates osteogenic and adipogenic differentiation using primary adipose-derived stem cells. The effects of biochanin A (at a physiologically relevant concentration of 0.1–1 μM) were assessed in vitro using various approaches, including Oil red O staining, Nile red staining, alizarin red S staining, alkaline phosphatase (ALP) activity, flow cytometry, RT-PCR, and western blotting. The results showed that biochanin A significantly suppressed adipocyte differentiation, as demonstrated by the inhibition of cytoplasmic lipid droplet accumulation, along with the inhibition of peroxisome proliferator-activated receptor gamma (PPARγ), lipoprotein lipase (LPL), and leptin and osteopontin (OPN) mRNA expression, in a dose-dependent manner. On the other hand, treatment of cells with 0.3 μM biochanin A increased the mineralization and ALP activity, and stimulated the expression of the osteogenic marker genes ALP and osteocalcin (OCN). Furthermore, biochanin A induced the expression of runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG), and Ras homolog gene family, member A (RhoA) proteins. These observations suggest that biochanin A prevents adipogenesis, enhances osteoblast differentiation in mesenchymal stem cells, and has beneficial regulatory effects in bone formation. PMID:23843885

  15. Enhanced Osteogenicity of Bioactive Composites with Biomimetic Treatment

    PubMed Central

    Meretoja, Ville V.; Tirri, Teemu; Seppälä, Jukka V.; Närhi, Timo O.

    2014-01-01

    Purpose. This study aimed to explore if initiation of biomimetic apatite nucleation can be used to enhance osteoblast response to biodegradable tissue regeneration composite membranes. Materials and Methods. Bioactive thermoplastic composites consisting of poly(ε-caprolactone/DL-lactide) and bioactive glass (BAG) were prepared at different stages of biomimetic calcium phosphate deposition by immersion in simulated body fluid (SBF). The modulation of the BAG dissolution and the osteogenic response of rat mesenchymal stem cells (MSCs) were analyzed. Results. SBF treatment resulted in a gradual calcium phosphate deposition on the composites and decreased BAG reactivity in the subsequent cell cultures. Untreated composites and composites covered by thick calcium phosphate layer (14 days in SBF) expedited MSC mineralization in comparison to neat polymers without BAG, whereas other osteogenic markers—alkaline phosphatase activity, bone sialoprotein, and osteocalcin expression—were initially decreased. In contrast, surfaces with only small calcium phosphate aggregates (five days in SBF) had similar early response than neat polymers but still demonstrated enhanced mineralization. Conclusion. A short biomimetic treatment enhances osteoblast response to bioactive composite membranes. PMID:24812608

  16. 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

  17. Iron overload inhibits osteogenic commitment and differentiation of mesenchymal stem cells via the induction of ferritin.

    PubMed

    Balogh, Enikő; Tolnai, Emese; Nagy, Béla; Nagy, Béla; Balla, György; Balla, József; Jeney, Viktória

    2016-09-01

    Osteogenic differentiation of multipotent mesenchymal stem cells (MSCs) plays a crucial role in bone remodeling. Numerous studies have described the deleterious effect of iron overload on bone density and microarchitecture. Excess iron decreases osteoblast activity, leading to impaired extracellular matrix (ECM) mineralization. Additionally, iron overload facilitates osteoclast differentiation and bone resorption. These processes contribute to iron overload-associated bone loss. In this study we investigated the effect of iron on osteogenic differentiation of human bone marrow MSCs (BMSCs), the third player in bone remodeling. We induced osteogenic differentiation of BMSCs in the presence or absence of iron (0-50μmol/L) and examined ECM mineralization, Ca content of the ECM, mRNA and protein expressions of the osteogenic transcription factor runt-related transcription factor 2 (Runx2), and its targets osteocalcin (OCN) and alkaline phosphatase (ALP). Iron dose-dependently attenuated ECM mineralization and decreased the expressions of Runx2 and OCN. Iron accomplished complete inhibition of osteogenic differentiation of BMSCs at 50μmol/L concentration. We demonstrated that in response to iron BMSCs upregulated the expression of ferritin. Administration of exogenous ferritin mimicked the anti-osteogenic effect of iron, and blocked the upregulation of Runx2, OCN and ALP. Iron overload in mice was associated with elevated ferritin and decreased Runx2 mRNA levels in compact bone osteoprogenitor cells. The inhibitory effect of iron is specific toward osteogenic differentiation of MSCs as neither chondrogenesis nor adipogenesis were influenced by excess iron. We concluded that iron and ferritin specifically inhibit osteogenic commitment and differentiation of BMSCs both in vitro and in vivo. PMID:27287253

  18. Low-Intensity Pulsed Ultrasound Stimulation Facilitates Osteogenic Differentiation of Human Periodontal Ligament Cells

    PubMed Central

    Hu, Bo; Zhang, Yuanyuan; Zhou, Jie; Li, Jing; Deng, Feng; Wang, Zhibiao; Song, Jinlin

    2014-01-01

    Human periodontal ligament cells (hPDLCs) possess stem cell properties, which play a key role in periodontal regeneration. Physical stimulation at appropriate intensities such as low-intensity pulsed ultrasound (LIPUS) enhances cell proliferation and osteogenic differentiation of mesechymal stem cells. However, the impacts of LIPUS on osteogenic differentiation of hPDLCs in vitro and its molecular mechanism are unknown. This study was undertaken to investigate the effects of LIPUS on osteogenic differentiation of hPDLCs. HPDLCs were isolated from premolars of adolescents for orthodontic reasons, and exposed to LIPUS at different intensities to determine an optimal LIPUS treatment dosage. Dynamic changes of alkaline phosphatase (ALP) activities in the cultured cells and supernatants, and osteocalcin production in the supernatants after treatment were analyzed. Runx2 and integrin β1 mRNA levels were assessed by reverse transcription polymerase chain reaction analysis after LIPUS stimulation. Blocking antibody against integrinβ1 was used to assess the effects of integrinβ1 inhibitor on LIPUS-induced ALP activity, osteocalcin production as well as calcium deposition. Our data showed that LIPUS at the intensity of 90 mW/cm2 with 20 min/day was more effective. The ALP activities in lysates and supernatants of LIPUS-treated cells started to increase at days 3 and 7, respectively, and peaked at day 11. LIPUS treatment significantly augmented the production of osteocalcin after day 5. LIPUS caused a significant increase in the mRNA expression of Runx2 and integrin β1, while a significant decline when the integrinβ1 inhibitor was used. Moreover, ALP activity, osteocalcin production as well as calcium nodules of cells treated with both daily LIPUS stimulation and integrinβ1 antibody were less than those in the LIPUS-treated group. In conclusion, LIPUS promotes osteogenic differentiation of hPDLCs, which is associated with upregulation of Runx2 and integrin β1, which

  19. Dose effect of tumor necrosis factor-alpha on in vitro osteogenic differentiation of mesenchymal stem cells on biodegradable polymeric microfiber scaffolds.

    PubMed

    Mountziaris, Paschalia M; Tzouanas, Stephanie N; Mikos, Antonios G

    2010-03-01

    This study presents a first step in the development of a bone tissue engineering strategy to trigger enhanced osteogenesis by modulating inflammation. This work focused on characterizing the effects of the concentration of a pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-alpha), on osteogenic differentiation of mesenchymal stem cells (MSCs) grown in a 3D culture system. MSC osteogenic differentiation is typically achieved in vitro through a combination of osteogenic supplements that include the anti-inflammatory corticosteroid dexamethasone. Although simple, the use of dexamethasone is not clinically realistic, and also hampers in vitro studies of the role of inflammatory mediators in wound healing. In this study, MSCs were pre-treated with dexamethasone to induce osteogenic differentiation, and then cultured in biodegradable electrospun poly(epsilon-caprolactone) (PCL) scaffolds, which supported continued MSC osteogenic differentiation in the absence of dexamethasone. Continuous delivery of 0.1 ng/mL of recombinant rat TNF-alpha suppressed osteogenic differentiation of rat MSCs over 16 days, which was likely the result of residual dexamethasone antagonizing TNF-alpha signaling. Continuous delivery of a higher dose, 5 ng/mL TNF-alpha, stimulated osteogenic differentiation for a few days, and 50 ng/mL TNF-alpha resulted in significant mineralized matrix deposition over the course of the study. These findings suggest that the pro-inflammatory cytokine TNF-alpha stimulates osteogenic differentiation of MSCs, an effect that can be blocked by the presence of anti-inflammatory agents like dexamethasone, with significant implications on the interplay between inflammation and tissue regeneration. PMID:19963268

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

    PubMed

    Qu, Bo; Ma, Yuan; Yan, Ming; Gong, Kai; Liang, Feng; Deng, Shaolin; Jiang, Kai; Ma, Zehui; Pan, Xianming

    2016-09-01

    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(+))-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 downregulation 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

  1. 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

  2. A combinatorial cell-laden gel microarray for inducing osteogenic differentiation of human mesenchymal stem cells

    PubMed Central

    Dolatshahi-Pirouz, Alireza; Nikkhah, Mehdi; Gaharwar, Akhilesh K.; Hashmi, Basma; Guermani, Enrico; Aliabadi, Hamed; Camci-Unal, Gulden; Ferrante, Thomas; Foss, Morten; Ingber, Donald E.; Khademhosseini, Ali

    2014-01-01

    Development of three dimensional (3D) microenvironments that direct stem cell differentiation into functional cell types remains a major challenge in the field of regenerative medicine. Here, we describe a new platform to address this challenge by utilizing a robotic microarray spotter for testing stem cell fates inside various miniaturized cell-laden gels in a systematic manner. To demonstrate the feasibility of our platform, we evaluated the osteogenic differentiation of human mesenchymal stem cells (hMSCs) within combinatorial 3D niches. We were able to identify specific combinations, that enhanced the expression of osteogenic markers. Notably, these ‘hit' combinations directed hMSCs to form mineralized tissue when conditions were translated to 3D macroscale hydrogels, indicating that the miniaturization of the experimental system did not alter stem cell fate. Overall, our findings confirmed that the 3D cell-laden gel microarray can be used for screening of different conditions in a rapid, cost-effective, and multiplexed manner for a broad range of tissue engineering applications. PMID:24473466

  3. 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

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

    PubMed

    Huszar, Jessica M; Payne, Christopher J

    2014-05-01

    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

  5. Osteogenic differentiation of GFP-labeled human umbilical cord blood derived mesenchymal stem cells after cryopreservation.

    PubMed

    Liu, Guangpeng; Ye, Xinhai; Zhu, Yuchang; Li, Yulin; Sun, Jian; Cui, Lei; Cao, Yilin

    2011-10-01

    The osteogenic capacity of human umbilical cord blood derived mesenchymal stem cells (UCB-MSCs) has been demonstrated both in vitro and in vivo. Therefore, cell labeling and storage are becoming necessary for researching the potential therapeutic use of UCB-MSCs for bone tissue engineering. The aim of this study was to determine the effect of cryopreservation on the osteogenic differentiation of green fluorescent protein (GFP)-marked UCB-MSCs in vitro. MSCs were isolated from full-term human UCB, expanded, transfected with the GFP gene, and then cryopreserved in liquid nitrogen for 4 weeks. After thawing, cell surface antigen markers and osteogenic potential were analyzed, and the luminescence of these cells was observed by fluorescence microscopy. The results demonstrate that cryopreservation has no effect on the cell phenotype, GFP expression or osteogenic differentiation of UCB-MSCs, showing that cryopreserved GFP-labeled UCB-MSCs might be applied for bone tissue engineering. PMID:21684270

  6. Small Buccal Fat Pad Cells Have High Osteogenic Differentiation Potential.

    PubMed

    Tsurumachi, Niina; Akita, Daisuke; Kano, Koichiro; Matsumoto, Taro; Toriumi, Taku; Kazama, Tomohiko; Oki, Yoshinao; Tamura, Yoko; Tonogi, Morio; Isokawa, Keitaro; Shimizu, Noriyoshi; Honda, Masaki

    2016-03-01

    Dedifferentiated fat (DFAT) cells derived from mature adipocytes have mesenchymal stem cells' (MSCs) characteristics. Generally, mature adipocytes are 60-110 μm in diameter; however, association between adipocyte size and dedifferentiation efficiency is still unknown. This study, therefore, investigated the dedifferentiation efficiency of adipocytes based on cell diameter. Buccal fat pad was harvested from five human donors and dissociated by collagenase digestion. After exclusion of unwanted stromal cells by centrifugation, floating adipocytes were collected and their size distribution was analyzed. The floating adipocytes were then separated into two groups depending on cell size using 40- and 100-μm nylon mesh filters: cell diameters less than 40 μm (small adipocytes: S-adipocytes) and cell diameters of 40-100 μm (large adipocytes: L-adipocytes). Finally, we evaluated the efficiency of adipocyte dedifferentiation and then characterized the resultant DFAT cells. The S-adipocytes showed a higher capacity to dedifferentiate into DFAT cells (S-DFAT cells) compared to the L-adipocytes (L-DFAT cells). The S-DFAT cells also showed a relatively higher proportion of CD146-positive cells than L-DFAT cells, and exhibited more osteogenic differentiation ability based on the alkaline phosphatase activity and amount of calcium deposition. These results suggested that the S- and L-DFAT cells had distinct characteristics, and that the higher dedifferentiation potential of S-adipocytes compared to L-adipocytes gives the former group an advantage in yielding DFAT cells. PMID:26651216

  7. Gene expression of markers of osteogenic differentiation of human mesenchymal cells on collagen I-modified microrough titanium surfaces.

    PubMed

    Morra, M; Cassinelli, C; Cascardo, G; Bollati, D; Baena, R Rodriguez Y

    2011-02-01

    Microrough, doubly acid etched titanium surfaces (Ti) were further modified by amination and covalent coupling of fibrillar collagen type I (ColTi). Human Mesenchymal Cells (HMC) adhesion and growth, and relevant osteogenic differentiation in nonosteogenic (basal) medium were evaluated by fluorescence microscopy, scanning electron microscopy, and RT-PCR for a three-week period. Results show strongly enhanced HMC adhesion and cell density at short experimental time on ColTi, together with complete spreading of the cell body over the microrough surface topography. RT-PCR analysis of several genes involved in osteogenesis indicate, since the first week of culturing, significant progression of HMC on ColTi along the osteogenic pathway. These results indicate that the adopted process of surface immobilization of collagen, mandatory to impart collagenase resistance in implant sites, does not impair biospecific interactions between HMC and collagen. Thus, it is possible to upgrade properties arising from the control of Ti surfaces topography by surface-chemistry driven enhanced recruitment of precursor osteogenic cells and pro-osteogenic stimula. PMID:21171164

  8. 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. PMID:26765285

  9. An EWS-FLI1-Induced Osteosarcoma Model Unveiled a Crucial Role of Impaired Osteogenic Differentiation on Osteosarcoma Development

    PubMed Central

    Komura, Shingo; Semi, Katsunori; Itakura, Fumiaki; Shibata, Hirofumi; Ohno, Takatoshi; Hotta, Akitsu; Woltjen, Knut; Yamamoto, Takuya; Akiyama, Haruhiko; Yamada, Yasuhiro

    2016-01-01

    Summary EWS-FLI1, a multi-functional fusion oncogene, is exclusively detected in Ewing sarcomas. However, previous studies reported that rare varieties of osteosarcomas also harbor EWS-ETS family fusion. Here, using the doxycycline-inducible EWS-FLI1 system, we established an EWS-FLI1-dependent osteosarcoma model from murine bone marrow stromal cells. We revealed that the withdrawal of EWS-FLI1 expression enhances the osteogenic differentiation of sarcoma cells, leading to mature bone formation. Taking advantage of induced pluripotent stem cell (iPSC) technology, we also show that sarcoma-derived iPSCs with cancer-related genetic abnormalities exhibited an impaired differentiation program of osteogenic lineage irrespective of the EWS-FLI1 expression. Finally, we demonstrate that EWS-FLI1 contributed to secondary sarcoma development from the sarcoma iPSCs after osteogenic differentiation. These findings demonstrate that modulating cellular differentiation is a fundamental principle of EWS-FLI1-induced osteosarcoma development. This in vitro cancer model using sarcoma iPSCs should provide a unique platform for dissecting relationships between the cancer genome and cellular differentiation. PMID:26997645

  10. An EWS-FLI1-Induced Osteosarcoma Model Unveiled a Crucial Role of Impaired Osteogenic Differentiation on Osteosarcoma Development.

    PubMed

    Komura, Shingo; Semi, Katsunori; Itakura, Fumiaki; Shibata, Hirofumi; Ohno, Takatoshi; Hotta, Akitsu; Woltjen, Knut; Yamamoto, Takuya; Akiyama, Haruhiko; Yamada, Yasuhiro

    2016-04-12

    EWS-FLI1, a multi-functional fusion oncogene, is exclusively detected in Ewing sarcomas. However, previous studies reported that rare varieties of osteosarcomas also harbor EWS-ETS family fusion. Here, using the doxycycline-inducible EWS-FLI1 system, we established an EWS-FLI1-dependent osteosarcoma model from murine bone marrow stromal cells. We revealed that the withdrawal of EWS-FLI1 expression enhances the osteogenic differentiation of sarcoma cells, leading to mature bone formation. Taking advantage of induced pluripotent stem cell (iPSC) technology, we also show that sarcoma-derived iPSCs with cancer-related genetic abnormalities exhibited an impaired differentiation program of osteogenic lineage irrespective of the EWS-FLI1 expression. Finally, we demonstrate that EWS-FLI1 contributed to secondary sarcoma development from the sarcoma iPSCs after osteogenic differentiation. These findings demonstrate that modulating cellular differentiation is a fundamental principle of EWS-FLI1-induced osteosarcoma development. This in vitro cancer model using sarcoma iPSCs should provide a unique platform for dissecting relationships between the cancer genome and cellular differentiation. PMID:26997645

  11. Osteogenic differentiation of human lens epithelial cells might contribute to lens calcification.

    PubMed

    Balogh, Enikő; Tóth, Andrea; Tolnai, Emese; Bodó, Tímea; Bányai, Emese; Szabó, Dóra Júlia; Petrovski, Goran; Jeney, Viktória

    2016-09-01

    Calcification of the human lens has been described in senile cataracts and in young patients with congenital cataract or chronic uveitis. Lens calcification is also a major complication of cataract surgery and plays a role in the opacification of intraocular lenses. A cell-mediated process has been suggested in the background of lens calcification, but so far the exact mechanism remained unexplored. Lens calcification shares remarkable similarities with vascular calcification; in both pathological processes hydroxyapatite accumulates in the soft tissue. Vascular calcification is a regulated, cell-mediated process in which vascular cells undergo osteogenic differentiation. Our objective was to investigate whether human lens epithelial cells (HuLECs) can undergo osteogenic transition in vitro, and whether this process contributes to lens calcification. We used inorganic phosphate (Pi) and Ca to stimulate osteogenic differentiation of HuLECs. Osteogenic stimuli (2.5mmol/L Pi and 1.2mmol/L Ca) induced extracellular matrix mineralization and Ca deposition in HuLECs with the critical involvement of active Pi uptake. Osteogenic stimuli almost doubled mRNA expressions of osteo-/chondrogenic transcription factors Runx2 and Sox9, which was accompanied by a 1.9-fold increase in Runx2 and a 5.5-fold increase in Sox9 protein expressions. Osteogenic stimuli induced mRNA and protein expressions of alkaline phosphatase and osteocalcin in HuLEC. Ca content was higher in human cataractous lenses, compared to non-cataractous controls (n=10). Osteocalcin, an osteoblast-specific protein, was expressed in 2 out of 10 cataractous lenses. We conclude that osteogenic stimuli induce osteogenic differentiation of HuLECs and propose that this mechanism might play a role in lens calcification. PMID:27318027

  12. 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

  13. 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.

  14. 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

    2016-01-01

    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 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. PMID:27225821

  15. Transcriptome changes during TNF-α promoted osteogenic differentiation of dental pulp stem cells (DPSCs).

    PubMed

    Liu, Ya-Ke; Zhou, Zhen-Yu; Liu, Fan

    2016-08-01

    Dental pulp stem cells (DPSCs), due to the ease of isolation and their capacities of multi-lineage differentiation, are considered as attractive resources for regenerative medicine. In a previous study, we showed that TNF-α promoted the osteogenic differentiation of DPSCs via the NF-κB signaling pathway. However, the mechanisms of such differentiation were largely unknown. Here, we examined the gene expression profiles between undifferentiated, partially differentiated and fully differentiated DPSCs induced by TNF-α by performing the next-generation sequencing technique (RNA-Seq). Our results revealed a continuous transition of the transcriptome changes during TNF-α promoted osteogenic differentiation of DPSC. Bioinformatics analysis revealed a relatively general to specific transformation of the involved signaling pathways from the early to late stages of differentiation. Gene regulatory network analysis highlighted novel, key genes that are essential for osteogenic differentiation at different time points. These results were further validated by quantitative RT-PCR, confirming the high reliability of the RNA-Seq. Our data therefore will not only provide novel insights into the molecular mechanisms that drive the osteogenic differentiation of DPSCs, but also promote the studies of bone tissue engineering that utilizes DPSCs as a crucial resource. PMID:27237976

  16. Crosstalk between Wnt/β-Catenin and Estrogen Receptor Signaling Synergistically Promotes Osteogenic Differentiation of Mesenchymal Progenitor Cells

    PubMed Central

    Gao, Yanhong; Huang, Enyi; Zhang, Hongmei; Wang, Jinhua; Wu, Ningning; Chen, Xian; Wang, Ning; Wen, Sheng; Nan, Guoxin; Deng, Fang; Liao, Zhan; Wu, Di; Zhang, Bosi; Zhang, Junhui; Haydon, Rex C.; Luu, Hue H.; Shi, Lewis L.; He, Tong-Chuan

    2013-01-01

    Osteogenic differentiation from mesenchymal progenitor cells (MPCs) are initiated and regulated by a cascade of signaling events. Either Wnt/β-catenin or estrogen signaling pathway has been shown to play an important role in regulating skeletal development and maintaining adult tissue homeostasis. Here, we investigate the potential crosstalk and synergy of these two signaling pathways in regulating osteogenic differentiation of MPCs. We find that the activation of estrogen receptor (ER) signaling by estradiol (E2) or exogenously expressed ERα in MPCs synergistically enhances Wnt3A-induced early and late osteogenic markers, as well as matrix mineralization. The E2 or ERα-mediated synergy can be effectively blocked by ERα antagonist tamoxifen. E2 stimulation can enhance endochondral ossification of Wnt3A-transduced mouse fetal limb explants. Furthermore, exogenously expressed ERα significantly enhances the maturity and mineralization of Wnt3A-induced subcutaneous and intramuscular ectopic bone formation. Mechanistically, we demonstrate that E2 does not exert any detectable effect on β-catenin/Tcf reporter activity. However, ERα expression is up-regulated within the first 48h in AdWnt3A-transduced MPCs, whereas ERβ expression is significantly inhibited within 24h. Moreover, the key enzyme for the biosynthesis of estrogens aromatase is modulated by Wnt3A in a biphasic manner, up-regulated at 24h but reduced after 48h. Our results demonstrate that, while ER signaling acts synergistically with Wnt3A in promoting osteogenic differentiation, Wnt3A may crosstalk with ER signaling by up-regulating ERα expression and down-regulating ERβ expression in MPCs. Thus, the signaling crosstalk and synergy between these two pathways should be further explored as a potential therapeutic approach to combating bone and skeletal disorders, such as fracture healing and osteoporosis. PMID:24340027

  17. Epigallocatechin-3-gallate Protects against Hydrogen Peroxide-Induced Inhibition of Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

    PubMed Central

    Wang, Dawei; Wang, Yonghui; Xu, Shihong; Wang, Fu; Wang, Bomin; Han, Ke; Sun, Daqing; Li, Lianxin

    2016-01-01

    Oxidative stress induces bone loss and osteoporosis, and epigallocatechin-3-gallate (EGCG) may be used to combat these diseases due to its antioxidative property. Herein, oxidative stress in human bone marrow-derived mesenchymal stem cells (BM-MSCs) was induced by H2O2, resulting in an adverse effect on their osteogenic differentiation. However, this H2O2-induced adverse effect was nullified when the cells were treated with EGCG. In addition, treatment of BM-MSCs with EGCG alone also resulted in the enhancement of osteogenic differentiation of BM-MSCs. After EGCG treatment, expressions of β-catenin and cyclin D1 were upregulated, suggesting that the Wnt pathway was involved in the effects of EGCG on the osteogenic differentiation of BM-MSCs. This was also confirmed by the fact that the Wnt pathway inhibitor, Dickkopf-1 (DKK-1), can nullify the EGCG-induced enhancement effect on BM-MSC's osteogenic differentiation. Hence, our results suggested that EGCG can reduce the effects of oxidative stress on Wnt pathway in osteogenic cells, which supported a potentially promising therapy of bone disorders induced by oxidative stress. Considering its positive effects on BM-MSCs, EGCG may also be beneficial for stem cell-based bone repair. PMID:26977159

  18. Osteogenic Differentiation of Human Amniotic Epithelial Cells and Its Application in Alveolar Defect Restoration

    PubMed Central

    Jiawen, Si; Jianjun, Zhang; Jiewen, Dai; Dedong, Yu; Hongbo, Yu; Jun, Shi; Xudong, Wang; Shen, Steve G.F.

    2014-01-01

    The present study investigated the detailed in vitro osteogenic differentiation process and in vivo bone regenerative property of human amniotic epithelial cells (hAECs). The in vitro osteogenic differentiation process of hAECs was evaluated by biochemical staining, real-time polymerase chain reaction, and immunofluorescence. Next, β-tricalcium phosphate (β-TCP) scaffolds alone or loaded with hAECs were implanted into the alveolar defects of rats. Micro-computed tomography evaluation and histologic studies were conducted. Our results validated the in vitro osteogenic capacity of hAECs by upregulation of Runx2, osterix, alkaline phosphatase, collagen I, and osteopontin, with positive biochemical staining for osteoblasts. An epithelial-mesenchymal transformation process might be involved in the osteogenic differentiation of hAECs by increased expression of transforming growth factor-β1. Our data also demonstrated that in vivo implantation of hAECs loaded on β-TCP scaffolds, not only improved bone regeneration by direct participation, but also reduced the early host immune response to the scaffolds. The presented data indicate that hAECs possess proper osteogenic differentiation potential and a modulatory influence on the early tissue remodeling process, making these cells a potential source of progenitor cells for clinical restoration of the alveolar defect. PMID:25368378

  19. Immobilizing osteogenic growth peptide with and without fibronectin on a titanium surface: effects of loading methods on mesenchymal stem cell differentiation

    PubMed Central

    Chen, Cen; Li, Han; Kong, Xiangdong; Zhang, Sheng-Min; Lee, In-Seop

    2015-01-01

    In this study, to improve the osseointegration of implants, osteogenic growth peptide (OGP) and fibronectin (FN) were loaded within mineral, which was formed on titanium, through adsorption and coprecipitation methods. The release profiles of OGP loaded by either adsorption or coprecipitation and the effects of the loading methods to immobilize OGP with and without FN on rat mesenchymal stem cell (rMSC) osteogenic differentiation were studied. The coprecipitation approach slightly reduced the initial burst release, while the adsorption approach provided a more sustained release. Dual loading of OGP and FN further improved cell attachments compared with either OGP or FN alone. Dually loaded OGP and FN also had a positive impact on rMSC proliferation and osteogenic differentiation. The difference in methods of loading OGP with and without FN also had some effects on osteogenic differentiation. Compared with coprecipitated OGP alone, adsorbed OGP enhanced later differentiation, such as osteocalcin secretion and matrix mineralization. Simultaneously adsorbed OGP and FN led to higher proliferation and higher osteogenic differentiation in both early and late stages compared with sequentially loaded OGP and FN. rMSC culture clearly indicated that simultaneously adsorbed OGP and FN could improve osseointegration, and this treatment represents a potential method for effective surface modification of dental and orthopedic implants. PMID:25678785

  20. Characterization of Notch Signaling During Osteogenic Differentiation in Human Osteosarcoma Cell Line MG63.

    PubMed

    Ongaro, Alessia; Pellati, Agnese; Bagheri, Leila; Rizzo, Paola; Caliceti, Cristiana; Massari, Leo; De Mattei, Monica

    2016-12-01

    Osteogenic differentiation is a multi-step process controlled by a complex molecular framework. Notch is an evolutionarily conserved intercellular signaling pathway playing a prominent role in cell fate and differentiation, although the mechanisms by which this pathway regulates osteogenesis remain controversial. This study aimed to investigate, in vitro, the involvement of Notch pathway during all the developmental stages of osteogenic differentiation in human osteosarcoma cell line MG63. Cells were cultured in basal condition (control) and in osteoinductive medium (OM). Notch inhibitors were also added in OM to block Notch pathway. During osteogenic differentiation, early (alkaline phosphatase activity and collagen type I) and late osteogenic markers (osteocalcin levels and matrix mineralization), as well as the gene expression of the main osteogenic transcription factors (Runx2, Osterix, and Dlx5) increased. Time dependent changes in the expression of specific Notch receptors were identified in OM versus control with a significant reduction in the expression of Notch1 and Notch3 receptors in the early phase of differentiation, and an increase of Notch2 and Notch4 receptors in the late phase. Among Notch nuclear target genes, Hey1 expression was significantly higher in OM than control, while Hes5 expression decreased. Osteogenic markers were reduced and Hey1 was significantly inhibited by Notch inhibitors, suggesting a role for Notch through the canonical pathway. In conclusion, Notch pathway might be involved with a dual role in osteogenesis of MG63, through the activation of Notch2, Notch4, and Hey1, inducing osteoblast differentiation and the depression of Notch1, Notch3, and Hes5, maintaining an undifferentiated status. J. Cell. Physiol. 231: 2652-2663, 2016. © 2016 Wiley Periodicals, Inc. PMID:26946465

  1. 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

  2. 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

  3. 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. PMID:26369111

  4. Effect of molecular weight and concentration of hyaluronan on cell proliferation and osteogenic differentiation in vitro.

    PubMed

    Zhao, Ningbo; Wang, Xin; Qin, Lei; Guo, Zhengze; Li, Dehua

    2015-09-25

    Hyaluronan (HA), the simplest glycosaminoglycan and a major component of the extracellular matrix, exists in various tissues. It is involved in some critical biological procedures, including cellular signaling, cell adhesion and proliferation, and cell differentiation. The effect of molecular weight (MW) and concentration of HA on cell proliferation and differentiation was controversial. In this study, we investigated the effect of MW and concentration of HA on the proliferation and osteogenic differentiation of rabbit bone marrow-derived stem cells in vitro. Results showed that high MW HA decreased the cell adhesion rate in a concentration-dependant manner. The cell adhesion rate was decreased by increasing MW of HA. Cell proliferation was significantly enhanced by low MW HA (P < 0.05). The factorial analysis indicated that MW and concentration had an interactive effect on the cell adhesion rate and cell proliferation (P < 0.05). High MW HA increased the mRNA expressions of ALP, RUNX-2 and OCN. The higher the MW was, the higher the mRNA expressions were. The factorial analysis indicated that MW and concentration had an interactive effect on ALP mRNA expression (P < 0.05). HA of higher MW and higher concentration promoted bone formation. These findings provide some useful information in understanding the mechanism underlying the effect of MW and concentration of HA on cell proliferation and differentiation. PMID:26284973

  5. Stimulated Osteogenic Differentiation of Human Mesenchymal Stem Cells by Reduced Graphene Oxide.

    PubMed

    Jin, Linhua; Lee, Jong Ho; Jin, Oh Seong; Shin, Yong Cheol; Kim, Min Jeong; Hong, Suck Won; Lee, Mi Hee; Park, Jong-Chul; Han, Dong-Wook

    2015-10-01

    Osteoprogenitor cells play a significant role in the growth or repair of bones, and have great potential as cell sources for regenerative medicine and bone tissue engineering, but control of their specific differentiation into bone cells remains a challenge. Graphene-based nanomaterials are attractive candidates for biomedical applications as substrates for stem cell (SC) differentiation, scaffolds in tissue engineering, and components of implant devices owing to their biocompatible, transferable and implantable properties. This study examined the enhanced osteogenic differentiation of human mesenchymal stem cells (hMSCs) by reduced graphene oxide (rGO) nanoparticles (NPs), and rGO NPs was prepared by reducing graphene oxide (GO) with a hydrazine treatment followed by annealing in argon and hydrogen. The cytotoxicity profile of each particle was examined using a water-soluble tetrazolium-8 (WST-8) assay. At different time-points, a WST-8 assay, alkaline phosphatase (ALP) activity assay and alizarin red S (ARS) staining were used to determine the effects of rGO NPs on proliferation, differentiation and mineralization, respectively. The results suggest that graphene-based materials have potential as a platform for stem cells culture and biomedical applications. PMID:26726448

  6. Hepcidin promotes osteogenic differentiation through the bone morphogenetic protein 2/small mothers against decapentaplegic and mitogen-activated protein kinase/P38 signaling pathways in mesenchymal stem cells

    PubMed Central

    LU, HUADING; LIAN, LIYI; SHI, DEHAI; ZHAO, HUIQING; DAI, YUHU

    2015-01-01

    The ability of mesenchymal stem cells (MSCs) to differentiate into osteogenic lineages requires management for their future use in treating bone destruction and osteoporosis. Hepcidin is closely associated with bone metabolism, however, it remains to be elucidated whether hepcidin affects osteogenic differentiation in MSCs. The present study demonstrated that hepcidin enhanced osteoblastic differentiation and mineralization, which was manifested by an upregulation in the differentiation markers alkaline phosphatase and osteogenic genes. Furthermore, the expression levels of bone morphogenetic proteins and small mothers against decapentaplegic homologs were concomitantly increased following hepcidin treatment. In addition, the p38 mitogen-activated protein kinase may be an upstream kinase for osteoblastic differentiation. Thus, hepcidin may be important in the osteogenic differentiation of MSCs and may be considered as a target in the development of therapies for pathological bone loss. PMID:25351366

  7. Influence of scaffold composition over in vitro osteogenic differentiation of hBMSCs and in vivo inflammatory response.

    PubMed

    Costa-Pinto, Ana R; Vargel, I; Tuzlakoglu, K; Correlo, V M; Sol, P C; Faria, S; Piskin, E; Reis, R L; Neves, Nuno M

    2014-05-01

    To understand the role of chitosan in chitosan-poly(butylene succinate) scaffolds (50% wt), 50%, 25%, and 0% of chitosan were used to produce different scaffolds. These scaffolds were in vitro seeded and cultured with human bone marrow stromal cells in osteogenic conditions, revealing that higher percentage of chitosan showed enhanced cell viability over time, adhesion, proliferation, and osteogenic differentiation. Scaffolds were also implanted in cranial defects and iliac submuscular region in Wistar rats, and the results evidenced that chitosan-containing scaffolds displayed mild inflammatory response and good integration with surrounding tissues, showed by connective tissue colonization and the presence of new blood vessels. Scaffolds without chitosan-evidenced necrotic tissue in scaffolds' interior, proving that chitosan exerts a positive effect over cell behavior and displays a milder host inflammatory response in vivo. PMID:24255003

  8. Intercellular Adhesion Molecule-1 Inhibits Osteogenic Differentiation of Mesenchymal Stem Cells and Impairs Bio-Scaffold-Mediated Bone Regeneration In Vivo

    PubMed Central

    Xu, Fen-Fen; Li, Xi-Mei; Yang, Fei; Chen, Ji-De; Tang, Bo; Sun, Hong-Guang; Chu, Ya-Nan; Zheng, Rong-Xiu; Liu, Yuan-Lin

    2014-01-01

    Mesenchymal stem cell (MSC) loaded bio-scaffold transplantation is a promising therapeutic approach for bone regeneration and repair. However, growing evidence shows that pro-inflammatory mediators from injured tissues suppress osteogenic differentiation and impair bone formation. To improve MSC-based bone regeneration, it is important to understand the mechanism of inflammation mediated osteogenic suppression. In the present study, we found that synovial fluid from rheumatoid arthritis patients and pro-inflammatory cytokines including interleukin-1α, interleukin-1β, and tumor necrosis factor α, stimulated intercellular adhesion molecule-1(ICAM-1) expression and impaired osteogenic differentiation of MSCs. Interestingly, overexpression of ICAM-1 in MSCs using a genetic approach also inhibited osteogenesis. In contrast, ICAM-1 knockdown significantly reversed the osteogenic suppression. In addition, after transplanting a traceable MSC-poly(lactic-co-glycolic acid) construct in rat calvarial defects, we found that ICAM-1 suppressed MSC osteogenic differentiation and matrix mineralization in vivo. Mechanistically, we found that ICAM-1 enhances MSC proliferation but causes stem cell marker loss. Furthermore, overexpression of ICAM-1 stably activated the MAPK and NF-κB pathways but suppressed the PI3K/AKT pathway in MSCs. More importantly, specific inhibition of the ERK/MAPK and NF-κB pathways or activation of the PI3K/AKT pathway partially rescued osteogenic differentiation, while inhibition of the p38/MAPK and PI3K/AKT pathway caused more serious osteogenic suppression. In summary, our findings reveal a novel function of ICAM-1 in osteogenesis and suggest a new molecular target to improve bone regeneration and repair in inflammatory microenvironments. PMID:24702024

  9. T cell subsets differently regulate osteogenic differentiation of human mesenchymal stromal cells in vitro.

    PubMed

    Grassi, Francesco; Cattini, Luca; Gambari, Laura; Manferdini, Cristina; Piacentini, Anna; Gabusi, Elena; Facchini, Andrea; Lisignoli, Gina

    2016-04-01

    T lymphocytes play a key role in the regulation of bone homeostasis and bone healing. The inflammatory response at the site of bone injury is essential to the initiation of the bone repair program; however, an uncontrolled exposure to inflammatory environment has a negative effect on tissue regeneration - indeed, activated T cells were shown to inhibit osteogenic differentiation on human mesenchymal stromal cells (MSCs). Whether resting T cells can induce osteogenic differentiation of MSCs and what role specific T cells subset play in this process is still elusive. In this study, we sought to analyse the osteogenic gene expression profile of whole T cells, CD4 and CD8 T cells isolated from healthy donors and investigated whether secreted factors from each group modulate osteogenic differentiation of human MSCs. Gene expression profiling identified a pool of 51 genes involved at various stages in bone growth which are expressed above detectable levels in CD4 and CD8 T cells. Most genes of this pool were expressed at higher levels in the CD4 subset. In vitro mineralization assays revealed that conditioned medium from CD4 T cells, but not from CD8 cells, significantly increased mineralization in osteogenic cultures of human MSCs; furthermore, mRNA expression of Runt-related transcription factor 2 (RUNX-2), osteocalcin (OC), bone sialoprotein (BSP) and alkaline phosphatase (ALP) in MSCs was significantly upregulated in the presence of CD4-conditioned medium but not with that obtained from CD8. The results show a differential role for CD4 and CD8 T cells in supporting bone formation and identify an osteogenic gene signature of each subset. Copyright © 2013 John Wiley & Sons, Ltd. PMID:23653421

  10. Protein isoprenylation regulates osteogenic differentiation of mesenchymal stem cells: effect of alendronate, and farnesyl and geranylgeranyl transferase inhibitors

    PubMed Central

    Duque, G; Vidal, C; Rivas, D

    2011-01-01

    BACKGROUND AND PURPOSE Protein isoprenylation is an important step in the intracellular signalling pathway conducting cell growth and differentiation. In bone, protein isoprenylation is required for osteoclast differentiation and activation. However, its role in osteoblast differentiation and function remains unknown. In this study, we assessed the role of protein isoprenylation in osteoblastogenesis in a model of mesenchymal stem cells (MSC) differentiation. EXPERIMENTAL APPROACH We tested the effect of an inhibitor of farnesylation [farnesyl transferase inhibitor-277 (FTI-277)] and one of geranylgeranylation [geranylgeranyltransferase inhibitor-298 (GGTI-298)] on osteoblast differentiating MSC. In addition, we tested the effect of alendronate on protein isoprenylation in this model either alone or in combination with other inhibitors of isoprenylation. KEY RESULTS Initially, we found that levels of unfarnesylated proteins (prelamin A and HDJ-2) increased after treatment with FTI-277 concomitantly affecting osteoblastogenesis and increasing nuclear morphological changes without affecting cell survival. Furthermore, inhibition of geranylgeranylation by GGTI-298 alone increased osteoblastogenesis. This effect was enhanced by the combination of GGTI-298 and alendronate in the osteogenic media. CONCLUSIONS AND IMPLICATIONS Our data indicate that both farnesylation and geranylgeranylation play a role in osteoblastogenesis. In addition, a new mechanism of action for alendronate on protein isoprenylation in osteogenic differentiating MSC in vitro was found. In conclusion, protein isoprenylation is an important component of the osteoblast differentiation process that could constitute a new therapeutic target for osteoporosis in the future. PMID:21077849

  11. Differential effects of tyrosine-rich amelogenin peptide on chondrogenic and osteogenic differentiation of adult chondrocytes.

    PubMed

    Amin, H D; Ethier, C R

    2016-04-01

    Current approaches to treat osteoarthritis (OA) are insufficient. Autologous chondrocyte implantation (ACI) has been used for the past decade to treat patients with OA or focal cartilage defects. However, a number of complications have been reported post-ACI, including athrofibrosis and symptomatic hypertrophy. Thus, a long-term ACI strategy should ideally incorporate methods to 'prime' autologous chondrocytes to form a cartilage-specific matrix and suppress hypertrophic mineralization. The objective of this study is to examine the effects of tyrosine-rich amelogenin peptide (TRAP; an isoform of the developmental protein amelogenin) on human articular cartilage cell (HAC) chondrogenic differentiation and hypertrophic mineralization in vitro. Effects of chemically synthesized TRAP on HAC chondrogenic differentiation were determined by assessing: (1) sGAG production; (2) Alcian blue staining for proteoglycans; (3) collagen type II immunostaining; and (4) expression of the chondrogenic genes SOX9, ACAN and COL2A1. Hypertrophic mineralization was assayed by: (1) ALP expression; (2) Alizarin red staining for Ca(+2)-rich bone nodules; (3) OC immunostaining; and (4) expression of the osteogenic/hypertrophic genes Ihh and BSP. Chemically synthesized TRAP was found to suppress terminal osteogenic differentiation of HACs cultured in hypertrophic mineralization-like conditions, an effect mediated via down-regulation of the Ihh gene. Moreover, TRAP was found to augment chondrogenic differentiation of HACs via induction of SOX9 gene expression when cells were cultured in pro-chondrogenic media. The results obtained from this proof-of-concept study motivate further studies on the use of TRAP as part of a preconditioning regimen in autologous chondrocyte implantation procedures for OA patients and patients suffering from focal cartilage defects. PMID:26404401

  12. Synergistic effect of exogeneous and endogeneous electrostimulation on osteogenic differentiation of human mesenchymal stem cells seeded on silk scaffolds.

    PubMed

    Çakmak, Anıl S; Çakmak, Soner; White, James D; Raja, Waseem K; Kim, Kyungsook; Yiğit, Sezin; Kaplan, David L; Gümüşderelioğlu, Menemşe

    2016-04-01

    Bioelectrical regulation of bone fracture healing is important for many cellular events such as proliferation, migration, and differentiation. The aim of this study was to investigate the osteogenic differentiation potential of human mesenchymal stem cells (hMSCs) cultivated on silk scaffolds in response to different modes of electrostimulation (e.g., exogeneous and/or endogeneous). Endogeneous electrophysiology was altered through the use of monensin (10 nM) and glibenclamide (10 μM), along with external electrostimulation (60 kHz; 100-500 mV). Monensin enhanced the expression of early osteogenic markers such as alkaline phosphatase (ALP) and runt-related transcription factor 2 (RUNX-2). When exogeneous electrostimulation was combined with glibenclamide, more mature osteogenic marker upregulation based on bone sialoprotein expression (BSP) and mineralization was found. These results suggest the potential to exploit both exogeneous and endogeneous biophysical control of cell functions towards tissue-specific goals. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:581-590, 2016. PMID:26419698

  13. Modulation of osteogenic differentiation in hMSCs cells by submicron topographically-patterned ridges and grooves.

    PubMed

    Watari, Shinya; Hayashi, Kei; Wood, Joshua A; Russell, Paul; Nealey, Paul F; Murphy, Christopher J; Genetos, Damian C

    2012-01-01

    Recent studies have shown that nanoscale and submicron topographic cues modulate a menu of fundamental cell behaviors, and the use of topographic cues is an expanding area of study in tissue engineering. We used topographically-patterned substrates containing anisotropically ordered ridges and grooves to investigate the effects of topographic cues on mesenchymal stem cell morphology, proliferation, and osteogenic differentiation. We found that human mesenchymal stem cells cultured on 1400 or 4000 nm pitches showed greater elongation and alignment relative to 400 nm pitch or planar control. Cells cultured on 400 nm pitch demonstrated significant increases in RUNX2 and BGLAP expression relative to cells cultured on 1400 or 4000 nm pitch or planar control. Four-hundred nanometer pitch enhanced extracellular calcium deposition. Cells cultured in osteoinductive medium revealed combinatory effects of topography and chemical cues on 400 nm pitch as well as up-regulation of expression of ID1, a target of the BMP pathway. Our data demonstrate that a specific size scale of topographic cue promotes osteogenic differentiation with or without osteogenic agents. These data demonstrate that the integration of topographic cues may be useful for the fabrication of orthopedic implants. PMID:21982295

  14. Modulation of osteogenic differentiation in hMSCs cells by submicron topographically-patterned ridges and grooves

    PubMed Central

    Watari, Shinya; Hayashi, Kei; Wood, Joshua A.; Russell, Paul; Nealey, Paul F; Murphy, Christopher J.; Genetos, Damian C.

    2011-01-01

    Recent studies have shown that nanoscale and submicron topographic cues modulate a menu of fundamental cell behaviors, and the use of topographic cues is an expanding area of study in tissue engineering. We used topographically-patterned substrates containing anisotropically-ordered ridges and grooves to investigate the effects of topographic cues on mesenchymal stem cell morphology, proliferation, and osteogenic differentiation. We found that human mesenchymal stem cells cultured on 1400 or 4000 nm pitches showed greater elongation and alignment relative to 400 nm pitch or planar control. Cells cultured on 400 nm pitch demonstrated significant increases in RUNX2 and BGLAP expression relative to cells cultured on 1400 or 4000 nm pitch or planar control. 400 nm pitch enhanced extracellular calcium deposition. Cells cultured in osteoinductive medium revealed combinatory effects of topography and chemical cues on 400 nm pitch as well as up-regulation of expression of ID1, a target of the BMP pathway. Our data demonstrate that a specific size scale of topographic cue promotes osteogenic differentiation with or without osteogenic agents. These data demonstrate that the integration of topographic cues may be useful for the fabrication of orthopedic implants. PMID:21982295

  15. Spatiotemporal release of BMP-2 and VEGF enhances osteogenic and vasculogenic differentiation of human mesenchymal stem cells and endothelial colony-forming cells co-encapsulated in a patterned hydrogel.

    PubMed

    Barati, Danial; Shariati, Seyed Ramin Pajoum; Moeinzadeh, Seyedsina; Melero-Martin, Juan M; Khademhosseini, Ali; Jabbari, Esmaiel

    2016-02-10

    Reconstruction of large bone defects is limited by insufficient vascularization and slow bone regeneration. The objective of this work was to investigate the effect of spatial and temporal release of recombinant human bone morphogenetic protein-2 (BMP2) and vascular endothelial growth factor (VEGF) on the extent of osteogenic and vasculogenic differentiation of human mesenchymal stem cells (hMSCs) and endothelial colony-forming cells (ECFCs) encapsulated in a patterned hydrogel. Nanogels (NGs) based on polyethylene glycol (PEG) macromers chain-extended with short lactide (L) and glycolide (G) segments were used for grafting and timed-release of BMP2 and VEGF. NGs with 12kDa PEG molecular weight (MW), 24 LG segment length, and 60/40L/G ratio (P12-II, NG(10)) released the grafted VEGF in 10days. NGs with 8kDa PEG MW, 26 LG segment length, and 60/40L/G ratio (P8-I, NG(21)) released the grafted BMP2 in 21days. hMSCs and NG-BMP2 were encapsulated in a patterned matrix based on acrylate-functionalized lactide-chain-extended star polyethylene glycol (SPELA) hydrogel and microchannel patterns filled with a suspension of hMSCs+ECFCs and NG-VEGF in a crosslinked gelatin methacryloyl (GelMA) hydrogel. Groups included patterned constructs without BMP2/VEGF (None), with directly added BMP2/VEGF, and NG-BMP2/NG-VEGF. Based on the results, timed-release of VEGF in the microchannels in 10days from NG(10) and BMP2 in the matrix in 21days from NG(21) resulted in highest extent of osteogenic and vasculogenic differentiation of the encapsulated hMSCs and ECFCs compared to direct addition of VEGF and BMP2. Further, timed-release of VEGF from NG(10) in hMSC+ECFC encapsulating microchannels and BMP2 from NG(21) in hMSC encapsulating matrix sharply increased bFGF expression in the patterned constructs. The results suggest that mineralization and vascularization are coupled by localized secretion of paracrine signaling factors by the differentiating hMSCs and ECFCs. PMID:26721447

  16. Homeobox B7 promotes the osteogenic differentiation potential of mesenchymal stem cells by activating RUNX2 and transcript of BSP

    PubMed Central

    Gao, Run-Tao; Zhan, Li-Ping; Meng, Cen; Zhang, Ning; Chang, Shi-Min; Yao, Rui; Li, Chong

    2015-01-01

    Mesenchymal stem cells (MSCs) are a reliable cell source for tissue regeneration. However, the molecular mechanisms underlying the directed differentiation of MSCs remain unclear; thus, their use is limited. Here, we investigate HOXB7 function in the osteogenic differentiation potentials of MSCs using stem cells from apical papilla (SCAPs) and bone marrow stem cells (BMSCs). The HOXB7 gene is highly expressed in BMSCs compared with dental tissue-derived MSCs. We found that, in vitro, over-expression of HOXB7 in SCAPs enhanced alkaline phosphatase (ALP) activity and mineralization. HOXB7 over-expression affected the mRNA expression of osteonectin (ON), collagen alpha-2(I) chain (COL1A2), bone sialoprotein (BSP), and osteocalcin (OCN), led to the expression of the key transcription factor, runt-related transcription factor 2 (RUNX2), and promoted SCAP osteogenic differentiation in vitro. The knock-down of HOXB7 inhibited ALP activity, mineralization, and the expression of ON, BSP, COL1A2, OCN, and RUNX2 in BMSCs in vitro. In addition, transplant experiments in nude mice confirmed that SCAP osteogenesis was triggered when HOXB7 was activated. Furthermore, Over-expression of HOXB7 significantly increased the levels of HOXB7 associated with the BSP promoter by ChIP assays. Taken together, these results indicate that HOXB7 enhances SCAP osteogenic differentiation by up-regulating RUNX2 and directly activating transcript of BSP. Thus, the activation of HOXB7 signaling might improve tissue regeneration mediated by MSCs. These results provide insight into the mechanism underlying the directed differentiation of MSCs. PMID:26379836

  17. Mineralized gelatin methacrylate-based matrices induce osteogenic differentiation of human induced pluripotent stem cells

    PubMed Central

    Kang, Heemin; Shih, Yu-Ru V.; Hwang, Yongsung; Wen, Cai; Rao, Vikram; Seo, Timothy; Varghese, Shyni

    2014-01-01

    Human induced pluripotent stem cells (hiPSCs) are a promising cell source with pluripotency and self-renewal properties. Design of simple and robust biomaterials with an innate ability to induce lineage-specificity of hiPSCs is desirable to realize their applications in regenerative medicine. In this study, we investigated the potential of biomaterials containing calcium phosphate minerals to induce osteogenic differentiation of hiPSCs. hiPSCs cultured using mineralized gelatin methacrylate-based matrices underwent osteogenic differentiation ex vivo, both in two- dimensional (2-D) and three-dimensional (3-D) cultures, in growth medium devoid of any osteogenic-inducing chemical components or growth factors. Our findings that osteogenic differentiation of hiPSCs can be achieved through biomaterial-based cues alone present new avenues for personalized regenerative medicine. Such biomaterials that could not only act as structural scaffolds, but could also provide tissue-specific functions such as directing stem cell differentiation commitment, have great potential in bone tissue engineering. PMID:25153779

  18. 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. PMID:23749732

  19. Osteogenic differentiation associated with x-ray therapy for adenocarcinoma of the prostate gland

    SciTech Connect

    Locke, J.R.; Soloway, M.S.; Evans, J.; Murphy, W.M.

    1986-03-01

    A case of osteogenic differentiation in a prostate gland associated with high-dose x-irradiation for adenocarcinoma is reported. Heterologous cancerous elements in adults are extremely unusual in the prostate and their occurrence after treatment has rarely been documented. The relationship of this lesion to the primary glandular neoplasm is discussed.

  20. Effect of calcium phosphate ceramic substrate geometry on mesenchymal stromal cell organization and osteogenic differentiation.

    PubMed

    Urquia Edreira, Eva R; Hayrapetyan, Astghik; Wolke, Joop G C; Croes, Huib J E; Klymov, Alexey; Jansen, John A; van den Beucken, Jeroen J J P

    2016-01-01

    The composition of calcium phosphate (CaP) ceramics in combination with surface features have been shown to influence biological performance, and micro- and nano-scale topography is known to stimulate osteogenic differentiation of mesenchymal stromal cells (MSCs). In view of this, adipose tissue derived MSCs were cultured on CaP disks featuring hemispherical concavities of various sizes (440, 800 or 1800 μm diameter). It was hypothesized that (i) surface concavities would promote cell proliferation, cellular organization within the concavities, and osteogenic differentiation, as a result of a more pronounced 3D micro-environment and CaP nucleation in concavities, and (ii) MSC proliferation and osteogenic differentiation would increase with smaller concavity size due to more rapidly occurring 3D cell-cell interactions. We found that concavities indeed affect cell proliferation, with 440 μm concavities increasing cell proliferation to a larger extent compared to 800 and 1800 μm concavities as well as planar surfaces. Additionally, concavity size influenced 3D cellular organization within the concavity volume. Interestingly, concavity size promoted osteogenic differentiation of cells, as evidenced by increased osteocalcin gene expression in 440 μm concavities, and osteocalcin staining predominantly for 440 and 800 μm concavities, but not for 1800 μm concavities and only slightly for planar surface controls. PMID:27150445

  1. Dimethyloxaloylglycine Increases the Bone Healing Capacity of Adipose-Derived Stem Cells by Promoting Osteogenic Differentiation and Angiogenic Potential

    PubMed Central

    Ding, Hao; Gao, You-Shui; Wang, Yang; Hu, Chen

    2014-01-01

    Hypoxia inducible factor-1α (HIF-1α) plays an important role in angiogenesis-osteogenesis coupling during bone regeneration, which can enhance the bone healing capacity of mesenchymal stem cells (MSCs) by improving their osteogenic and angiogenic activities. Previous studies transduced the HIF-1α gene into MSCs with lentivirus vectors to improve their bone healing capacity. However, the risks due to lentivirus vectors, such as tumorigenesis, should be considered before clinical application. Dimethyloxaloylglycine (DMOG) is a cell-permeable prolyl-4-hydroxylase inhibitor, which can activate the expression of HIF-1α in cells at normal oxygen tension. Therefore, DMOG is expected to be an alternative strategy for enhancing HIF-1α expression in cells. In this study, we explored the osteogenic and angiogenic activities of adipose-derived stem cells (ASCs) treated with different concentrations of DMOG in vitro, and the bone healing capacity of DMOG-treated ASCs combined with hydrogels for treating critical-sized calvarial defects in rats. The results showed that DMOG had no obvious cytotoxic effects on ASCs and could inhibit the death of ASCs induced by serum deprivation. DMOG markedly increased vascular endothelial growth factor production in ASCs in a dose-dependent manner and improved the osteogenic differentiation potential of ASCs by activating the expression of HIF-1α. Rats with critical-sized calvarial defects treated with hydrogels containing DMOG-treated ASCs had more bone regeneration and new vessel formation than the other groups. Therefore, we believe that DMOG enhanced the angiogenic and osteogenic activity of ASCs by activating the expression of HIF-1α, thereby improving the bone healing capacity of ASCs in rat critical-sized calvarial defects. PMID:24328551

  2. Collagen-Hydroxyapatite Scaffolds Induce Human Adipose Derived Stem Cells Osteogenic Differentiation In Vitro.

    PubMed

    Calabrese, Giovanna; Giuffrida, Raffaella; Fabbi, Claudia; Figallo, Elisa; Lo Furno, Debora; Gulino, Rosario; Colarossi, Cristina; Fullone, Francesco; Giuffrida, Rosario; Parenti, Rosalba; Memeo, Lorenzo; Forte, Stefano

    2016-01-01

    Mesenchymal stem cells (MSCs) play a crucial role in regulating normal skeletal homeostasis and, in case of injury, in bone healing and reestablishment of skeletal integrity. Recent scientific literature is focused on the development of bone regeneration models where MSCs are combined with biomimetic three-dimensional scaffolds able to direct MSC osteogenesis. In this work the osteogenic potential of human MSCs isolated from adipose tissue (hADSCs) has been evaluated in vitro in combination with collagen/Mg doped hydroxyapatite scaffolds. Results demonstrate the high osteogenic potential of hADSCs when cultured in specific differentiation induction medium, as revealed by the Alizarin Red S staining and gene expression profile analysis. In combination with collagen/hydroxyapatite scaffold, hADSCs differentiate into mature osteoblasts even in the absence of specific inducing factors; nevertheless, the supplement of the factors markedly accelerates the osteogenic process, as confirmed by the expression of specific markers of pre-osteoblast and mature osteoblast stages, such as osterix, osteopontin (also known as bone sialoprotein I), osteocalcin and specific markers of extracellular matrix maturation and mineralization stages, such as ALPL and osteonectin. Hence, the present work demonstrates that the scaffold per se is able to induce hADSCs differentiation, while the addition of osteo-inductive factors produces a significant acceleration of the osteogenic process. This observation makes the use of our model potentially interesting in the field of regenerative medicine for the treatment of bone defects. PMID:26982592

  3. Effects of substrate stiffness on adipogenic and osteogenic differentiation of human mesenchymal stem cells.

    PubMed

    Zhao, Wen; Li, Xiaowei; Liu, Xiaoyan; Zhang, Ning; Wen, Xuejun

    2014-07-01

    Substrate mechanical properties, in addition to biochemical signals, have been shown to modulate cell phenotype. In this study, we inspected the effects of substrate stiffness on human mesenchymal stem cells (hMSCs) derived from adult human bone marrow differentiation into adipogenic and osteogenic cells. A chemically modified extracellular matrix derived and highly biocompatible hydrogel, based on thiol functionalized hyaluronic acid (HA-SH) and thiol functionalized recombinant human gelatin (Gtn-SH), which can be crosslinked by poly (ethylene glycol) tetra-acrylate (PEGTA), was used as a model system. The stiffness of the hydrogel was controlled by adjusting the crosslinking density. Human bone marrow MSCs were cultured on the hydrogels with different stiffness under adipogenic and osteogenic conditions. Oil Red O staining and F-actin staining were applied to assess the change of cell morphologies under adipogenic and osteogenic differentiation, respectively. Gene expression of cells was determined with reverse transcription polymerase chain reaction (RT-PCR) as a function of hydrogel stiffness. Results support the hypothesis that adipogenic and osteogenic differentiation of hMSCs are inclined to occur on substrate with stiffness similar to their in vivo microenvironments. PMID:24857499

  4. Collagen-Hydroxyapatite Scaffolds Induce Human Adipose Derived Stem Cells Osteogenic Differentiation In Vitro

    PubMed Central

    Fabbi, Claudia; Figallo, Elisa; Lo Furno, Debora; Gulino, Rosario; Colarossi, Cristina; Fullone, Francesco; Giuffrida, Rosario; Parenti, Rosalba; Memeo, Lorenzo; Forte, Stefano

    2016-01-01

    Mesenchymal stem cells (MSCs) play a crucial role in regulating normal skeletal homeostasis and, in case of injury, in bone healing and reestablishment of skeletal integrity. Recent scientific literature is focused on the development of bone regeneration models where MSCs are combined with biomimetic three-dimensional scaffolds able to direct MSC osteogenesis. In this work the osteogenic potential of human MSCs isolated from adipose tissue (hADSCs) has been evaluated in vitro in combination with collagen/Mg doped hydroxyapatite scaffolds. Results demonstrate the high osteogenic potential of hADSCs when cultured in specific differentiation induction medium, as revealed by the Alizarin Red S staining and gene expression profile analysis. In combination with collagen/hydroxyapatite scaffold, hADSCs differentiate into mature osteoblasts even in the absence of specific inducing factors; nevertheless, the supplement of the factors markedly accelerates the osteogenic process, as confirmed by the expression of specific markers of pre-osteoblast and mature osteoblast stages, such as osterix, osteopontin (also known as bone sialoprotein I), osteocalcin and specific markers of extracellular matrix maturation and mineralization stages, such as ALPL and osteonectin. Hence, the present work demonstrates that the scaffold per se is able to induce hADSCs differentiation, while the addition of osteo-inductive factors produces a significant acceleration of the osteogenic process. This observation makes the use of our model potentially interesting in the field of regenerative medicine for the treatment of bone defects. PMID:26982592

  5. The Immunologic Properties of Bone Morphogenic Protein Receptor IB Positive Subpopulation before and after Osteogenic Differentiation in Mouse Dermis

    PubMed Central

    Wang, Tao; Xu, Hua; Zhang, Yi; Dong, Jia-Sheng

    2016-01-01

    We have previously reported that human dermal bone morphogenic protein receptor (BMPR) IB positive subpopulation had a high osteogenic differentiation potential and may be a promising cell source for allogeneic bone tissue engineering. In this study, the immunologic properties of dermal BMPR-IB+ subpopulation before and after osteogenic differentiation were reported. The results confirmed that dermal BMPR-IB+ cells possessed a similar osteogenic differentiation potential with bone marrow mesenchymal stromal cells in a mouse model. Furthermore, the expression of immune rejection-related surface antigens such as major histocompatibility class II and co-stimulatory proteins (CD40, CD80, and CD86) were absent on dermal BMPRIB+ cells. Dermal BMPRIB+ cells elicited no proliferation of allogeneic splenocytes and suppressed the proliferation of stimulated immune cells. Interestingly, osteogenic differentiation in vitro had no adverse effect on the immunological features of these cells. Most importantly, inducible NO synthase (iNOS) was involved in immunoregulatory effects by undifferentiated BMPRIB+ fibroblasts, whereas indoleamine 2,3-dioxygenase (IDO) activity was related to mediating immunomodulatory function by osteogenic differentiated BMPRIB+ fibroblasts. In conclusion, dermal BMPRIB+ cells have a low immunogenicity and possess immunosuppressive capacity before and after osteogenic differentiation in vitro, which would facilitate the allotransplantation in the future. However, mechanisms mediating immunoregulatory property between undifferentiated and osteogenic differentiated BMPRIB+ fibroblasts may be different and need further investigation. PMID:27552226

  6. The Immunologic Properties of Bone Morphogenic Protein Receptor IB Positive Subpopulation before and after Osteogenic Differentiation in Mouse Dermis.

    PubMed

    He, Jin-Guang; Wang, Ting-Liang; Wang, Tao; Xu, Hua; Zhang, Yi; Dong, Jia-Sheng

    2016-01-01

    We have previously reported that human dermal bone morphogenic protein receptor (BMPR) IB positive subpopulation had a high osteogenic differentiation potential and may be a promising cell source for allogeneic bone tissue engineering. In this study, the immunologic properties of dermal BMPR-IB+ subpopulation before and after osteogenic differentiation were reported. The results confirmed that dermal BMPR-IB+ cells possessed a similar osteogenic differentiation potential with bone marrow mesenchymal stromal cells in a mouse model. Furthermore, the expression of immune rejection-related surface antigens such as major histocompatibility class II and co-stimulatory proteins (CD40, CD80, and CD86) were absent on dermal BMPRIB+ cells. Dermal BMPRIB+ cells elicited no proliferation of allogeneic splenocytes and suppressed the proliferation of stimulated immune cells. Interestingly, osteogenic differentiation in vitro had no adverse effect on the immunological features of these cells. Most importantly, inducible NO synthase (iNOS) was involved in immunoregulatory effects by undifferentiated BMPRIB+ fibroblasts, whereas indoleamine 2,3-dioxygenase (IDO) activity was related to mediating immunomodulatory function by osteogenic differentiated BMPRIB+ fibroblasts. In conclusion, dermal BMPRIB+ cells have a low immunogenicity and possess immunosuppressive capacity before and after osteogenic differentiation in vitro, which would facilitate the allotransplantation in the future. However, mechanisms mediating immunoregulatory property between undifferentiated and osteogenic differentiated BMPRIB+ fibroblasts may be different and need further investigation. PMID:27552226

  7. Osteogenic differentiation of mouse mesenchymal progenitor cell, Kusa-A1 is promoted by mammalian transcriptional repressor Rbpj

    SciTech Connect

    Wang, Shengchao; Kawashima, Nobuyuki; Sakamoto, Kei; Katsube, Ken-ichi; Umezawa, Akihiro; Suda, Hideaki

    2010-09-10

    Research highlights: {yields} High Rbpj mRNA expression was observed in mesenchymal cells surrounding the bone of mouse embryos. {yields} Overexpression of Rbpj depressed Notch-Hes1/Hey1 signaling. {yields} Rbpj upregulated promoter activities of Runx2 and Ose2. {yields} Rbpj promoted osteoblastic differentiation/maturation in Kusa-A1 cells. -- Abstract: Pluripotent mesenchymal stem cells possess the ability to differentiate into many cell types, but the precise mechanisms of differentiation are still unclear. Here, we provide evidence that Rbpj (recombination signal-binding protein for immunoglobulin kappa j region) protein, the primary nuclear mediator of Notch, is involved in osteogenesis. Overexpression of Rbpj promoted osteogenic differentiation of mouse Kusa-A1 cells in vitro and in vivo. Transient transfection of an Rbpj expression vector into Kusa-A1 cells upregulated promoter activities of Runx2 and Ose2. Enhanced osteogenic potentials including high alkaline phosphatase activity, rapid calcium deposition, and increased calcified nodule formation, were observed in established stable Rbpj-overexpressing Kusa-A1 (Kusa-A1/Rbpj) cell line. In vivo mineralization by Kusa-A1/Rbpj was promoted compared to that by Kusa-A1 host cells. Histological findings revealed that expression of Rbpj was primarily observed in osteoblasts. These results suggest that Rbpj may play essential roles in osteoblast differentiation.

  8. Steroid regulation of proliferation and osteogenic differentiation of bone marrow stromal cells: a gender difference.

    PubMed

    Hong, Liu; Sultana, Habiba; Paulius, Karina; Zhang, Guoquan

    2009-04-01

    Bone marrow mesenchymal stem cells (MSCs) are considered a potential cell source for stem cell-based bone tissue engineering. However, noticeable limitations of insufficient supply and reduction of differentiation potential impact the feasibility of their clinical application. This study investigated the in vitro function of steroids and gender differences on the proliferation and differentiation of rat MSCs. Bone marrow MSCs of age-matched rats were exposed to proliferation and osteogenic differentiation media supplements with various concentrations of 17beta-estradiol (E2) and dexamethasone. Cell proliferation was measured by MTS assay; osteogenic markers and steroid-associated growth factors and receptors were evaluated by ELISA and real-time PCR. The results revealed that supplements of E2 and dexamethasone increase MSC proliferation in a biphasic manner. The optimal dose and interaction of steroids required to improve MSC proliferation effectively varied depending on the gender of donors. Supplementation of E2 effectively improves osteogenic differentiation markers including ALP, osteocalcin and calcium levels for MSCs isolated from both male and female donors. The mRNA of TGF-beta1 and BMP-7 are also up-regulated. However, effective doses to maximally improve osteogenic potentials and growth factors for MSCs are different between male and female donors. The relationship between steroid receptors, osteogenic markers and cytokines are also varied by genders. The outcomes of the present study strongly indicate that steroids potentially function as an effective modulator to improve the capacity of MSCs in bone regeneration. It provides crucial information for improving and optimizing MSCs for future clinical application of bone regeneration. PMID:19429449

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

    PubMed Central

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

    2016-01-01

    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. PMID:26905619

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

    PubMed

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

    2016-01-01

    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. PMID:26905619

  11. 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.

  12. DKK1 rescues osteogenic differentiation of mesenchymal stem cells isolated from periodontal ligaments of patients with diabetes mellitus induced periodontitis

    PubMed Central

    Liu, Qi; Hu, Cheng-Hu; Zhou, Cui-Hong; Cui, Xiao-Xia; Yang, Kun; Deng, Chao; Xia, Jia-Jia; Wu, Yan; Liu, Lu-Chuan; Jin, Yan

    2015-01-01

    Multiple studies have shown that diabetes mellitus is an established risk factor for periodontitis. Recently mesenchymal stem cells derived from periodontal ligament (PDLSCs) have been utilized to reconstruct tissues destroyed by chronic inflammation. However, impact of periodontitis with diabetes mellitus on PDLSCs and mechanisms mediating effects of complex microenvironments remain poorly understood. In this study, we found multiple differentiation potential of PDLSCs from chronic periodontitis with diabetes mellitus donors (D-PDLSCs) was damaged significantly. Inhibition of NF-κB signaling could rescue osteogenic potential of PDLSCs from simple chronic periodontitis patients (P-PDLSCs), whereas did not promote D-PDLSCs osteogenesis. In addition, we found expression of DKK1 in D-PDLSCs did not respond to osteogenic signal and decreased osteogenic potential of D-PDLSCs treated with DKK1 could be reversed. To further elucidate different character between P-PDLSCs and D-PDLSCs, we treated PDLSCs with TNF-α and advanced glycation end products (AGEs), and find out AGEs which enhance effect of TNF-α in PDLSCs might mediate special personality of D-PDLSCs. The adverse effect of AGEs in PDLSCs could be reversed when PDLSCs were treated with DKK1. These results suggested DKK1 mediating WNT signaling might be a therapy target to rescue potential of PDLSCs in periodontitis with diabetes mellitus. PMID:26278788

  13. Electro-magnetic field promotes osteogenic differentiation of BM-hMSCs through a selective action on Ca2+-related mechanisms

    PubMed Central

    Petecchia, Loredana; Sbrana, Francesca; Utzeri, Roberto; Vercellino, Marco; Usai, Cesare; Visai, Livia; Vassalli, Massimo; Gavazzo, Paola

    2015-01-01

    Exposure to Pulsed Electromagnetic Field (PEMF) has been shown to affect proliferation and differentiation of human mesenchymal stem cells derived from bone marrow stroma (BM-hMSC). These cells offer considerable promise in the field of regenerative medicine, but their clinical application is hampered by major limitations such as poor availability and the time required to differentiate up to a stage suitable for implantation. For this reason, several research efforts are focusing on identifying strategies to speed up the differentiation process. In this work we investigated the in vitro effect of PEMF on Ca2+-related mechanisms promoting the osteogenic differentiation of BM-hMSC. Cells were daily exposed to PEMF while subjected to osteogenic differentiation and various Ca2+-related mechanisms were monitored using multiple approaches for identifying functional and structural modifications related to this process. The results indicate that PEMF exposure promotes chemically induced osteogenesis by mechanisms that mainly interfere with some of the calcium-related osteogenic pathways, such as permeation and regulation of cytosolic concentration, leaving others, such as extracellular deposition, unaffected. The PEMF effect is primarily associated to early enhancement of intracellular calcium concentration, which is proposed here as a reliable hallmark of the osteogenic developmental stage. PMID:26364969

  14. Electro-magnetic field promotes osteogenic differentiation of BM-hMSCs through a selective action on Ca(2+)-related mechanisms.

    PubMed

    Petecchia, Loredana; Sbrana, Francesca; Utzeri, Roberto; Vercellino, Marco; Usai, Cesare; Visai, Livia; Vassalli, Massimo; Gavazzo, Paola

    2015-01-01

    Exposure to Pulsed Electromagnetic Field (PEMF) has been shown to affect proliferation and differentiation of human mesenchymal stem cells derived from bone marrow stroma (BM-hMSC). These cells offer considerable promise in the field of regenerative medicine, but their clinical application is hampered by major limitations such as poor availability and the time required to differentiate up to a stage suitable for implantation. For this reason, several research efforts are focusing on identifying strategies to speed up the differentiation process. In this work we investigated the in vitro effect of PEMF on Ca(2+)-related mechanisms promoting the osteogenic differentiation of BM-hMSC. Cells were daily exposed to PEMF while subjected to osteogenic differentiation and various Ca(2+)-related mechanisms were monitored using multiple approaches for identifying functional and structural modifications related to this process. The results indicate that PEMF exposure promotes chemically induced osteogenesis by mechanisms that mainly interfere with some of the calcium-related osteogenic pathways, such as permeation and regulation of cytosolic concentration, leaving others, such as extracellular deposition, unaffected. The PEMF effect is primarily associated to early enhancement of intracellular calcium concentration, which is proposed here as a reliable hallmark of the osteogenic developmental stage. PMID:26364969

  15. Activation of the Extracellular Signal-Regulated Kinase Signaling Is Critical for Human Umbilical Cord Mesenchymal Stem Cell Osteogenic Differentiation

    PubMed Central

    Li, Chen-Shuang; Zheng, Zhong; Su, Xiao-Xia; Wang, Fei; Ling, Michelle; Zou, Min; Zhou, Hong

    2016-01-01

    Human umbilical cord mesenchymal stem cells (hUCMSCs) are recognized as candidate progenitor cells for bone regeneration. However, the mechanism of hUCMSC osteogenesis remains unclear. In this study, we revealed that mitogen-activated protein kinases (MAPKs) signaling is involved in hUCMSC osteogenic differentiation in vitro. Particularly, the activation of c-Jun N-terminal kinases (JNK) and p38 signaling pathways maintained a consistent level in hUCMSCs through the entire 21-day osteogenic differentiation period. At the same time, the activation of extracellular signal-regulated kinases (ERK) signaling significantly increased from day 5, peaked at day 9, and declined thereafter. Moreover, gene profiling of osteogenic markers, alkaline phosphatase (ALP) activity measurement, and alizarin red staining demonstrated that the application of U0126, a specific inhibitor for ERK activation, completely prohibited hUCMSC osteogenic differentiation. However, when U0126 was removed from the culture at day 9, ERK activation and osteogenic differentiation of hUCMSCs were partially recovered. Together, these findings demonstrate that the activation of ERK signaling is essential for hUCMSC osteogenic differentiation, which points out the significance of ERK signaling pathway to regulate the osteogenic differentiation of hUCMSCs as an alternative cell source for bone tissue engineering. PMID:26989682

  16. Human Placenta-Derived CD146-Positive Mesenchymal Stromal Cells Display a Distinct Osteogenic Differentiation Potential.

    PubMed

    Ulrich, Christine; Abruzzese, Tanja; Maerz, Jan K; Ruh, Manuel; Amend, Bastian; Benz, Karin; Rolauffs, Bernd; Abele, Harald; Hart, Melanie L; Aicher, Wilhelm K

    2015-07-01

    Mesenchymal stromal cells (MSCs) are multipotent cells that can be differentiated in vitro into a variety of cell types, including adipocytes or osteoblasts. Our recent studies indicated that a high expression of CD146 on MSCs from bone marrow correlates with their robust osteogenic differentiation potential. We therefore investigated if expression of CD146 on MSCs from the placenta correlates with a similar osteogenic differentiation potential. The MSCs were isolated specifically from the endometrial and fetal parts of human term placenta and expanded in separate cultures and compared with MSCs from bone marrow as controls. The expression of cell surface antigens was investigated by flow cytometry. Differentiation of MSCs was documented by cytochemistry and analysis of typical lineage marker genes. CD146-positive MSCs were separated from CD146-negative cells by magnet-assisted cell sorts (MACS). We report that the expression of CD146 is associated with a higher osteogenic differentiation potential in human placenta-derived MSCs (pMSCs) and the CD146(pos) pMSCs generated a mineralized extracellular matrix, whereas the CD146(neg) pMSCs failed to do so. In contrast, adipogenic and chondrogenic differentiation of pMSCs was not different in CD146(pos) compared with CD146(neg) pMSCs. Upon enrichment of pMSCs by MACS, the CD146(neg) and CD146(pos) populations maintained their expression levels for this antigen for several passages in vitro. We conclude that CD146(pos) pMSCs either respond to osteogenic stimuli more vividly or, alternatively, CD146(pos) pMSCs present a pMSC subset that is predetermined to differentiate into osteoblasts. PMID:25743703

  17. Magnesium ion implantation on a micro/nanostructured titanium surface promotes its bioactivity and osteogenic differentiation function

    PubMed Central

    Wang, Guifang; Li, Jinhua; Zhang, Wenjie; Xu, Lianyi; Pan, Hongya; Wen, Jin; Wu, Qianju; She, Wenjun; Jiao, Ting; Liu, Xuanyong; Jiang, Xinquan

    2014-01-01

    As one of the important ions associated with bone osseointegration, magnesium was incorporated into a micro/nanostructured titanium surface using a magnesium plasma immersion ion-implantation method. Hierarchical hybrid micro/nanostructured titanium surfaces followed by magnesium ion implantation for 30 minutes (Mg30) and hierarchical hybrid micro/nanostructured titanium surfaces followed by magnesium ion implantation for 60 minutes (Mg60) were used as test groups. The surface morphology, chemical properties, and amount of magnesium ions released were evaluated by field-emission scanning electron microscopy, energy dispersive X-ray spectroscopy, field-emission transmission electron microscopy, and inductively coupled plasma-optical emission spectrometry. Rat bone marrow mesenchymal stem cells (rBMMSCs) were used to evaluate cell responses, including proliferation, spreading, and osteogenic differentiation on the surface of the material or in their medium extraction. Greater increases in the spreading and proliferation ability of rBMMSCs were observed on the surfaces of magnesium-implanted micro/nanostructures compared with the control plates. Furthermore, the osteocalcin (OCN), osteopontin (OPN), and alkaline phosphatase (ALP) genes were upregulated on both surfaces and in their medium extractions. The enhanced cell responses were correlated with increasing concentrations of magnesium ions, indicating that the osteoblastic differentiation of rBMMSCs was stimulated through the magnesium ion function. The magnesium ion-implanted micro/nanostructured titanium surfaces could enhance the proliferation, spreading, and osteogenic differentiation activity of rBMMSCs, suggesting they have potential application in improving bone-titanium integration. PMID:24940056

  18. Effects of hTERT immortalization on osteogenic and adipogenic differentiation of dental pulp stem cells.

    PubMed

    Ikbale, El-Ayachi; Goorha, Sarita; Reiter, Lawrence T; Miranda-Carboni, Gustavo A

    2016-03-01

    These data relate to the differentiation of human dental pulp stem cells (DPSC) and DPSC immortalized by constitutively expressing human telomerase reverse transcriptase (hTERT) through both osteogenic and adipogenic lineages (i.e. to make bone producing and fat producing cells from these dental pulp stem cells). The data augment another study to characterize immortalized DPSC for the study of neurogenetic "Characterization of neurons from immortalized dental pulp stem cells for the study of neurogenetic disorders" [1]. Two copies of one typical control cell line (technical replicates) were used in this study. The data represent the differentiation of primary DPSC into osteoblast cells approximately 60% more effectively than hTERT immortalized DPSC. Conversely, both primary and immortalized DPSC are poorly differentiated into adipocytes. The mRNA expression levels for both early and late adipogenic and osteogenic gene markers are shown. PMID:26958627

  19. The induction of cellular senescence in dental follicle cells inhibits the osteogenic differentiation.

    PubMed

    Morsczeck, Christian; Gresser, Jan; Ettl, Tobias

    2016-06-01

    Dental stem cells such as human dental follicle cells (DFCs) have opened new promising treatment alternatives for today's dental health issues such as periodontal tissue regeneration. However, cellular senescence represents a restricting factor to cultured stem cells, resulting in limited lifespan and reduced cell differentiation potential. Therefore, this study evaluated if and how DFCs exhibit features of cellular senescence after being expanded in cell culture. The cell proliferation of DFCs decreased, while the cell size increased during prolonged cell culture. Moreover, DFCs expressed the senescence-associated β-galactosidase after a prolonged cell culture. The onset of senescence inhibited both the induction of osteoblast markers RUNX2 and osteopontin and the biomineralization of DFCs after stimulation of the osteogenic differentiation. In conclusion, we showed that a prolonged cell culture induces cellular senescence and inhibits the osteogenic differentiation in DFCs. PMID:27165403

  20. Effects of hTERT immortalization on osteogenic and adipogenic differentiation of dental pulp stem cells

    PubMed Central

    Ikbale, El-Ayachi; Goorha, Sarita; Reiter, Lawrence T.; Miranda-Carboni, Gustavo A.

    2016-01-01

    These data relate to the differentiation of human dental pulp stem cells (DPSC) and DPSC immortalized by constitutively expressing human telomerase reverse transcriptase (hTERT) through both osteogenic and adipogenic lineages (i.e. to make bone producing and fat producing cells from these dental pulp stem cells). The data augment another study to characterize immortalized DPSC for the study of neurogenetic “Characterization of neurons from immortalized dental pulp stem cells for the study of neurogenetic disorders” [1]. Two copies of one typical control cell line (technical replicates) were used in this study. The data represent the differentiation of primary DPSC into osteoblast cells approximately 60% more effectively than hTERT immortalized DPSC. Conversely, both primary and immortalized DPSC are poorly differentiated into adipocytes. The mRNA expression levels for both early and late adipogenic and osteogenic gene markers are shown. PMID:26958627

  1. [Bone and Stem Cells. The mechanism of osteogenic differentiation from mesenchymal stem cell].

    PubMed

    Ohata, Yasuhisa; Ozono, Keiichi

    2014-04-01

    Osteoblasts and osteocytes originate from pluripotent mesenchymal stem cells. Mesenchymal stem cells commit to osteogenic lineage and differentiate into mature osteoblasts and osteocytes through osteoprogenitor cells and preosteoblasts in response to multiple stimuli. The osteoblast commitment, differentiation, and functions are governed by several transcription factors. Among these transcription factors, runt-related transcription factor 2 (Runx2) is a crucial factor in osteoblast differentiation and controls bone formation. Differentiation toward these osteogenic lineage is controlled by a multitude of cytokines including WNTs, bone morphogenetic protein (BMP) , transforming growth factor-β (TGF-β) , hedgehog, parathyroid hormone (PTH) /parathyroid hormone related protein (PTHrP) , insulin-like growth factor-1 (IGF-1) , fibroblast growth factor (FGF) , and Notch. Although regulation of Runx2 activity is a point of convergence of many of the signal transduction routes, there is also a high degree of cross-talk between these pathways. Thus, the combined action of the signal transduction pathways induced by some cytokines determines the commitment and differentiation of mesenchymal stem cells toward the osteogenic lineage. PMID:24681495

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

    SciTech Connect

    Lee, Hye-Lim; Park, Hyun-Jung; Kwon, Arang; Baek, Kyunghwa; Woo, Kyung Mi; Ryoo, Hyun-Mo; Kim, Gwan-Shik; Baek, Jeong-Hwa

    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, 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.

  3. Ginsenoside Rg1 promotes osteogenic differentiation of rBMSCs and healing of rat tibial fractures through regulation of GR-dependent BMP-2/SMAD signaling.

    PubMed

    Gu, Yanqing; Zhou, Jinchun; Wang, Qin; Fan, Weimin; Yin, Guoyong

    2016-01-01

    Fracture healing is closely related to the number and activity of bone marrow mesenchymal stem cells (BMSCs) near the fracture site. The present study was to investigate the effect of Rg1 on osteogenic differentiation of cultured BMSCs and related mechanisms and on the fracture healing in a fracture model. In vitro experiments showed that Rg1 promoted the proliferation and osteogenic differentiation of BMSCs. Western blot analyses demonstrated that Rg1 promoted osteogenic differentiation of BMSCs through the glucocorticoid receptor (GR)-dependent BMP-2/Smad signaling pathway. In vivo, X-ray examination showed that callus growth in rats treated with Rg1 was substantially faster than that in control rats after fracture. The results of H&E and Safranin-O/Fast Green staining revealed that, compared with controls, rats in the Rg1 treatment group had a significantly higher proportion of trabecular bone but a much lower proportion of fibers and cartilage components inside the callus. Micro-CT suggested that bone mineral density (BMD), percent bone volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) were significantly increased in the treatment group, whereas trabecular separation (Tb.Sp) was significantly reduced. Thus, Rg1 promotes osteogenic differentiation by activating the GR/BMP-2 signaling pathway, enhances bone calcification, and ultimately accelerates the fracture healing in rats. PMID:27141994

  4. 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. PMID:26999642

  5. Ginsenoside Rg1 promotes osteogenic differentiation of rBMSCs and healing of rat tibial fractures through regulation of GR-dependent BMP-2/SMAD signaling

    PubMed Central

    Gu, Yanqing; Zhou, Jinchun; Wang, Qin; Fan, Weimin; Yin, Guoyong

    2016-01-01

    Fracture healing is closely related to the number and activity of bone marrow mesenchymal stem cells (BMSCs) near the fracture site. The present study was to investigate the effect of Rg1 on osteogenic differentiation of cultured BMSCs and related mechanisms and on the fracture healing in a fracture model. In vitro experiments showed that Rg1 promoted the proliferation and osteogenic differentiation of BMSCs. Western blot analyses demonstrated that Rg1 promoted osteogenic differentiation of BMSCs through the glucocorticoid receptor (GR)-dependent BMP-2/Smad signaling pathway. In vivo, X-ray examination showed that callus growth in rats treated with Rg1 was substantially faster than that in control rats after fracture. The results of H&E and Safranin-O/Fast Green staining revealed that, compared with controls, rats in the Rg1 treatment group had a significantly higher proportion of trabecular bone but a much lower proportion of fibers and cartilage components inside the callus. Micro-CT suggested that bone mineral density (BMD), percent bone volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) were significantly increased in the treatment group, whereas trabecular separation (Tb.Sp) was significantly reduced. Thus, Rg1 promotes osteogenic differentiation by activating the GR/BMP-2 signaling pathway, enhances bone calcification, and ultimately accelerates the fracture healing in rats. PMID:27141994

  6. Depletion of MEIS2 inhibits osteogenic differentiation potential of human dental stem cells

    PubMed Central

    Wu, Zhifang; Wang, Jinsong; Dong, Rui; Wang, Liping; Fan, Zhipeng; Liu, Dayong; Wang, Songlin

    2015-01-01

    Dental mesenchymal stem cells (MSCs) are a reliable and promising cell source for the regeneration of tooth,bone and other tissues . However, the molecular mechanisms underlying their differentiation are still largely unknown, which restricts their further wide application. Here, we investigate regulatory function of homeobox gene MEIS2 in the osteogenic differentiation potential of MSCs using stem cells from apical papilla (SCAPs) and dental pulp stem cells (DPSCs) by loss-of-function experiments. Our findings demonstrated that knockdown of MEIS2 in SCAPs and DPSCs decreased alkaline phosphatase (ALP) activity and mineralization, and inhibited the mRNA expression of ALP, bone sialoprotein (BSP), and osteocalcin (OCN). Besides, depletion of MEIS2 resulted in reduced expression of the key osteogenesis-related transcription factor, osterix (OSX) but not in the expression of runt-related transcription factor 2 (RUNX2). Furthermore, MEIS2 expression significantly increased during osteogenic induction and was strongly upregulated by BMP4 stimulation. Taken together, these results indicated that MEIS2 played an essential role in maintaining osteogenic differentiation potential of dental tissue- derived MSCs. These findings will provide new insights into the mechanisms underlying directed differentiation of MSCs, and identify a potential target gene in dental tissues derived MSCs for promoting the tissue regeneration. PMID:26221261

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

    SciTech Connect

    Salamon, Achim; Jonitz-Heincke, Anika; Adam, Stefanie; Rychly, Joachim; Müller-Hilke, Brigitte; Bader, Rainer; Lochner, Katrin; Peters, Kirsten

    2013-11-01

    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, and 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

  8. Effect of low-level diode laser on proliferation and osteogenic differentiation of dental pulp stem cells

    NASA Astrophysics Data System (ADS)

    Tabatabaei, Fahimeh S.; Torshabi, Maryam; Mojahedi Nasab, Masoud; Khosraviani, Keikhosro; Khojasteh, Arash

    2015-09-01

    This study assessed the effect of low-level laser irradiation (LLLI) on the proliferation and osteogenic differentiation of dental pulp stem cells (DPSCs). DPSCs were exposed to 810 nm laser light (0.1, 0.2, or 0.3 J cm-2) for 7 d (60 s daily). The negative control group (cells in regular medium) and positive control group (cells in osteogenic medium (OM)) were not lased. One group of cells in OM was irradiated with laser operated at 0.2 J cm-2. Cell viability was evaluated at 24 h and one week after the last day of laser irradiation using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Osteogenic differentiation was assessed using real-time reverse transcriptase polymerase chain reaction (RT-PCR) and alizarin Red S staining. Cell proliferation was not affected by laser irradiation at 24 h except in one group (cells in OM exposed to laser at 0.2 J cm-2). However, one week after the last day of laser irradiation, it was significantly increased in groups exposed to laser at 0.1 or 0.2 J cm-2 and decreased in groups containing OM (P  <  0.05). Osteoblast marker expression was observed in groups containing OM. LLLI at 0.2 J cm-2 dramatically enhanced cell differentiation. Laser at 0.3 J cm-2 increased bone sialoprotein (BSP) and decreased alkaline phosphatase (ALP). Mineralized nodules were only observed in groups containing OM. Considering these findings, LLLI may be used as a novel approach for preconditioning of DPSCs in vitro prior to bone tissue engineering.

  9. BMP‐9‐induced osteogenic differentiation of mesenchymal progenitors requires functional canonical Wnt/β‐catenin signalling

    PubMed Central

    Tang, Ni; Song, Wen‐Xin; Luo, Jinyong; Luo, Xiaoji; Chen, Jin; Sharff, Katie A.; Bi, Yang; He, Bai‐Cheng; Huang, Jia‐Yi; Zhu, Gao‐Hui; Su, Yu‐Xi; Jiang, Wei; Tang, Min; He, Yun; Wang, Yi; Chen, Liang; Zuo, Guo‐Wei; Shen, Jikun; Pan, Xiaochuan; Reid, Russell R.; Luu, Hue H.; Haydon, Rex C.

    2008-01-01

    Abstract Bone morphogenetic protein 9 (BMP‐9) is a member of the transforming growth factor (TGF)‐β/BMP superfamily, and we have demonstrated that it is one of the most potent BMPs to induce osteoblast differentiation of mesenchymal stem cells (MSCs). Here, we sought to investigate if canonical Wnt/β‐catenin signalling plays an important role in BMP‐9‐induced osteogenic differentiation of MSCs. Wnt3A and BMP‐9 enhanced each other’s ability to induce alkaline phosphatase (ALP) in MSCs and mouse embryonic fibroblasts (MEFs). Wnt antagonist FrzB was shown to inhibit BMP‐9‐induced ALP activity more effectively than Dkk1, whereas a secreted form of LPR‐5 or low‐density lipoprotein receptor‐related protein (LRP)‐6 exerted no inhibitory effect on BMP‐9‐induced ALP activity. β‐Catenin knockdown in MSCs and MEFs diminished BMP‐9‐induced ALP activity, and led to a decrease in BMP‐9‐induced osteocalcin reporter activity and BMP‐9‐induced expression of late osteogenic markers. Furthermore, β‐catenin knockdown or FrzB overexpression inhibited BMP‐9‐induced mineralization in vitro and ectopic bone formation in vivo, resulting in immature osteogenesis and the formation of chondrogenic matrix. Chromatin immunoprecipitation (ChIP) analysis indicated that BMP‐9 induced recruitment of both Runx2 and β‐catenin to the osteocalcin promoter. Thus, we have demonstrated that canonical Wnt signalling, possibly through interactions between β‐catenin and Runx2, plays an important role in BMP‐9‐induced osteogenic differentiation of MSCs. PMID:19175684

  10. Intermittent parathyroid hormone (1–34) application regulates cAMP-response element binding protein activity to promote the proliferation and osteogenic differentiation of bone mesenchymal stromal cells, via the cAMP/PKA signaling pathway

    PubMed Central

    CHEN, BAILING; LIN, TAO; YANG, XIAOXI; LI, YIQIANG; XIE, DENGHUI; CUI, HAOWEN

    2016-01-01

    The potential effects of intermittent parathyroid hormone (1–34) [PTH (1–34)] administration on bone formation have previously been investigated. A number of studies have suggested that the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway is associated with PTH-induced osteogenic differentiation. However, the precise signaling pathways and molecular mechanism by which PTH (1–34) induces the osteogenic differentiation of bone mesenchymal stromal cells (BMSCs) remain elusive. The purpose of the present study was to investigate the mechanism underlying the effect of intermittent PTH (1–34) application on the proliferation and osteogenic differentiation of BMSCs. BMSCs were randomly divided into four groups, as follows: Osteogenic medium (control group); osteogenic medium and intermittent PTH (1–34); osteogenic medium and intermittent PTH (1–34) plus the adenylyl cyclase activator forskolin; and osteogenic medium and intermittent PTH (1–34) plus the PKA inhibitor H-89. A cell proliferation assay revealed that PTH (1–34) stimulates BMSC proliferation via the cAMP/PKA pathway. Furthermore, reverse transcription-quantitative polymerase chain reaction, alkaline phosphatase activity testing and cell examination using Alizarin Red S staining demonstrated that PTH (1–34) administration promotes osteogenic differentiation and mineralization, mediated by the cAMP/PKA pathway. Crucially, the results of western blot analyses suggested that PTH (1–34) treatment and, to a greater degree, PTH (1–34) plus forskolin treatment caused an increase in phosphorylated cAMP response element binding protein (p-CREB) expression, but the effect of PTH on p-CREB expression was blocked by H-89. In conclusion, the current study demonstrated that intermittent PTH (1–34) administration regulates downstream proteins, particularly p-CREB, in the cAMP/PKA signaling pathway, to enhance the proliferation, osteogenic differentiation and mineralization of BMSCs

  11. [BMAL1 gene regulates the osteogenic differentiation of bone marrow mesenchymal stem cells].

    PubMed

    Xiaoguang, Li; Xiao-long, Guo; Bin, Guo

    2016-06-01

    Periodontitis is a chronic infective disease characterized as the destruction of the supporting tissues of the teeth. Bone marrow mesenchymal stem cells, which are ideal adult stem cells for the regeneration of supporting tissues, may play important roles in restoring the structure and function of the periodontium and in promoting the treatment of periodontal disease. As a consequence, the characteristics, especially osteogenic differentiation mechanism, of these stem cells have been extensively investigated. The regulation of the physiological behavior of these stem cells is associated with BMAL1 gene. This gene is a potential treatment target for periodontal disease, although the specific mechanism remains inconclusive. This study aimed to describe the characteristics of BMAL1 gene and its ability to regulate the osteogenic differentiation of stem cells. PMID:27526460

  12. Nanoclay-enriched poly(ɛ-caprolactone) electrospun scaffolds for osteogenic differentiation of human mesenchymal stem cells.

    PubMed

    Gaharwar, Akhilesh K; Mukundan, Shilpaa; Karaca, Elif; Dolatshahi-Pirouz, Alireza; Patel, Alpesh; Rangarajan, Kaushik; Mihaila, Silvia M; Iviglia, Giorgio; Zhang, Hongbin; Khademhosseini, Ali

    2014-08-01

    Musculoskeletal tissue engineering aims at repairing and regenerating damaged tissues using biological tissue substitutes. One approach to achieve this aim is to develop osteoconductive scaffolds that facilitate the formation of functional bone tissue. We have fabricated nanoclay-enriched electrospun poly(ɛ-caprolactone) (PCL) scaffolds for osteogenic differentiation of human mesenchymal stem cells (hMSCs). A range of electrospun scaffolds is fabricated by varying the nanoclay concentrations within the PCL scaffolds. The addition of nanoclay decreases fiber diameter and increases surface roughness of electrospun fibers. The enrichment of PCL scaffold with nanoclay promotes in vitro biomineralization when subjected to simulated body fluid (SBF), indicating bioactive characteristics of the hybrid scaffolds. The degradation rate of PCL increases due to the addition of nanoclay. In addition, a significant increase in crystallization temperature of PCL is also observed due to enhanced surface interactions between PCL and nanoclay. The effect of nanoclay on the mechanical properties of electrospun fibers is also evaluated. The feasibility of using nanoclay-enriched PCL scaffolds for tissue engineering applications is investigated in vitro using hMSCs. The nanoclay-enriched electrospun PCL scaffolds support hMSCs adhesion and proliferation. The addition of nanoclay significantly enhances osteogenic differentiation of hMSCs on the electrospun scaffolds as evident by an increase in alkaline phosphates activity of hMSCs and higher deposition of mineralized extracellular matrix compared to PCL scaffolds. Given its unique bioactive characteristics, nanoclay-enriched PCL fibrous scaffold may be used for musculoskeletal tissue engineering. PMID:24842693

  13. Nanoclay-Enriched Poly(ɛ-caprolactone) Electrospun Scaffolds for Osteogenic Differentiation of Human Mesenchymal Stem Cells

    PubMed Central

    Gaharwar, Akhilesh K.; Mukundan, Shilpaa; Karaca, Elif; Dolatshahi-Pirouz, Alireza; Patel, Alpesh; Rangarajan, Kaushik; Mihaila, Silvia M.; Iviglia, Giorgio; Zhang, Hongbin

    2014-01-01

    Musculoskeletal tissue engineering aims at repairing and regenerating damaged tissues using biological tissue substitutes. One approach to achieve this aim is to develop osteoconductive scaffolds that facilitate the formation of functional bone tissue. We have fabricated nanoclay-enriched electrospun poly(ɛ-caprolactone) (PCL) scaffolds for osteogenic differentiation of human mesenchymal stem cells (hMSCs). A range of electrospun scaffolds is fabricated by varying the nanoclay concentrations within the PCL scaffolds. The addition of nanoclay decreases fiber diameter and increases surface roughness of electrospun fibers. The enrichment of PCL scaffold with nanoclay promotes in vitro biomineralization when subjected to simulated body fluid (SBF), indicating bioactive characteristics of the hybrid scaffolds. The degradation rate of PCL increases due to the addition of nanoclay. In addition, a significant increase in crystallization temperature of PCL is also observed due to enhanced surface interactions between PCL and nanoclay. The effect of nanoclay on the mechanical properties of electrospun fibers is also evaluated. The feasibility of using nanoclay-enriched PCL scaffolds for tissue engineering applications is investigated in vitro using hMSCs. The nanoclay-enriched electrospun PCL scaffolds support hMSCs adhesion and proliferation. The addition of nanoclay significantly enhances osteogenic differentiation of hMSCs on the electrospun scaffolds as evident by an increase in alkaline phosphates activity of hMSCs and higher deposition of mineralized extracellular matrix compared to PCL scaffolds. Given its unique bioactive characteristics, nanoclay-enriched PCL fibrous scaffold may be used for musculoskeletal tissue engineering. PMID:24842693

  14. Hypoxia Suppresses Spontaneous Mineralization and Osteogenic Differentiation of Mesenchymal Stem Cells via IGFBP3 Up-Regulation.

    PubMed

    Kim, Ji Hye; Yoon, Sei Mee; Song, Sun U; Park, Sang Gyu; Kim, Won-Serk; Park, In Guk; Lee, Jinu; Sung, Jong-Hyuk

    2016-01-01

    Hypoxia has diverse stimulatory effects on human adipose-derived stem cells (ASCs). In the present study, we investigated whether hypoxic culture conditions (2% O₂) suppress spontaneous mineralization and osteogenic differentiation of ASCs. We also investigated signaling pathways and molecular mechanisms involved in this process. We found that hypoxia suppressed spontaneous mineralization and osteogenic differentiation of ASCs, and up-regulated mRNA and protein expression of Insulin-like growth factor binding proteins (IGFBPs) in ASCs. Although treatment with recombinant IGFBPs did not affect osteogenic differentiation of ASCs, siRNA-mediated inhibition of IGFBP3 attenuated hypoxia-suppressed osteogenic differentiation of ASCs. In contrast, overexpression of IGFBP3 via lentiviral vectors inhibited ASC osteogenic differentiation. These results indicate that hypoxia suppresses spontaneous mineralization and osteogenic differentiation of ASCs via intracellular IGFBP3 up-regulation. We determined that reactive oxygen species (ROS) generation followed by activation of the MAPK and PI3K/Akt pathways play pivotal roles in IGFBP3 expression under hypoxia. For example, ROS scavengers and inhibitors for MAPK and PI3K/Akt pathways attenuated the hypoxia-induced IGFBP3 expression. Inhibition of Elk1 and NF-κB through siRNA transfection also led to down-regulation of IGFBP3 mRNA expression. We next addressed the proliferative potential of ASCs with overexpressed IGFBP3, but IGFBP3 overexpression reduced the proliferation of ASCs. In addition, hypoxia reduced the osteogenic differentiation of bone marrow-derived clonal mesenchymal stem cells. Collectively, our results indicate that hypoxia suppresses the osteogenic differentiation of mesenchymal stem cells via IGFBP3 up-regulation. PMID:27563882

  15. Silver nanoparticles promote osteogenic differentiation of human urine-derived stem cells at noncytotoxic concentrations

    PubMed Central

    Qin, Hui; Zhu, Chen; An, Zhiquan; Jiang, Yao; Zhao, Yaochao; Wang, Jiaxin; Liu, Xin; Hui, Bing; Zhang, Xianlong; Wang, Yang

    2014-01-01

    In tissue engineering, urine-derived stem cells are ideal seed cells and silver nanoparticles (AgNPs) are perfect antimicrobial agents. Due to a distinct lack of information on the effects of AgNPs on urine-derived stem cells, a study was conducted to evaluate the effects of silver ions and AgNPs upon the cytotoxicity and osteogenic differentiation of urine-derived stem cells. Initially, AgNPs or AgNO3 were exposed to urine-derived stem cells for 24 hours. Cytotoxicity was measured using the Cell Counting kit-8 (CCK-8) test. The effects of AgNPs or AgNO3 at the maximum safety concentration determined by the CCK-8 test on osteogenic differentiation of urine-derived stem cells were assessed by alkaline phosphatase activity, Alizarin Red S staining, and the quantitative reverse transcription polymerase chain reaction. Lastly, the effects of AgNPs or AgNO3 on “urine-derived stem cell actin cytoskeleton organization” and RhoA activity were assessed by rhodamine-phalloidin staining and Western blotting. Concentration-dependent toxicity was observed starting at an AgNO3 concentration of 2 μg/mL and at an AgNP concentration of 4 μg/mL. At these concentrations, AgNPs were observed to promote osteogenic differentiation of urine-derived stem cells, induce actin polymerization and increase cytoskeletal tension, and activate RhoA; AgNO3 had no such effects. In conclusion, AgNPs can promote osteogenic differentiation of urine-derived stem cells at a suitable concentration, independently of silver ions, and are suitable for incorporation into tissue-engineered scaffolds that utilize urine-derived stem cells as seed cells. PMID:24899804

  16. Differentially circulating miRNAs after recent osteoporotic fractures can influence osteogenic differentiation.

    PubMed

    Weilner, Sylvia; Skalicky, Susanna; Salzer, Benjamin; Keider, Verena; Wagner, Michael; Hildner, Florian; Gabriel, Christian; Dovjak, Peter; Pietschmann, Peter; Grillari-Voglauer, Regina; Grillari, Johannes; Hackl, Matthias

    2015-10-01

    Osteoporosis is the consequence of altered bone metabolism resulting in the systemic reduction of bone strength and increased risk of fragility fractures. MicroRNAs (miRNAs) regulate gene expression on a post-transcriptional level and are known to take part in the control of bone formation and bone resorption. In addition, it is known that miRNAs are secreted by many cell types and can transfer "messages" to recipient cells. Thus, circulating miRNAs might not only be useful as surrogate biomarkers for the diagnosis or prognosis of pathological conditions, but could be actively modulating tissue physiology. Therefore, the aim of this study was to test whether circulating miRNAs that exhibit changes in recent osteoporotic fracture patients could be causally related to bone metabolism. In the first step we performed an explorative analysis of 175 miRNAs in serum samples obtained from 7 female patients with recent osteoporotic fractures at the femoral neck, and 7 age-matched female controls. Unsupervised cluster analysis revealed a high discriminatory power of the top 10 circulating miRNAs for patients with recent osteoporotic fractures. In total 6 miRNAs, miR-10a-5p, miR-10b-5p, miR-133b, miR-22-3p, miR-328-3p, and let-7g-5p exhibited significantly different serum levels in response to fracture (adjusted p-value<0.05). These miRNAs were subsequently analyzed in a validation cohort of 23 patients (11 control, 12 fracture), which confirmed significant regulation for miR-22-3p, miR-328-3p, and let-7g-5p. A set of these and of other miRNAs known to change in the context of osteoporotic fractures were subsequently tested for their effects on osteogenic differentiation of human mesenchymal stem cells (MSCs) in vitro. The results show that 5 out of 7 tested miRNAs can modulate osteogenic differentiation of MSCs in vitro. Overall, these data suggest that levels of specific circulating miRNAs change in the context of recent osteoporotic fractures and that such perturbations of

  17. Calcium phosphate-bearing matrices induce osteogenic differentiation of stem cells through adenosine signaling

    PubMed Central

    Shih, Yu-Ru V.; Hwang, YongSung; Phadke, Ameya; Kang, Heemin; Hwang, Nathaniel S.; Caro, Eduardo J.; Nguyen, Steven; Siu, Michael; Theodorakis, Emmanuel A.; Gianneschi, Nathan C.; Vecchio, Kenneth S.; Chien, Shu; Lee, Oscar K.; Varghese, Shyni

    2014-01-01

    Synthetic matrices emulating the physicochemical properties of tissue-specific ECMs are being developed at a rapid pace to regulate stem cell fate. Biomaterials containing calcium phosphate (CaP) moieties have been shown to support osteogenic differentiation of stem and progenitor cells and bone tissue formation. By using a mineralized synthetic matrix mimicking a CaP-rich bone microenvironment, we examine a molecular mechanism through which CaP minerals induce osteogenesis of human mesenchymal stem cells with an emphasis on phosphate metabolism. Our studies show that extracellular phosphate uptake through solute carrier family 20 (phosphate transporter), member 1 (SLC20a1) supports osteogenic differentiation of human mesenchymal stem cells via adenosine, an ATP metabolite, which acts as an autocrine/paracrine signaling molecule through A2b adenosine receptor. Perturbation of SLC20a1 abrogates osteogenic differentiation by decreasing intramitochondrial phosphate and ATP synthesis. Collectively, this study offers the demonstration of a previously unknown mechanism for the beneficial role of CaP biomaterials in bone repair and the role of phosphate ions in bone physiology and regeneration. These findings also begin to shed light on the role of ATP metabolism in bone homeostasis, which may be exploited to treat bone metabolic diseases. PMID:24395775

  18. 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. PMID:27157722

  19. 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

  20. 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. PMID:26219349

  1. Simulated microgravity inhibits osteogenic differentiation of mesenchymal stem cells through down regulating the transcriptional co-activator TAZ.

    PubMed

    Chen, Zhe; Luo, Qing; Lin, Chuanchuan; Song, Guanbin

    Microgravity induces observed bone loss in space flight or simulated experiments, while the mechanism underlying it is still obscure. Here, we utilized a clinostat to model simulated microgravity (SMG) and found that SMG obviously inhibited osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs). We detected that SMG dramatically inhibited the expression of the transcriptional coactivator with PDZ-binding motif (TAZ), which acts as a vital regulator of osteogenesis. Interestingly, we found that lysophosphatidic acid (LPA) could activate TAZ and retain osteogenic differentiation of BMSCs under SMG. Our data further demonstrated that depletion of TAZ by siRNA blocked the LPA-induced increase in osteogenic differentiation of BMSCs under SMG. Moreover, Y27632 (the Rock inhibitor) abrogated the activation of TAZ and the increased osteogenic differentiation induced by LPA. Taken together, we propose that microgravity inhibits osteogenic differentiation of BMSCs due to decreased TAZ expression and that LPA can efficiently reverse the reduced osteogenic differentiation via the Rock-TAZ pathway. PMID:26549225

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

    PubMed

    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

  3. 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.

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

    PubMed

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

    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

  5. 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

  6. Evaluation of the growth and osteogenic differentiation of ASCs cultured with PL and seeded on PLGA scaffolds.

    PubMed

    Awidi, Abdalla; Ababneh, Nidaa; Alkilani, Hussein; Salah, Bariqa; Nazzal, Shymaa; Zoghool, Maisaa; Shomaf, Maha

    2015-02-01

    Scaffold serves as an important component of tissue engineering, which facilitates cell attachment, proliferation and differentiation of cultured cells. In this study we aimed to use platelet lysates as a substitute for FBS in culturing and proliferation of human adipose tissue-derived stromal cells (ASCs), which constitute a promising source for cell therapy. We characterized ASCs in the presence of PL, and then we seeded them onto poly(lactic-co-glycolic acid) (PLGA) scaffolds, osteogenic media was used to induce their proliferation and osteogenic differentiation. Gene expression analysis revealed higher expression of osteogenic related genes, immunohistochemical staining showed proper cell attachment, growth and collagen matrix formation with the ability to induce vascularization. In conclusion, expansion of ASCs in PL-supplemented medium could promote cell proliferation and osteogenic differentiation of cells seeded on PLGA scaffolds, therefore it could be considered as a suitable and effective substitute for FBS to be used in clinical applications. PMID:25644098

  7. Titania-hydroxyapatite nanocomposite coatings support human mesenchymal stem cells osteogenic differentiation.

    PubMed

    Dimitrievska, Sashka; Bureau, Martin N; Antoniou, John; Mwale, Fackson; Petit, Alain; Lima, Rogerio S; Marple, Basil R

    2011-09-15

    In addition to mechanical and chemical stability, the third design goal of the ideal bone-implant coating is the ability to support osteogenic differentiation of mesenchymal stem cells (MSCs). Plasma-sprayed TiO(2)-based bone-implant coatings exhibit excellent long-term mechanical properties, but their applications in bone implants are limited by their bioinertness. We have successfully produced a TiO(2) nanostructured (grain size <50 nm) based coating charged with 10% wt hydroxyapatite (TiO(2)-HA) sprayed by high-velocity oxy-fuel. On Ti64 substrates, the novel TiO(2)-HA coating bond 153× stronger and has a cohesive strength 4× higher than HA coatings. The HA micro- and nano-sized particles covering the TiO(2)-HA coating surface are chemically bound to the TiO(2) coating matrix, producing chemically stable coatings under high mechanical solicitations. In this study, we elucidated the TiO(2)-HA nanocomposite coating surface chemistry, and in vitro osteoinductive potential by culturing human MSCs (hMSCs) in basal and in osteogenic medium (hMSC-ob). We assessed the following hMSCs and hMSC-ob parameters over a 3-week period: (i) proliferation; (ii) cytoskeleton organization and cell-substrate adhesion; (iii) coating-cellular interaction morphology and growth; and (iv) cellular mineralization. The TiO(2) -HA nanocomposite coatings demonstrated 3× higher hydrophilicity than HA coatings, a TiO(2)-nanostructured surface in addition to the chemically bound HA micron- and nano-sized rod to the surface. hMSCs and hMSC-ob demonstrated increased proliferation and osteoblastic differentiation on the nanostructured TiO(2)-HA coatings, suggesting the TiO(2)-HA coatings nanostructure surface properties induce osteogenic differentiation of hMSC and support hMSC-ob osteogenic potential better than our current golden standard HA coating. PMID:21702080

  8. Commitment of the teratocarcinoma-derived mesodermal clone C1 towards terminal osteogenic differentiation.

    PubMed

    Poliard, A; Lamblin, D; Marie, P J; Buc-Caron, M H; Kellermann, O

    1993-10-01

    The mesodermal clone C1 was derived from the multipotent embryonal carcinoma 1003 cell line transformed with the plasmid pK4 carrying SV40 oncogenes under the control of the adenovirus E1A promoter. We have shown that the C1 clone becomes committed to the osteogenic pathway when cultured in aggregates in the presence of mediators of the osteogenic differentiation. To further validate C1 as a model with which to study osteogenesis in vitro the kinetics of its differentiation was studied, focusing on the histology of the aggregates and on the expression of a set of genes corresponding to representative bone matrix proteins. The presence of ascorbic acid and beta- glycerophosphate specifically leads to mineralization in almost 100% of the aggregates. Transcription of the above genes, silent in exponentially growing cells, specifically occurred with the establishment of cell-cell contacts independently of the presence of ascorbic acid and inorganic phosphate. The latter, however, were absolutely required for matrix deposition and mineralization. In their presence, one observed an overall decline in type I collagen and alkaline phosphatase transcripts while osteocalcin and osteopontin transcripts preferentially accumulated in cells lining the mineralizing foci. Concomitantly, type I collagen and osteocalcin became extracellularly deposited. The osteogenic differentiation of C1 occurred while cells were still proliferating. The C1 clone thus behaves as a mesodermal stem cell, becoming committed to the osteogenic pathway upon: firstly, establishment of cellular contacts; and secondly, addition of ascorbate and beta-glycerophosphate. It therefore appears to be a promising in vitro system for deciphering the molecular basis of osteoblast ontogeny.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8282757

  9. Simvastatin induces the osteogenic differentiation of human periodontal ligament stem cells.

    PubMed

    Zhao, Bing-jiao; Liu, Yue-hua

    2014-10-01

    Periodontal ligament stem cells (PDLSCs) are considered as potential mesenchymal stem cell sources for future clinical applications in periodontal regeneration therapy. Simvastation, widely used for lowering serum cholesterol, is known to have a bone stimulatory effect. However, it is not clear whether simvastation affects the differentiation of PDLSCs. This study examined the effects of simvastatin on human PDLSCs in vitro and in vivo. Using the limiting dilution technique, human PDLSCs were isolated and expanded. PDLSCs were cultured with simvastatin (0.01-10 μM), and the proliferation was measured. The osteogenic differentiation was characterized by alkaline phosphatase (ALP) activity and Alizarin Red-S staining for calcium deposition. The gene expression levels of osteogenic markers were evaluated by RT-PCR. In addition, PDLSCs were transplanted into nude mice with ceramic bovine bone powders as carriers to observe the capacity of mineralized tissue formation in vivo. Simvastatin at concentrations <1 μM did not suppress the proliferation of PDLSCs. After the administration of 0.1 μM simvastatin, the expression of ALP, bone sialoprotein, and bone morphogenetic protein-2 genes were significantly upregulated, and the ALP activity and mineralized nodule formation were significantly higher in the simvastatin-treated cells than the control cells. In addition, the in vivo transplantation results showed that simvastatin treatment promoted the degree of mineralized tissue formation. Collectively, simvastatin has positive effects on osteogenic differentiation of human PDLSCs in vitro and in vivo. This suggests that simvastatin might be a useful osteogenic induction agent for periodontal bone regeneration. PMID:24112098

  10. Titanium phosphate glass microcarriers induce enhanced osteogenic cell proliferation and human mesenchymal stem cell protein expression

    PubMed Central

    Lakhkar, Nilay J; M Day, Richard; Kim, Hae-Won; Ludka, Katarzyna; Mordan, Nicola J; Salih, Vehid; Knowles, Jonathan C

    2015-01-01

    In this study, we have developed 50- to 100-µm-sized titanium phosphate glass microcarriers (denoted as Ti5) that show enhanced proliferation of human mesenchymal stem cells and MG63 osteosarcoma cells, as well as enhanced human mesenchymal stem cell expression of bone differentiation markers, in comparison with commercially available glass microspheres at all time points. We also demonstrate that these microcarriers provide superior human mesenchymal stem cell proliferation with conventional Dulbecco’s Modified Eagle medium than with a specially developed commercial stem cell medium. The microcarrier proliferative capacity is revealed by a 24-fold increase in MG63 cell numbers in spinner flask bioreactor studies performed over a 7-day period, versus only a 6-fold increase in control microspheres under the same conditions; the corresponding values of Ti5 and control microspheres under static culture are 8-fold and 7-fold, respectively. The capability of guided osteogenic differentiation is confirmed by ELISAs for bone morphogenetic protein-2 and osteopontin, which reveal significantly greater expression of these markers, especially osteopontin, by human mesenchymal stem cells on the Ti5 microspheres than on the control. Scanning electron microscopy and confocal laser scanning microscopy images reveal favorable MG63 and human mesenchymal stem cell adhesion on the Ti5 microsphere surfaces. Thus, the results demonstrate the suitability of the developed microspheres for use as microcarriers in bone tissue engineering applications. PMID:26668711

  11. PPARγ and Wnt Signaling in Adipogenic and Osteogenic Differentiation of Mesenchymal Stem Cells.

    PubMed

    Yuan, Zongyi; Li, Qing; Luo, Shihong; Liu, Zhi; Luo, Daowen; Zhang, Bo; Zhang, Dongdong; Rao, Pengcheng; Xiao, Jingang

    2016-01-01

    Mesenchymal stem cells (MSCs) arise from a variety of tissues, including bone marrow and adipose tissue and, accordingly, have the potential to differentiate into multiple cell types, including osteoblasts and adipocytes. Research on MSCs to date has demonstrated that a large number of transcription factors and ectocytic or intrastitial signaling pathways regulate adipogenic and osteogenic differentiation. A theoretical inverse relationship exists in adipogenic and osteogenic lineage commitment and differentiation, such that signaling pathways induce adipogenesis at the expense of osteogenesis and vice versa. For example, peroxisome proliferator-activated receptor γ(PPARγ), which belongs to the nuclear hormone receptor superfamily of ligand-activated transcription factors, is known to function as a master transcriptional regulator of adipocyte differentiation, and inhibit osteoblast differentiation. Moreover, recent studies have demonstrated that inducers of osteogenic differentiation, such as bone morphogenetic protein (BMP) and Wnt, inhibit the function of PPARγ transactivation during MSC differentiation towards adipocytes through a variety of mechanisms. To illustrate this, the canonical Wnt/β-catenin pathway represses expression of PPARγ mRNA, whereas the noncanonical Wnt pathway activates histone methyltransferases that inhibit PPARγ transactivation via histone H3 lysine 9 (H3K9) methylation of its target genes. The role of microRNAs (miRNAs) in adipogenesis and osteoblastogenesis is garnering increased attention, and studies in this area have shed light on the integration of miRNAs with Wnt signaling and transcription factors such as Runx2 and PPARγ. This review summarizes our current understanding of the mechanistic basis of these signaling pathways, and indicates future clinical applications for stem cell-based cell transplantation and regenerative therapy. PMID:25986621

  12. Reconciling the effects of inflammatory cytokines on mesenchymal cell osteogenic differentiation

    PubMed Central

    Deshpande, Sagar; James, Aaron W.; Blough, Jordan; Donneys, Alexis; Wang, Stewart C.; Cederna, Paul S.; Buchman, Steven R.; Levi, Benjamin

    2015-01-01

    Therapies using mesenchymal stem cells are a popular current avenue for development and utilization, especially in the fields of de novo tissue engineering (Sanchez-Ramos J, Song S, Cardozo-Pelaez F, et al. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol 2000;164:247.) or tissue regeneration after physical injury (Kitoh H, Kitakoji T, Tsuchiya H, et al. Transplantation of marrow-derived mesenchymal stem cells and platelet-rich plasma during distraction osteogenesis—a preliminary result of three cases. Bone 2004;35:892; Shumakov VI, Onishchenko NA, Rasulov MF, Krasheninnikov ME, Zaidenov VA. Mesenchymal bone marrow stem cells more effectively stimulate regeneration of deep burn wounds than embryonic fibroblasts. Bull Exp Biol Med 2003;136:192; Bruder SP, Fink DJ, Caplan AI. Mesenchymal stem cells in bone development, bone repair, and skeletal regeneration therapy. J Cell Biochem 1994;56:283.). The osteogenic potential of these cells is of particular interest, given their recent usage for the closure of critical-sized bone defects and other nonhealing bone scenarios such as a nonunion. Recent literature suggests that inflammatory cytokines can significantly impact the osteogenic potential of these cells. A review of relevant, recent literature is presented regarding the impact of the inflammatory cascade on the osteogenic differentiation of these cells and how this varies across species. Finally, we identify areas of conflicting or absent evidence regarding the behavior of mesenchymal stem cells in response to inflammatory cytokines. PMID:23972621

  13. MicroRNAs regulate signaling pathways in osteogenic differentiation of mesenchymal stem cells (Review)

    PubMed Central

    PENG, SHUPING; GAO, DAN; GAO, CHENGDE; WEI, PINGPIN; NIU, MAN; SHUAI, CIJUN

    2016-01-01

    Osteogenesis is a complex multi-step process involving the differentiation of mesenchymal stem cells (MSCs) into osteoblast progenitor cells, preosteoblasts, osteoblasts and osteocytes, and the crosstalk between multiple cell types for the formation and remodeling of bone. The signaling regulatory networks during osteogenesis include various components, including growth factors, transcription factors, micro (mi)RNAs and effectors, a number of which form feedback loops controlling the balance of osteogenic differentiation by positive or negative regulation. miRNAs have been found to be important regulators of osteogenic signaling pathways in multiple aspects and multiple signaling pathways. The present review focusses on the progress in elucidating the role of miRNA in the osteogenesis signaling networks of MSCs as a substitute for bone implantation the the field of bone tissue engineering. In particular, the review classifies which miRNAs promote or suppress the osteogenic process, and summarizes which signaling pathway these miRNAs are involved in. Improvements in knowledge of the characteristics of miRNAs in osteogenesis provide an important step for their application in translational investigations of bone tissue engineering and bone disease. PMID:27222009

  14. In vitro proliferation and osteogenic differentiation of mesenchymal stem cells on nanoporous alumina

    PubMed Central

    Song, Yuanhui; Ju, Yang; Song, Guanbin; Morita, Yasuyuki

    2013-01-01

    Cell adhesion, migration, and proliferation are significantly affected by the surface topography of the substrates on which the cells are cultured. Alumina is one of the most popular implant materials used in orthopedics, but few data are available concerning the cellular responses of mesenchymal stem cells (MSCs) grown on nanoporous structures. MSCs were cultured on smooth alumina substrates and nanoporous alumina substrates to investigate the interaction between surface topographies of nanoporous alumina and cellular behavior. Nanoporous alumina substrates with pore sizes of 20 nm and 100 nm were used to evaluate the effect of pore size on MSCs as measured by proliferation, morphology, expression of integrin β1, and osteogenic differentiation. An MTT assay was used to measure cell viability of MSCs on different substrates, and determined that cell viability decreased with increasing pore size. Scanning electron microscopy was used to investigate the effect of pore size on cell morphology. Extremely elongated cells and prominent cell membrane protrusions were observed in cells cultured on alumina with the larger pore size. The expression of integrin β1 was enhanced in MSCs cultured on porous alumina, revealing that porous alumina substrates were more favorable for cell growth than smooth alumina substrates. Higher levels of osteoblastic differentiation markers such as alkaline phosphatase, osteocalcin, and mineralization were detected in cells cultured on alumina with 100 nm pores compared with cells cultured on alumina with either 20 nm pores or smooth alumina. This work demonstrates that cellular behavior is affected by variation in pore size, providing new insight into the potential application of this novel biocompatible material for the developing field of tissue engineering. PMID:23935364

  15. Effect of molecular weight and concentration of hyaluronan on cell proliferation and osteogenic differentiation in vitro

    SciTech Connect

    Zhao, Ningbo Wang, Xin Qin, Lei Guo, Zhengze Li, Dehua

    2015-09-25

    Hyaluronan (HA), the simplest glycosaminoglycan and a major component of the extracellular matrix, exists in various tissues. It is involved in some critical biological procedures, including cellular signaling, cell adhesion and proliferation, and cell differentiation. The effect of molecular weight (MW) and concentration of HA on cell proliferation and differentiation was controversial. In this study, we investigated the effect of MW and concentration of HA on the proliferation and osteogenic differentiation of rabbit bone marrow-derived stem cells in vitro. Results showed that high MW HA decreased the cell adhesion rate in a concentration-dependant manner. The cell adhesion rate was decreased by increasing MW of HA. Cell proliferation was significantly enhanced by low MW HA (P < 0.05). The factorial analysis indicated that MW and concentration had an interactive effect on the cell adhesion rate and cell proliferation (P < 0.05). High MW HA increased the mRNA expressions of ALP, RUNX-2 and OCN. The higher the MW was, the higher the mRNA expressions were. The factorial analysis indicated that MW and concentration had an interactive effect on ALP mRNA expression (P < 0.05). HA of higher MW and higher concentration promoted bone formation. These findings provide some useful information in understanding the mechanism underlying the effect of MW and concentration of HA on cell proliferation and differentiation. - Highlights: • Effect of hyaluronan on cell proliferation and differentiation is evaluated in vitro. • Hyaluronan of low molecular weight increases cell proliferation. • Hyaluronan of high molecular weight promotes cell osteogenic differentiation. • Molecular weight and concentration of hyaluronan show interactive effect.

  16. γ-Secretase inhibitor reverts the Notch signaling attenuation of osteogenic differentiation in aged bone marrow mesenchymal stem cells.

    PubMed

    Tang, Zhaolong; Wei, Junjun; Yu, Yunbo; Zhang, Jiankang; Liu, Lei; Tang, Wei; Long, Jie; Zheng, Xiaohui; Jing, Wei

    2016-04-01

    The age-related changes in cell viability and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) play pivotal roles in the fracture healing process, especially in geriatric individuals. This study was designed to explore the age-related changes in murine BMSCs and the regulation of osteogenic differentiation in aged BMSCs in vitro. Notch signaling pathway took part in the regulation of osteogensis, while the relationship between Notch and the osteogenic differentiation in aged BMSCs has not been reported yet. BMSCs harvested from the bone marrow of young, adult, and aged C57BL/6 mice were cultured in osteogenic and adipogenic differentiation media. Histochemical staining results indicated that the osteogenic ability of BMSCs gradually decreased with aging, whereas the adipogenic ability increased. Cell activity assays showed that the proliferative and migrated capacity did not decline with aging significantly. According to real-time PCR and Western blotting results, the aged cells exhibited higher Notch signaling expression level than the younger ones did. After the aged BMSCs being treated with γ-secretase inhibitor, however, Notch activity was changed and the aging-imparied osteogenic ability reverted to a normal level. This study demonstrated that the decreased bone formation capacity in aged BMSCs had relationship with the transdifferentiation between osteogenesis and adipogenesis, which would be regulated by Notch signaling pathway and the attenuated osteogenesis in aged BMSCs could be promoted when the inhibition of Notch pathway. PMID:26801333

  17. Enhanced osteogenic activity of a poly(butylene succinate)/calcium phosphate composite by simple alkaline hydrolysis.

    PubMed

    Arphavasin, Suphakit; Singhatanadgit, Weerachai; Ngamviriyavong, Patcharee; Janvikul, Wanida; Meesap, Preeyapan; Patntirapong, Somying

    2013-10-01

    Bone engineering offers the prospect of alternative therapies for clinically relevant skeletal defects. Poly(butylene succinate) (PBSu) is a biodegradable and biocompatible polyester which may possess some limitations in clinical use due to its hydrophobicity. In order to overcome these limitations and increase the bioactivity, a simple and convenient surface hydrolysis of PBSu, PBSu/hydroxyapatite and PBSu/β-tricalcium phosphate (TCP) films was performed. The resulting surfaces (i.e., HPBSu, HPBSu/HA and HPBSu/TCP) were tested for their physicochemical property, biocompatibility and osteogenic potency. The results showed that surface hydrolysis significantly increased surface roughness and hydrophilicity of the composites, with the HPBSu/TCP possessing the most pronounced results. All the materials appeared to be biocompatible and supported in vitro growth and osteoblast differentiation of hMSCs, and the alkaline hydrolysis significantly enhanced the hMSC cell proliferation and the osteogenic potency of PBSu/TCP compared with the non-hydrolyzed sample. In conclusion, the HPBSu/TCP possessed better hydrophilicity, biocompatibility and osteogenic potency in vitro, suggesting that this simple and convenient alkaline hydrolysis could be used to augment the biological property of PBSu-based composites for bone engineering in vivo. PMID:24057872

  18. Hydroxyapatite nanoparticle injectable hydrogel scaffold to support osteogenic differentiation of human mesenchymal stem cells.

    PubMed

    Thorpe, A A; Creasey, S; Sammon, C; Le Maitre, C L

    2016-01-01

    Bone loss associated with degenerative disease and trauma is a clinical problem increasing with the aging population. Thus, effective bone augmentation strategies are required; however, many have the disadvantages that they require invasive surgery and often the addition of expensive growth factors to induce osteoblast differentiation. Here, we investigated a LaponiteÒ crosslinked, pNIPAM-DMAc copolymer (L-pNIPAM-co-DMAc) hydrogel with hydroxyapatite nanoparticles (HAPna), which can be maintained as a liquid ex vivo, injected via narrow-gauge needle into affected bone, followed by in situ gelation to deliver and induce osteogenic differentiation of human mesenchymal stem cells (hMSC). L-pNIPAM-co-DMAc hydrogels were synthesised and HAPna added post polymerisation. Commercial hMSCs from one donor (Lonza) were incorporated in liquid hydrogel, the mixture solidified and cultured for up to 6 weeks. Viability of hMSCs was maintained within hydrogel constructs containing 0.5 mg/mL HAPna. SEM analysis demonstrated matrix deposition in cellular hydrogels which were absent in acellular controls. A significant increase in storage modulus (G') was observed in cellular hydrogels with 0.5 mg/mL HAPna. Semi-quantitative immunohistochemistry and histological analysis demonstrated that bone differentiation markers and collagen deposition was induced within 48 h, with increased calcium deposition with time. The thermally triggered hydrogel system, described here, was sufficient without the need of additional growth factors or osteogenic media to induce osteogenic differentiation of commercial hMSCs. Preliminary data presented here will be expanded on multiple patient samples to ensure differentiation is seen in these samples. This system could potentially reduce treatment costs and simplify the treatment strategy for orthopaedic repair and regeneration. PMID:27377664

  19. Nanosensors for Continuous and Noninvasive Monitoring of Mesenchymal Stem Cell Osteogenic Differentiation.

    PubMed

    Wiraja, Christian; Yeo, David C; Chong, Mark S K; Xu, Chenjie

    2016-03-01

    Assessing mesenchymal stem cell (MSC) differentiation status is crucial to verify therapeutic efficacy and optimize treatment procedures. Currently, this involves destructive methods including antibody-based protein detection and polymerase chain reaction gene analysis, or laborious and technically challenging genetic reporters. Development of noninvasive methods for real-time differentiation status assessment can greatly benefit MSC-based therapies. This report introduces a nanoparticle-based sensing platform that encapsulates two molecular beacon (MB) probes within the same biodegradable polymeric nanoparticles. One MB targets housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference, while another detects alkaline phosphatase (ALP), a functional biomarker. Following internalization, MBs are gradually released as the nanoparticle degrades. GAPDH MBs provide a stable reference signal throughout the monitoring period (18 days) regardless of differentiation induction. Meanwhile, ALP mRNA undergoes well-defined dynamics with peak expression observed during early stages of osteogenic differentiation. By normalizing ALP-MB signal with GAPDH-MB, changes in ALP expression can be monitored, to noninvasively validate osteogenic differentiation. As proof-of-concept, a dual-colored nanosensor is applied to validate MSC osteogenesis on 2D culture and polycaprolactone films containing osteo-inductive tricalcium phospate. PMID:26756453

  20. Live-Cell, Temporal Gene Expression Analysis of Osteogenic Differentiation in Adipose-Derived Stem Cells

    PubMed Central

    Desai, Hetal V.; Voruganti, Indu S.; Jayasuriya, Chathuraka; Chen, Qian

    2014-01-01

    Adipose-derived stem cells (ASCs) are a widely investigated type of mesenchymal stem cells with great potential for musculoskeletal regeneration. However, the use of ASCs is complicated by their cellular heterogeneity, which exists at both the population and single-cell levels. This study demonstrates a live-cell assay to investigate gene expression in ASCs undergoing osteogenesis using fluorescently tagged DNA hybridization probes called molecular beacons. A molecular beacon was designed to target the mRNA sequence for alkaline phosphatase (ALPL), a gene characteristically expressed during early osteogenesis. The percentage of cells expressing this gene in a population was monitored daily to quantify the uniformity of the differentiation process. Differentiating ASC populations were repeatedly measured in a nondestructive fashion over a 10-day period to obtain temporal gene expression data. Results showed consistent expression patterns for the investigated osteogenic genes in response to induction medium. Peak signal level, indicating when the most cells expressed ALPL at once, was observed on days 3–5. The differentiation response of sample populations was generally uniform when assessed on a well-by-well basis over time. The expression of alkaline phosphatase is consistent with previous studies of osteogenic differentiation, suggesting that molecular beacons are a viable means of monitoring the spatiotemporal gene expression of live, differentiating ASCs. PMID:24367991

  1. A bioceramic with enhanced osteogenic properties to regulate the function of osteoblastic and osteocalastic cells for bone tissue regeneration.

    PubMed

    Roohani-Esfahani, Seyed-Iman; No, Young Jung; Lu, Zufu; Ng, Pei Ying; Chen, Yongjuan; Shi, Jeffrey; Pavlos, Nathan J; Zreiqat, Hala

    2016-01-01

    Bioceramics for regenerative medicine applications should have the ability to promote adhesion, proliferation and differentiation of osteoblast and osteoclast cells. Osteogenic properties of the material are essential for rapid bone regeneration and new bone formation. The aim of this study was to develop a silicate-based ceramic, gehlenite (GLN, Ca2Al2SiO7), and characterise its physiochemical, biocompatibility and osteogenic properties. A pure GLN powder was synthesised by a facile reactive sintering method and compacted to disc-shaped specimens. The sintering behaviour and degradation of the GLN discs in various buffer solutions were fully characterised. The cytotoxicity of GLN was evaluated by direct and indirect methods. In the indirect method, primary human osteoblast cells (HOBs) were exposed to diluted extracts (100, 50, 25, 12.5 and 6.25 mg ml(-1)) of fine GLN particles in culture medium. The results showed that the extracts did not cause any cytotoxic effect on the HOBs with the number of cells increasing significantly from day 1 to day 7. GLN-supported HOB attachment and proliferation, and significantly enhanced osteogenic gene expression levels (Runx2, osteocalcin, osteopontin and bone sialoprotein) were compared with biphasic calcium phosphate groups (BCP, a mixture of hydroxyapatite (60wt.%) and β-tricalcium phosphate(40wt.%)). We also demonstrated that in addition to supporting HOB attachment and proliferation, GLN promoted the formation of tartrate-acid resistance phosphatase (TRAP) positive multinucleated osteoclastic cells (OCs) derived from mouse bone marrow cells. Results also demonstrated the ability of GLN to support the polarisation of OCs, a prerequisite for their functional resorptive activity which is mainly influenced by the composition and degradability of biomaterials. Overall, the developed GLN is a prospective candidate to be used in bone regeneration applications due its effective osteogenic properties and biocompatibility. PMID

  2. Differential regulation of osteogenic marker gene expression by Wnt-3a in embryonic mesenchymal multipotential progenitor cells.

    PubMed

    Derfoul, Assia; Carlberg, Alyssa L; Tuan, Rocky S; Hall, David J

    2004-06-01

    The Wnt family of secreted glycoproteins plays an integral role in embryonic development and differentiation. To explore the role of Wnt's in one aspect of differentiation, namely osteogenesis, we employed a retroviral gene transfer approach to express Wnt-3a in the multipotent murine embryonic mesenchymal cell line C3H10T1/2. We found that expression of Wnt-3a in these cells had a significant, positive effect on cell growth in serum-containing medium, in that the cells grew to very high densities compared to the control cells. Additionally, apoptosis was markedly inhibited by Wnt-3a. However, when the cells were grown in serum-deficient medium, the Wnt-3a-expressing cells arrested efficiently in G1 phase, indicating that serum growth factors were needed in addition to Wnt-3a for enhanced proliferation. Wnt-3a-expressing cells exhibited high levels of alkaline phosphatase gene expression and enzymatic activity, but did not show any matrix mineralization. Unexpectedly, basal expression of bone sialoprotein, osteocalcin, and osteopontin were markedly inhibited by Wnt-3a, as were other known target genes of Wnt-3a, such as Brachyury, FGF-10, and Cdx1. When Wnt-3a-expressing cells were treated with osteogenic supplements in the presence of BMP-2, alkaline phosphatase gene expression and activity were further elevated. Additionally, BMP-2 was able to reverse the inhibitory effect of Wnt-3a on osteocalcin and osteopontin gene expression. These results indicate that while Wnt-3a represses basal expression of some osteogenic genes, this repression can be partially reversed by BMP-2. Finally, the enhanced gene expression of alkaline phosphatase induced by Wnt-3a could be effectively suppressed by the combined action of dexamethasone and 1,25-dihydroxyvitamin D(3). These data show for the first time that Wnt-3a has an unusual effect on multipotential embryonic cells, in that it enhances cellular proliferation and expression of alkaline phosphatase, while it represses most

  3. Osteogenic differentiation of human mesenchymal stem cells promotes mineralization within a biodegradable peptide hydrogel.

    PubMed

    Castillo Diaz, Luis A; Elsawy, Mohamed; Saiani, Alberto; Gough, Julie E; Miller, Aline F

    2016-01-01

    An attractive strategy for the regeneration of tissues has been the use of extracellular matrix analogous biomaterials. Peptide-based fibrillar hydrogels have been shown to mimic the structure of extracellular matrix offering cells a niche to undertake their physiological functions. In this study, the capability of an ionic-complementary peptide FEFEFKFK (F, E, and K are phenylalanine, glutamic acid, and lysine, respectively) hydrogel to host human mesenchymal stem cells in three dimensions and induce their osteogenic differentiation is demonstrated. Assays showed sustained cell viability and proliferation throughout the hydrogel over 12 days of culture and these human mesenchymal stem cells differentiated into osteoblasts simply upon addition of osteogenic stimulation. Differentiated osteoblasts synthesized key bone proteins, including collagen-1 (Col-1), osteocalcin, and alkaline phosphatase. Moreover, mineralization occurred within the hydrogel. The peptide hydrogel is a naturally biodegradable material as shown by oscillatory rheology and reversed-phase high-performance liquid chromatography, where both viscoelastic properties and the degradation of the hydrogel were monitored over time, respectively. These findings demonstrate that a biodegradable octapeptide hydrogel can host and induce the differentiation of stem cells and has the potential for the regeneration of hard tissues such as alveolar bone. PMID:27493714

  4. Bushen-Qiangdu-Zhilv decoction inhibits osteogenic differentiation of rat fibroblasts by regulating connexin 43

    PubMed Central

    ZHOU, YING-YAN; HUANG, RUN-YUE; LIN, JIE-HUA; XU, YONG-YUE; HE, XIAO-HONG; HE, YI-TING

    2016-01-01

    Bushen-Qiangdu-Zhilv (BQZ) decoction is a traditional Chinese medicinal compound widely used for treating ankylosing spondylitis (AS). However, the mechanisms underlying effects of BQZ remain largely unknown. Osteoblast differentiation of fibroblasts plays an important role in heterotopic ossification (HO) of AS, and connexin 43 (Cx43) is crucially involved in the osteoblast differentiation of fibroblasts. The aim of the present study was to evaluate the effects of BQZ on the osteogenic differentiation of fibroblasts by regulating Cx43. Rat fibroblasts were treated with freeze-dried powder of BQZ, in the presence or absence of recombinant human bone morphogenetic protein-2 (rhBMP-2). MTS assays were performed to examine the inhibitory effects of BQZ on fibroblast proliferation. Western blot assays were conducted to detect the protein expression of core-binding factor alpha 1 (Cbfα1), Cx43 and phosphorylated Cx43 (pCx43). BQZ appeared to inhibit fibroblast proliferation in a dose-dependent manner. Furthermore, the expression of Cbfα1 and Cx43/pCx43 was significantly suppressed by BQZ, with or without rhBMP-2 stimulation. Therefore, the present results indicate that BQZ may exert an anti-AS effect by suppressing the osteogenic differentiation of fibroblasts via Cx43 regulation. PMID:27347061

  5. Osteogenic differentiation of human mesenchymal stem cells promotes mineralization within a biodegradable peptide hydrogel

    PubMed Central

    Castillo Diaz, Luis A; Elsawy, Mohamed; Saiani, Alberto; Gough, Julie E; Miller, Aline F

    2016-01-01

    An attractive strategy for the regeneration of tissues has been the use of extracellular matrix analogous biomaterials. Peptide-based fibrillar hydrogels have been shown to mimic the structure of extracellular matrix offering cells a niche to undertake their physiological functions. In this study, the capability of an ionic-complementary peptide FEFEFKFK (F, E, and K are phenylalanine, glutamic acid, and lysine, respectively) hydrogel to host human mesenchymal stem cells in three dimensions and induce their osteogenic differentiation is demonstrated. Assays showed sustained cell viability and proliferation throughout the hydrogel over 12 days of culture and these human mesenchymal stem cells differentiated into osteoblasts simply upon addition of osteogenic stimulation. Differentiated osteoblasts synthesized key bone proteins, including collagen-1 (Col-1), osteocalcin, and alkaline phosphatase. Moreover, mineralization occurred within the hydrogel. The peptide hydrogel is a naturally biodegradable material as shown by oscillatory rheology and reversed-phase high-performance liquid chromatography, where both viscoelastic properties and the degradation of the hydrogel were monitored over time, respectively. These findings demonstrate that a biodegradable octapeptide hydrogel can host and induce the differentiation of stem cells and has the potential for the regeneration of hard tissues such as alveolar bone. PMID:27493714

  6. Osteogenic cell differentiation on H-terminated and O-terminated nanocrystalline diamond films

    PubMed Central

    Liskova, Jana; Babchenko, Oleg; Varga, Marian; Kromka, Alexander; Hadraba, Daniel; Svindrych, Zdenek; Burdikova, Zuzana; Bacakova, Lucie

    2015-01-01

    Nanocrystalline diamond (NCD) films are promising materials for bone implant coatings because of their biocompatibility, chemical resistance, and mechanical hardness. Moreover, NCD wettability can be tailored by grafting specific atoms. The NCD films used in this study were grown on silicon substrates by microwave plasma-enhanced chemical vapor deposition and grafted by hydrogen atoms (H-termination) or oxygen atoms (O-termination). Human osteoblast-like Saos-2 cells were used for biological studies on H-terminated and O-terminated NCD films. The adhesion, growth, and subsequent differentiation of the osteoblasts on NCD films were examined, and the extracellular matrix production and composition were quantified. The osteoblasts that had been cultivated on the O-terminated NCD films exhibited a higher growth rate than those grown on the H-terminated NCD films. The mature collagen fibers were detected in Saos-2 cells on both the H-terminated and O-terminated NCD films; however, the quantity of total collagen in the extracellular matrix was higher on the O-terminated NCD films, as were the amounts of calcium deposition and alkaline phosphatase activity. Nevertheless, the expression of genes for osteogenic markers – type I collagen, alkaline phosphatase, and osteocalcin – was either comparable on the H-terminated and O-terminated films or even lower on the O-terminated films. In conclusion, the higher wettability of the O-terminated NCD films is promising for adhesion and growth of osteoblasts. In addition, the O-terminated surface also seems to support the deposition of extracellular matrix proteins and extracellular matrix mineralization, and this is promising for better osteoconductivity of potential bone implant coatings. PMID:25670900

  7. Alkaline Phosphatase-Mimicking Peptide Nanofibers for Osteogenic Differentiation.

    PubMed

    Gulseren, Gulcihan; Yasa, I Ceren; Ustahuseyin, Oya; Tekin, E Deniz; Tekinay, Ayse B; Guler, Mustafa O

    2015-07-13

    Recognition of molecules and regulation of extracellular matrix synthesis are some of the functions of enzymes in addition to their catalytic activity. While a diverse array of enzyme-like materials have been developed, these efforts have largely been confined to the imitation of the chemical structure and catalytic activity of the enzymes, and it is unclear whether enzyme-mimetic molecules can also be used to replicate the matrix-regulatory roles ordinarily performed by natural enzymes. Self-assembled peptide nanofibers can provide multifunctional enzyme-mimetic properties, as the active sequences of the target enzymes can be directly incorporated into the peptides. Here, we report enhanced bone regeneration efficiency through peptide nanofibers carrying both catalytic and matrix-regulatory functions of alkaline phosphatase, a versatile enzyme that plays a critical role in bone formation by regulating phosphate homeostasis and calcifiable bone matrix formation. Histidine presenting peptide nanostructures were developed to function as phosphatases. These molecules are able to catalyze phosphate hydrolysis and serve as bone-like nodule inducing scaffolds. Alkaline phosphatase-like peptide nanofibers enabled osteogenesis for both osteoblast-like and mesenchymal cell lines. PMID:26039144

  8. Novel SCRG1/BST1 axis regulates self-renewal, migration, and osteogenic differentiation potential in mesenchymal stem cells

    PubMed Central

    Aomatsu, Emiko; Takahashi, Noriko; Sawada, Shunsuke; Okubo, Naoto; Hasegawa, Tomokazu; Taira, Masayuki; Miura, Hiroyuki; Ishisaki, Akira; Chosa, Naoyuki

    2014-01-01

    Human mesenchymal stem cells (hMSCs) remodel or regenerate various tissues through several mechanisms. Here, we identified the hMSC-secreted protein SCRG1 and its receptor BST1 as a positive regulator of self-renewal, migration, and osteogenic differentiation. SCRG1 and BST1 gene expression decreased during osteogenic differentiation of hMSCs. Intriguingly, SCRG1 maintained stem cell marker expression (Oct-4 and CD271/LNGFR) and the potentials of self-renewal, migration, and osteogenic differentiation, even at high passage numbers. Thus, the novel SCRG1/BST1 axis determines the fate of hMSCs by regulating their kinetic and differentiation potentials. Our findings provide a new perspective on methods for ex vivo expansion of hMSCs that maintain native stem cell potentials for bone-forming cell therapy. PMID:24413464

  9. Influence of platelet-rich plasma (PRP) on osteogenic differentiation of rat bone marrow stromal cells. An in vitro study.

    PubMed

    Arpornmaeklong, P; Kochel, M; Depprich, R; Kübler, N R; Würzler, K K

    2004-01-01

    Recent clinical reports suggest that the application of an autologous blood plasma enriched with thrombocytes by centrifugal concentration (platelet-rich plasma: PRP) can enhance the formation of new bone. There are very fewin vitro or in vivo studies published on the efficiency of PRP. In this project a three dimensional cell culture system was used to compare PRP and rhBMP-2 in vitro. Marrow derived bone forming cells from Spraque-Dawley (SD) rats were seeded on porous collagenous carriers (d=5mm, h=3mm) at a density of 4 x 10(4) cells/carrier and exposed to different concentrations of PRP (platelet counts from 2.5 x 10(8)-1.6 x 10(7) platelets/culture), rhBMP-2 (300 ng) or plasma poor in thrombocytes (platelet-poor plasma, PPP). Cultures without additional supplements were used as controls. During a culture period of 21 days cell proliferation, alkaline phosphatase activity (ALP) and calcium content (days 18, 21) were measured in 3 day intervals.PRP showed a dose dependent stimulation of cell proliferation, while reducing ALP activity and calcium deposition in the culture. BMP-2 led to an opposite cell response and induced the highest ALP activity and mineral deposition. These data suggest that PRP inhibited osteogenic differentiation of marrow derived pre-osteoblasts in a dose dependent manner. PRP is not a substitute for BMP-2 in osteogenic induction. PMID:14690661

  10. GCN5 modulates osteogenic differentiation of periodontal ligament stem cells through DKK1 acetylation in inflammatory microenvironment

    PubMed Central

    Li, Bei; Sun, Jin; Dong, Zhiwei; Xue, Peng; He, Xiaoning; Liao, Li; Yuan, Lin; Jin, Yan

    2016-01-01

    Periodontal ligament stem cells (PDLSCs) from periodontitis patients showed defective osteogenic differentiation. However, the mechanism of impaired osteogenic differentiation of PDLSCs in inflammatory microenvironments is still unclear. In this study, we found that inflammation in the microenvironment resulted in downregulation of histone acetyltransferase GCN5 expression and lack of GCN5 caused decreased osteogenic differentiation of PDLSCs. Previous study showed activated Wnt/β-cateinin pathway of PDLSCs resulted in defective osteogenic differentiation. Here we found knockdown of GCN5 decreased the expression of DKK1, an inhibitor of Wnt/β-cateinin pathway, thus activated Wnt/β-catenin pathway of PDLSCs. Mechanistically, GCN5 regulated DKK1 expression by acetylation of Histone H3 lysine 9 (H3K9) and Histone H3 lysine 14 (H3K14) at its promoter region. Interestingly, we found that in vivo injection of aspirin rescued the periodontitis of rats through inhibiting inflammation and upregulating GCN5 expression. Furthermore, aspirin treatment of PDLSCs upregulated GCN5 expression and increased osteogenic differentiation of PDLSCs. In conclusion, GCN5 plays a protective role in periodontitis through acetylation of DKK1 and applying drugs targeting GCN5, such as aspirin, could be a new approach for periodontitis treatment. PMID:27216891

  11. GCN5 modulates osteogenic differentiation of periodontal ligament stem cells through DKK1 acetylation in inflammatory microenvironment.

    PubMed

    Li, Bei; Sun, Jin; Dong, Zhiwei; Xue, Peng; He, Xiaoning; Liao, Li; Yuan, Lin; Jin, Yan

    2016-01-01

    Periodontal ligament stem cells (PDLSCs) from periodontitis patients showed defective osteogenic differentiation. However, the mechanism of impaired osteogenic differentiation of PDLSCs in inflammatory microenvironments is still unclear. In this study, we found that inflammation in the microenvironment resulted in downregulation of histone acetyltransferase GCN5 expression and lack of GCN5 caused decreased osteogenic differentiation of PDLSCs. Previous study showed activated Wnt/β-cateinin pathway of PDLSCs resulted in defective osteogenic differentiation. Here we found knockdown of GCN5 decreased the expression of DKK1, an inhibitor of Wnt/β-cateinin pathway, thus activated Wnt/β-catenin pathway of PDLSCs. Mechanistically, GCN5 regulated DKK1 expression by acetylation of Histone H3 lysine 9 (H3K9) and Histone H3 lysine 14 (H3K14) at its promoter region. Interestingly, we found that in vivo injection of aspirin rescued the periodontitis of rats through inhibiting inflammation and upregulating GCN5 expression. Furthermore, aspirin treatment of PDLSCs upregulated GCN5 expression and increased osteogenic differentiation of PDLSCs. In conclusion, GCN5 plays a protective role in periodontitis through acetylation of DKK1 and applying drugs targeting GCN5, such as aspirin, could be a new approach for periodontitis treatment. PMID:27216891

  12. Dense collagen matrix accelerates osteogenic differentiation and rescues the apoptotic response to MMP inhibition.

    PubMed

    Buxton, P G; Bitar, M; Gellynck, K; Parkar, M; Brown, R A; Young, A M; Knowles, J C; Nazhat, S N

    2008-08-01

    Bone is distinguished from other tissues by its mechanical properties, in particular stiffness. However, we know little of how osteoblasts react to the stiffness of their microenvironment; in this study we describe their response to a dense (>10 wt.%) collagenous 3D environment. Primary pre-osteoblasts were seeded within a novel form of native collagen, dense collagen, and cultured for up to 14 days in the presence and absence of osteogenic supplements: analysis was via Q-PCR, histology, fluorescent in situ zymography, MMP loss-of-function and tensile testing. Differentiation as measured through the up-regulation of Bsp (247-fold), Alp (14.2-fold), Col1A1 (4.5-fold), Mmp-13 (8.0-fold) and Runx2 (1.2-fold) transcripts was greatly accelerated compared to 2D plastic at 7 and 14 days in the same medium. The scale of this enhancement was confirmed through the use of growth factor stimulation on 2D via the addition of BMP-6 and the Hedgehog agonist purmorphamine. In concert, these molecules were capable of the same level of osteo-induction (measured by Bsp and Alp expression) as the dense collagen alone. Mineralisation was initially localised to remodelled pericellular regions, but by 14 days embedded cells were discernible within regions of apatite (confirmed by MicroRaman). Tensile testing of the matrices showed that this had resulted in a significant increase in Young's modulus at low strain values, consistent with a stiffening of the matrix. To determine the need for matrix remodelling in the mineralisation event the broad spectrum MMP Inhibitor Ilomastat was used. It was found that in its presence mineralisation could still occur (though serum-specific) and the apoptosis associated with MMP inhibition in hydrated collagen gels was abrogated. Analysis of gene expression indicated that this was due to the up-regulation of Mmp-13 in the presence of Ilomastat in dense collagen (400-fold), demonstrating a powerful feedback loop and a potential mechanism for the rescue

  13. Cell mediated contraction in 3D cell-matrix constructs leads to spatially regulated osteogenic differentiation

    PubMed Central

    Klumpers, Darinka D.; Zhao, Xuanhe; Mooney, David J.; Smit, Theo H.

    2013-01-01

    During embryonic development, morphogenetic processes give rise to a variety of shapes and patterns that lead to functional tissues and organs. While the impact of chemical signals in these processes is widely studied, the role of physical cues is less understood. The aim of this study was to test the hypothesis that the interplay of cell mediated contraction and mechanical boundary conditions alone can result in spatially regulated differentiation in simple 3D constructs. An experimental model consisting of a 3D cell-gel construct and a finite element (FE) model were used to study the effect of cellular traction exerted by mesenchymal stem cells (MSCs) on an initially homogeneous matrix under inhomogeneous boundary conditions. A robust shape change is observed due to contraction under time-varying mechanical boundary conditions, which is explained by the finite element model. Furthermore, distinct local differences of osteogenic differentiation are observed, with a spatial pattern independent of osteogenic factors in the culture medium. Regions that are predicted to have experienced relatively high shear stress at any time during contraction, correlate with the regions of distinct osteogenesis. Taken together, these results support the underlying hypothesis that cellular contractility and mechanical boundary conditions alone can result in spatially regulated differentiation. These results will have important implications for tissue engineering and regeneration. PMID:23925497

  14. Activation of GLP-1 Receptor Promotes Bone Marrow Stromal Cell Osteogenic Differentiation through β-Catenin

    PubMed Central

    Meng, Jingru; Ma, Xue; Wang, Ning; Jia, Min; Bi, Long; Wang, Yunying; Li, Mingkai; Zhang, Huinan; Xue, Xiaoyan; Hou, Zheng; Zhou, Ying; Yu, Zhibin; He, Gonghao; Luo, Xiaoxing

    2016-01-01

    Summary Glucagon-like peptide 1 (GLP-1) plays an important role in regulating bone remodeling, and GLP-1 receptor agonist shows a positive relationship with osteoblast activity. However, GLP-1 receptor is not found in osteoblast, and the mechanism of GLP-1 receptor agonist on regulating bone remodeling is unclear. Here, we show that the GLP-1 receptor agonist exendin-4 (Ex-4) promoted bone formation and increased bone mass and quality in a rat unloading-induced bone loss model. These functions were accompanied by an increase in osteoblast number and serum bone formation markers, while the adipocyte number was decreased. Furthermore, GLP-1 receptor was detected in bone marrow stromal cells (BMSCs), but not in osteoblast. Activation of GLP-1 receptor by Ex-4 promoted the osteogenic differentiation and inhibited BMSC adipogenic differentiation through regulating PKA/β-catenin and PKA/PI3K/AKT/GSK3β signaling. These findings reveal that GLP-1 receptor regulates BMSC osteogenic differentiation and provide a molecular basis for therapeutic potential of GLP-1 against osteoporosis. PMID:26947974

  15. Terbium promotes adhesion and osteogenic differentiation of mesenchymal stem cells via activation of the Smad-dependent TGF-β/BMP signaling pathway.

    PubMed

    Liu, Dan-Dan; Ge, Kun; Jin, Yi; Sun, Jing; Wang, Shu-Xiang; Yang, Meng-Su; Zhang, Jin-Chao

    2014-08-01

    With its special physical and chemical properties, terbium has been widely used, which has inevitably increased the chance of human exposure to terbium-based compounds. It was reported that terbium mainly deposited in bone after introduction into the human body. Although some studies revealed the effects of terbium on bone cell lines, there have been few reports about the potential effect of terbium on adhesion and differentiation of mesenchymal stem cells (MSCs). In this study, we investigated the effects of terbium on the adhesion and osteogenic and adipogenic differentiation of MSCs and the associated molecular mechanisms. Our data reveal that terbium promoted the osteogenic differentiation in a time-dependent manner and conversely inhibited the adipogenic differentiation of MSCs. Meanwhile, the cell-cell or cell-matrix interaction was enhanced by activating adherent-related key factors, which were evaluated by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Real-time RT-PCR and Western blot analysis were also performed to further detect osteogenic and adipogenic biomarkers of MSCs. The regulation of terbium on differentiation of MSCs led to the interaction between the transforming growth factor β/bone morphogenetic protein and peroxisome-proliferator-activated receptor γ (PPARγ) signaling pathways, resulting in upregulation of the osteogenic master transcription factors, such as Runt-related transcription factor 2, bone morphogenetic protein 2, collagen I, alkaline phosphatase, and osteocalcin, and downregulation of the adipogenic master transcription factors, such as PPARγ2. The results provide novel evidence to elucidate the mechanisms of bone metabolism by terbium and may be helpful for more rational application of terbium-based compounds in the future. PMID:24585101

  16. Effects of Plants on Osteogenic Differentiation and Mineralization of Periodontal Ligament Cells: A Systematic Review.

    PubMed

    Costa, Cláudio Rodrigues Rezende; Amorim, Bruna Rabelo; de Magalhães, Pérola; De Luca Canto, Graziela; Acevedo, Ana Carolina; Guerra, Eliete Neves Silva

    2016-04-01

    This systematic review aimed to evaluate the effects of plants on osteogenic differentiation and mineralization of human periodontal ligament cells. The included studies were selected using five different electronic databases. The reference list of the included studies was crosschecked, and a partial gray literature search was undertaken using Google Scholar and ProQuest. The methodology of the selected studies was evaluated using GRADE. After a two-step selection process, eight studies were identified. Six different types of plants were reported in the selected studies, which were Morinda citrifolia, Aloe vera, Fructus cnidii, Zanthoxylum schinifolium, Centella asiatica, and Epimedium species. They included five types of isolated plant components: acemannan, osthole, hesperetin, asiaticoside, and icariin. In addition, some active substances of these components were identified as polysaccharides, coumarins, flavonoids, and triterpenes. The studies demonstrated the potential effects of plants on osteogenic differentiation, cell proliferation, mineral deposition, and gene and protein expression. Four studies showed that periodontal ligament cells induce mineral deposition after plant treatment. Although there are few studies on the subject, current evidence suggests that plants are potentially useful for the treatment of periodontal diseases. However, further investigations are required to confirm the promising effect of these plants in regenerative treatments. PMID:26822584

  17. Osteogenic differentiation of human placenta-derived mesenchymal stem cells (PMSCs) on electrospun nanofiber meshes.

    PubMed

    Zhang, Dongmei; Tong, Aiping; Zhou, Liangxue; Fang, Fang; Guo, Gang

    2012-12-01

    Numerous challenges remain in the successful clinical translation of cell-based therapeutic studies for skeletal tissue repair, including appropriate cell sources and viable cell delivery systems. Poly(ethylene glycol)-poly(ε-caprolactone) (PEG-PCL) amphiphilic block copolymers have been extensively explored in microspheres preparation. Due to the introduction of hydrophilic PEG segments into PCL backbones, these copolymers have shown much more potentials in carrying protein, lipophilic drugs or genes than commonly used poly (ε-caprolactone) (PCL) and poly (lactic acid). The aim of this study is to investigate the attachment and osteogenic differentiation of human placenta derived mesenchymal stem cells (PMSCs) on PEG-PCL triblock copolymers nanofiber scaffolds. Here we demonstrated that PMSCs proliferate robustly and can be effectively differentiated into osteogenic-like cells on nanofiber scaffolds. This study provides evidence for the use of nanofiber scaffolds as an ideal supporting material for in vitro PMSCs culture and an in vivo cell delivery vehicle for bone repair. PMID:22526490

  18. Biomineralized matrix-assisted osteogenic differentiation of human embryonic stem cells

    PubMed Central

    Kang, Heemin; Wen, Cai; Hwang, Yongsung; Shih, Yu-Ru V.; Kar, Mrityunjoy; Seo, Sung Wook; Varghese, Shyni

    2014-01-01

    The physical and chemical properties of a matrix play an important role in determining various cellular behaviors, including lineage specificity. We demonstrate that the differentiation commitment of human embryonic stem cells (hESCs), both in vitro and in vivo, can be solely achieved through synthetic biomaterials. hESCs were cultured using mineralized synthetic matrices mimicking a calcium phosphate (CaP)-rich bone environment differentiated into osteoblasts in the absence of any osteogenic inducing supplements. When implanted in vivo, these hESC-laden mineralized matrices contributed to ectopic bone tissue formation. In contrast, cells within the corresponding non-mineralized matrices underwent either osteogenic or adipogenic fate depending upon the local cues present in the microenvironment. To our knowledge, this is the first demonstration where synthetic matrices are shown to induce terminal cell fate specification of hESCs exclusively by biomaterial-based cues both in vitro and in vivo. Technologies that utilize tissue specific cell-matrix interactions to control stem cell fate could be a powerful tool in regenerative medicine. Such approaches can be used as a tool to advance our basic understanding and assess the translational potential of stem cells. PMID:25114796

  19. Osteogenic differentiation of amniotic fluid mesenchymal stromal cells and their bone regeneration potential.

    PubMed

    Pipino, Caterina; Pandolfi, Assunta

    2015-05-26

    In orthopedics, tissue engineering approach using stem cells is a valid line of treatment for patients with bone defects. In this context, mesenchymal stromal cells of various origins have been extensively studied and continue to be a matter of debate. Although mesenchymal stromal cells from bone marrow are already clinically applied, recent evidence suggests that one may use mesenchymal stromal cells from extra-embryonic tissues, such as amniotic fluid, as an innovative and advantageous resource for bone regeneration. The use of cells from amniotic fluid does not raise ethical problems and provides a sufficient number of cells without invasive procedures. Furthermore, they do not develop into teratomas when transplanted, a consequence observed with pluripotent stem cells. In addition, their multipotent differentiation ability, low immunogenicity, and anti-inflammatory properties make them ideal candidates for bone regenerative medicine. We here present an overview of the features of amniotic fluid mesenchymal stromal cells and their potential in the osteogenic differentiation process. We have examined the papers actually available on this regard, with particular interest in the strategies applied to improve in vitro osteogenesis. Importantly, a detailed understanding of the behavior of amniotic fluid mesenchymal stromal cells and their osteogenic ability is desirable considering a feasible application in bone regenerative medicine. PMID:26029340

  20. The Effects of Simulated Microgravity on Gene Expression in Human Bone Marrow MSC's Under Osteogenic Differentiation

    NASA Astrophysics Data System (ADS)

    Buravkova, L. B.; Gershovich, J. G.; Gershovich, P. M.; Grigoriev, A. I.

    2013-02-01

    In this work it was found that the expression level of 144 genes significantly changed in human mesenchymal stem cells during their osteogenic differentiation after 20 days of exposure to simulated microgravity: the expression of 30 genes significantly increased (from 1.7 to 11.9 fold), and 114 - decreased (from 0.2 to 0.6 fold). Most of the revealed genes were attributed to the 11 major groups corresponding to its biological role in the cells. Additional group was formed from the genes which did not belong to these categories, or did not have a description in the known databases (such as Pubmed). The greatest number of genes with altered expression was found in the group “Matrix and Adhesion", while the lowest - in the "Apoptosis and the response to external stimuli" group. These findings suggest that cultured hMSCs, placed in non-standard conditions, maintain a high level of viability, but have significantly altered functional properties which could affect their efficiency to differentiate towards osteogenic direction.

  1. Hydroxyapatite coatings with oriented nanoplate and nanorod arrays: Fabrication, morphology, cytocompatibility and osteogenic differentiation.

    PubMed

    Chen, Wei; Tian, Bo; Lei, Yong; Ke, Qin-Fei; Zhu, Zhen-An; Guo, Ya-Ping

    2016-10-01

    Hydroxyapatite (HA) crystals exhibit rod-like shape with c-axis orientation and plate-like shape with a(b)-axis orientation in vertebrate bones and tooth enamel surfaces, respectively. Herein, we report the synthesis of HA coatings with the oriented nanorod arrays (RHACs) and HA coatings with oriented nanoplate arrays (PHACs) by using bioglass coatings as sacrificial templates. After soaking in simulated body fluid (SBF) at 120°C, the bioglass coatings are hydrothermally converted into the HA coatings via a dissolution-precipitation reaction. If the Ca/P ratios in SBF are 2.50 and 1.25, the HA crystals on the coatings are oriented nanorod arrays and oriented nanoplate arrays, respectively. Moreover, the bioglass coatings are treated with SBF at 37°C, plate-like HA coatings with a low crystallinity (SHACs) are prepared. As compared with the Ti6Al4V and SHACs, the human bone marrow stromal cells (hBMSCs) on the RHACs and PHACs have better cell adhesion, spreading, proliferation and osteogenic differentiation because of their moderately hydrophilic surfaces and similar chemical composition, morphology and crystal orientation to human hard tissues. Notably, the morphologies of HA crystals have no obvious effects on cytocompatibility and osteogenic differentiation. Hence, the HA coatings with oriented nanoplate arrays or oriented nanorod arrays have a great potential for orthopedic applications. PMID:27287136

  2. Osteogenic differentiation of amniotic fluid mesenchymal stromal cells and their bone regeneration potential

    PubMed Central

    Pipino, Caterina; Pandolfi, Assunta

    2015-01-01

    In orthopedics, tissue engineering approach using stem cells is a valid line of treatment for patients with bone defects. In this context, mesenchymal stromal cells of various origins have been extensively studied and continue to be a matter of debate. Although mesenchymal stromal cells from bone marrow are already clinically applied, recent evidence suggests that one may use mesenchymal stromal cells from extra-embryonic tissues, such as amniotic fluid, as an innovative and advantageous resource for bone regeneration. The use of cells from amniotic fluid does not raise ethical problems and provides a sufficient number of cells without invasive procedures. Furthermore, they do not develop into teratomas when transplanted, a consequence observed with pluripotent stem cells. In addition, their multipotent differentiation ability, low immunogenicity, and anti-inflammatory properties make them ideal candidates for bone regenerative medicine. We here present an overview of the features of amniotic fluid mesenchymal stromal cells and their potential in the osteogenic differentiation process. We have examined the papers actually available on this regard, with particular interest in the strategies applied to improve in vitro osteogenesis. Importantly, a detailed understanding of the behavior of amniotic fluid mesenchymal stromal cells and their osteogenic ability is desirable considering a feasible application in bone regenerative medicine. PMID:26029340

  3. Endothelial Progenitor Cell Fraction Contained in Bone Marrow-Derived Mesenchymal Stem Cell Populations Impairs Osteogenic Differentiation

    PubMed Central

    Duttenhoefer, Fabian; Lara de Freitas, Rafael; Loibl, Markus; Bittermann, Gido; Geoff Richards, R.; Alini, Mauro; Verrier, Sophie

    2015-01-01

    In bone tissue engineering (TE) endothelial cell-osteoblast cocultures are known to induce synergies of cell differentiation and activity. Bone marrow mononucleated cells (BMCs) are a rich source of mesenchymal stem cells (MSCs) able to develop an osteogenic phenotype. Endothelial progenitor cells (EPCs) are also present within BMC. In this study we investigate the effect of EPCs present in the BMC population on MSCs osteogenic differentiation. Human BMCs were isolated and separated into two populations. The MSC population was selected through plastic adhesion capacity. EPCs (CD34+ and CD133+) were removed from the BMC population and the resulting population was named depleted MSCs. Both populations were cultured over 28 days in osteogenic medium (Dex+) or medium containing platelet lysate (PL). MSC population grew faster than depleted MSCs in both media, and PL containing medium accelerated the proliferation for both populations. Cell differentiation was much higher in Dex+ medium in both cases. Real-time RT-PCR revealed upregulation of osteogenic marker genes in depleted MSCs. Higher values of ALP activity and matrix mineralization analyses confirmed these results. Our study advocates that absence of EPCs in the MSC population enables higher osteogenic gene expression and matrix mineralization and therefore may lead to advanced bone neoformation necessary for TE constructs. PMID:26491682

  4. Recombinant human bone morphogenetic protein-9 potently induces osteogenic differentiation of human periodontal ligament fibroblasts.

    PubMed

    Fuchigami, Sawako; Nakamura, Toshiaki; Furue, Kirara; Sena, Kotaro; Shinohara, Yukiya; Noguchi, Kazuyuki

    2016-04-01

    To accomplish effective periodontal regeneration for periodontal defects, several regenerative methods using growth and differentiation factors, including bone morphogenetic proteins (BMPs), have been developed. Bone morphogenetic protein-9 exhibits the most potent osteogenic activity of this growth factor family. However, it is unclear whether exogenous BMP-9 can induce osteogenic differentiation in human periodontal ligament (PDL) fibroblasts. Here, we examined the effects of recombinant human (rh) BMP-9 on osteoblastic differentiation in human PDL fibroblasts in vitro, compared with rhBMP-2. Recombinant human BMP-9 potently induced alkaline phosphatase (ALP) activity, mineralization, and increased expression of runt-related transcription factor-2/core binding factor alpha 1 (RUNX2/CBFA1), osterix, inhibitor of DNA binding/differentiation-1 (ID1), osteopontin, and bone sialoprotein genes, compared with rhBMP-2. The levels of rhBMP-9-induced osterix and ALP mRNA were significantly reduced in activin receptor-like kinase-1 and -2 small interfering RNA (siRNA)-transfected human PDL fibroblasts. Recombinant human BMP-9-induced ALP activity was not inhibited by noggin, in contrast to rhBMP-2 induced ALP activity, which was. Phosphorylation of SMAD1/5/8 in human PDL fibroblasts was induced by addition of rhBMP-9. Recombinant human BMP-9-induced ALP activity was suppressed by SB203580, SP600125, and U0126, which are inhibitors of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK1/2), respectively. Our data suggest that rhBMP-9 is a potent inducer of the differentiation of human PDL fibroblasts into osteoblast-like cells and that this may be mediated by the SMAD and mitogen-activated protein kinase (p38, ERK1/2, and JNK) pathways. PMID:26879145

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

    SciTech Connect

    Fiedler, Tomas; Salamon, Achim; Adam, Stefanie; Herzmann, Nicole; Taubenheim, Jan; Peters, Kirsten

    2013-11-01

    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 differentiation 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.

  6. Bone Progenitors Produced by Direct Osteogenic Differentiation of the Unprocessed Bone Marrow Demonstrate High Osteogenic Potential In Vitro and In Vivo

    PubMed Central

    Weinreb, Miron; Abramov, Natalie; Shinar, Doron; Merchav, Shoshana; Schwartz, Aharon; Shirvan, Mitchell

    2012-01-01

    Abstract Tissue-engineered bone grafts seeded with mesenchymal stem cells (MSCs) have been sought as a replacement for bone grafts currently used for bone repair. For production of osteogenic constructs, MSCs are isolated from bone marrow (BM) or other tissues, expanded in culture, then trypsinized, and seeded on a scaffold. Predifferentiation of seeded cells is often desired. We describe here bone progenitor cells (BPCs) obtained by direct osteogenic differentiation of unprocessed BM bypassing isolation of MSCs. Human BM aspirates were incubated for 2 weeks with a commonly used osteogenic medium (OM), except no fetal calf serum, serum substitutes, or growth factors were added, because responding stem and/or progenitor cells were present in the BM milieu. The adherent cells remaining after the culture medium and residual BM were washed out, expressed high levels of bone-specific alkaline phosphatase (ALP) on their surface, demonstrated high ALP activity, were capable of mineralization of the intercellular space, and expressed genes associated with osteogenesis. These parameters in BPCs were similar and even at higher levels compared to MSCs subjected to osteogenic differentiation for 2 weeks. The yield of BPCs per 1 mL BM was 0.71±0.39×106. In comparison, the yield of MSCs produced by adhesion of mononuclear cells derived from the same amount of BM and cultured in a commercial growth medium for 2 weeks was 0.3±0.17×106. When a scaffold was added to the BM-OM mixture, and the mixture was cultured in a simple rotational bioreactor; the resulting BPCs were obtained already seeded on the scaffold. BPCs seeded on scaffolds were capable of proliferation for at least 6 weeks, keeping high levels of ALP activity, expressing osteogenic genes, and mineralizing the scaffolds. Autologous rat BPCs seeded on various scaffolds were transplanted into critical-size calvarial defects. Six weeks after transplantation of polylactic acid/polyglycolic acid scaffolds, 76.1%±18

  7. Genistein induces adipogenic differentiation in human bone marrow mesenchymal stem cells and suppresses their osteogenic potential by upregulating PPARγ

    PubMed Central

    ZHANG, LI-YAN; XUE, HAO-GANG; CHEN, JI-YING; CHAI, WEI; NI, MING

    2016-01-01

    Genistein is a soy isoflavone that exists in the form of an aglycone. It is the primary active component in soy isoflavone and has a number of biological activities (anti-inflammatory and anti-oxidative). However, the specific effect of genistein on human bone marrow mesenchymal stem cells (BMSCs) remains unclear. In the present study, the mechanism underlying the effect of genistein on the suppression of BMSC adipogenic differentiation and the enhancement of osteogenic potential was investigated using an MTT assay. It was observed that genistein significantly increased BMSC cell proliferation in a time- and dose-dependent manner (P<0.01). In addition, reverse transcription-quantitative polymerase chain reaction revealed that genistein significantly inhibited the expression of runt-related transcription factor 2 (Runx2), type I collagen (Col I) and osteocalcin (OC; P<0.01). Furthermore, 20 µm genistein significantly inhibited the activity of alkaline phosphatase (ALP) and increased the activity of triglycerides (TGs) increased (P<0.01) as determined by an enzyme-linked immunosorbent assay. Finally, western blotting revealed that BMSC pretreatment with 20 µm genistein significantly increased peroxisome proliferator-activated receptor γ (PPARγ) protein expression (P<0.01). This suggests that the downregulation of PPARγ may significantly reduce the effect of genistein on cell proliferation, suppress the expression of Runx2, Col I and OC mRNA, and reduce ALP and promote TG activity in BMSCs. Thus, the results of the present study conclude that genistein induces adipogenic differentiation in human BMSCs and suppresses their osteogenic potential by upregulating the expression of PPARγ. In conclusion, genistein may be a promising candidate drug for treatment against osteogenesis. PMID:27168816

  8. The Effects of Secretion Factors from Umbilical Cord Derived Mesenchymal Stem Cells on Osteogenic Differentiation of Mesenchymal Stem Cells

    PubMed Central

    Wang, Kui-Xing; Xu, Liang-Liang; Rui, Yun-Feng; Huang, Shuo; Lin, Si-En; Xiong, Jiang-Hui; Li, Ying-Hui; Lee, Wayne Yuk-Wai; Li, Gang

    2015-01-01

    Factors synthesized by mesenchymal stem cells (MSCs) contain various growth factors, cytokines, exosomes and microRNAs, which may affect the differentiation abilities of MSCs. In the present study, we investigated the effects of secretion factors of human umbilical cord derived mesenchymal stem cells (hUCMSCs) on osteogenesis of human bone marrow derived MSCs (hBMSCs). The results showed that 20 μg/ml hUCMSCs secretion factors could initiate osteogenic differentiation of hBMSCs without osteogenic induction medium (OIM), and the amount of calcium deposit (stained by Alizarin Red) was significantly increased after the hUCMSCs secretion factors treatment. Real time quantitative reverse transcription-polymerase chain reaction (real time qRT-PCR) demonstrated that the expression of osteogenesis-related genes including ALP, BMP2, OCN, Osterix, Col1α and Runx2 were significantly up-regulated following hUCMSCs secretion factors treatment. In addition, we found that 10 μg hUCMSCs secretion factors together with 2×105 hBMSCs in the HA/TCP scaffolds promoted ectopic bone formation in nude mice. Local application of 10 μg hUCMSCs secretion factors with 50 μl 2% hyaluronic acid hydrogel and 1×105 rat bone marrow derived MSCs (rBMSCs) also significantly enhanced the bone repair of rat calvarial bone critical defect model at both 4 weeks and 8 weeks. Moreover, the group that received the hUCMSCs secretion factors treatment had more cartilage and bone regeneration in the defect areas than those in the control group. Taken together, these findings suggested that hUCMSCs secretion factors can initiate osteogenesis of bone marrow MSCs and promote bone repair. Our study indicates that hUCMSCs secretion factors may be potential sources for promoting bone regeneration. PMID:25799169

  9. Secreted microvesicular miR-31 inhibits osteogenic differentiation of mesenchymal stem cells.

    PubMed

    Weilner, Sylvia; Schraml, Elisabeth; Wieser, Matthias; Messner, Paul; Schneider, Karl; Wassermann, Klemens; Micutkova, Lucia; Fortschegger, Klaus; Maier, Andrea B; Westendorp, Rudi; Resch, Heinrich; Wolbank, Susanne; Redl, Heinz; Jansen-Dürr, Pidder; Pietschmann, Peter; Grillari-Voglauer, Regina; Grillari, Johannes

    2016-08-01

    Damage to cells and tissues is one of the driving forces of aging and age-related diseases. Various repair systems are in place to counteract this functional decline. In particular, the property of adult stem cells to self-renew and differentiate is essential for tissue homeostasis and regeneration. However, their functionality declines with age (Rando, 2006). One organ that is notably affected by the reduced differentiation capacity of stem cells with age is the skeleton. Here, we found that circulating microvesicles impact on the osteogenic differentiation capacity of mesenchymal stem cells in a donor-age-dependent way. While searching for factors mediating the inhibitory effect of elderly derived microvesicles on osteogenesis, we identified miR-31 as a crucial component. We demonstrated that miR-31 is present at elevated levels in the plasma of elderly and of osteoporosis patients. As a potential source of its secretion, we identified senescent endothelial cells, which are known to increase during aging in vivo (Erusalimsky, 2009). Endothelial miR-31 is secreted within senescent cell-derived microvesicles and taken up by mesenchymal stem cells where it inhibits osteogenic differentiation by knocking down its target Frizzled-3. Therefore, we suggest that microvesicular miR-31 in the plasma of elderly might play a role in the pathogenesis of age-related impaired bone formation and that miR-31 might be a valuable plasma-based biomarker for aging and for a systemic environment that does not favor cell-based therapies whenever osteogenesis is a limiting factor. PMID:27146333

  10. Linarin promotes osteogenic differentiation by activating the BMP-2/RUNX2 pathway via protein kinase A signaling.

    PubMed

    Li, Jia; Hao, Lingyu; Wu, Junhua; Zhang, Jiquan; Su, Jiansheng

    2016-04-01

    Linarin (LIN), a flavonoid which exerts both anti-inflammatory and antioxidative effects, has been found to promote osteogenic differentiation. However, the molecular mechanism of its effect on osteoblast differentiation was unclear. In the present study, LIN from Flos Chrysanthemi Indici (FCI) was isolated in order to investigate the underlying mechanisms of LIN on MC3T3-E1 cells (a mouse osteoblastic cell line) and the osteoprotective effect of LIN in mice which had undergone an ovariectomy (OVX). The results revealed that LIN enhanced osteoblast proliferation and differentiation in MC3T3-E1 cells dose‑dependently, with enhanced alkaline phosphatase (ALP) activity and mineralization of extracellular matrix. LIN upregulated osteogenesis-related gene expression, including that of ALP, runt‑related transcription factor 2 (RUNX2), osteocalcin (OCN), bone sialoprotein (BSP), and type I collagen (COL‑I). Pretreatment with noggin, a bone morphogenetic protein-2 (BMP-2) antagonist, meant that LIN-induced gene expression levels of COL-1, ALP, OCN, BSP and RUNX2 were significantly reduced, as shown by RT-qPCR. Western blot analysis showed that LIN dose‑dependently increased the protein levels of BMP-2 and RUNX2 and enhanced the phosphorylation of SMAD1/5. In addition, LIN dose‑dependently upregulated protein kinase A (PKA) expression. H-89 (a PKA inhibitor) partially blocked the LIN-induced protein increase in BMP-2, p-SMAD1/5 and RUNX2. We noted that LIN preserved the trabecular bone microarchitecture of ovariectomized mice in vivo. Moreover, pretreatment with LIN significantly lowered serum levels of ALP and OCN in ovariectomized mice. Our data indicated that LIN induced the osteogenic differentiation and mineralization of MC3T3-E1 osteoblastic cells by activating the BMP-2/RUNX2 pathway through PKA signaling in vitro and protected against OVX-induced bone loss in vivo. The results strongly suggest that LIN is a useful natural alternative for

  11. Linarin promotes osteogenic differentiation by activating the BMP-2/RUNX2 pathway via protein kinase A signaling

    PubMed Central

    LI, JIA; HAO, LINGYU; WU, JUNHUA; ZHANG, JIQUAN; SU, JIANSHENG

    2016-01-01

    Linarin (LIN), a flavonoid which exerts both anti-inflammatory and antioxidative effects, has been found to promote osteogenic differentiation. However, the molecular mechanism of its effect on osteoblast differentiation was unclear. In the present study, LIN from Flos Chrysanthemi Indici (FCI) was isolated in order to investigate the underlying mechanisms of LIN on MC3T3-E1 cells (a mouse osteoblastic cell line) and the osteoprotective effect of LIN in mice which had undergone an ovariectomy (OVX). The results revealed that LIN enhanced osteoblast proliferation and differentiation in MC3T3-E1 cells dose-dependently, with enhanced alkaline phosphatase (ALP) activity and mineralization of extracellular matrix. LIN upregulated osteogenesis-related gene expression, including that of ALP, runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), bone sialoprotein (BSP), and type I collagen (COL-I). Pretreatment with noggin, a bone morphogenetic protein-2 (BMP-2) antagonist, meant that LIN-induced gene expression levels of COL-1, ALP, OCN, BSP and RUNX2 were significantly reduced, as shown by RT-qPCR. Western blot analysis showed that LIN dose-dependently increased the protein levels of BMP-2 and RUNX2 and enhanced the phosphorylation of SMAD1/5. In addition, LIN dose-dependently upregulated protein kinase A (PKA) expression. H-89 (a PKA inhibitor) partially blocked the LIN-induced protein increase in BMP-2, p-SMAD1/5 and RUNX2. We noted that LIN preserved the trabecular bone microarchitecture of ovariectomized mice in vivo. Moreover, pretreatment with LIN significantly lowered serum levels of ALP and OCN in ovariectomized mice. Our data indicated that LIN induced the osteogenic differentiation and mineralization of MC3T3-E1 osteoblastic cells by activating the BMP-2/RUNX2 pathway through PKA signaling in vitro and protected against OVX-induced bone loss in vivo. The results strongly suggest that LIN is a useful natural alternative for the management of

  12. 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

  13. Abnormal osteogenic and chondrogenic differentiation of human mesenchymal stem cells from patients with adolescent idiopathic scoliosis in response to melatonin

    PubMed Central

    Chen, Chong; Xu, Caixia; Zhou, Taifeng; Gao, Bo; Zhou, Hang; Chen, Changhua; Zhang, Changli; Huang, Dongsheng; Su, Peiqiang

    2016-01-01

    Abnormalities of membranous and endochondral ossification in patients with adolescent idiopathic scoliosis (AIS) remain incompletely understood. To investigate abnormalities in the melatonin signaling pathway and cellular response to melatonin in AIS, a case-control study of osteogenic and chondrogenic differentiation was performed using human mesenchymal stem cells (hMSCs). AIS was diagnosed by physical and radiographic examination. hMSCs were isolated from the bone marrow of patients with AIS and control subjects (n=12 each), and purified by density gradient centrifugation. The expression levels of melatonin receptors (MTs) 1 and 2 were detected by western blotting. Osteogenic and chondrogenic differentiation was induced by culturing hMSCs in osteogenic and chondrogenic media containing vehicle or 50 nM melatonin. Alkaline phosphatase (ALP) activity assays, quantitative glycosaminoglycan (GAG) analysis, and reverse transcription-quantitative polymerase chain reaction analysis were performed. Compared with controls, MT2 demonstrated low expression in the AIS group. Melatonin increased ALP activity, GAG synthesis and upregulated the expression of genes involved in osteogenic and chondrogenic differentiation including, ALP, osteopontin, osteocalcin, runt-related transcription factor 2, collagen type II, collagen type X, aggrecan and sex-determining region Y-box 9 in the normal control hMSCs, but did not affect the AIS groups. Thus, AIS hMSCs exhibit abnormal cellular responses to melatonin during osteogenic and chondrogenic differentiation, which may be associated with abnormal membranous and endochondral ossification, and skeletal growth. These results indicate a potential modulating role of melatonin via the MT2 receptor on abnormal osteogenic and chondrogenic differentiaation in patients with AIS. PMID:27314307

  14. PARP Inhibitor PJ34 Suppresses Osteogenic Differentiation in Mouse Mesenchymal Stem Cells by Modulating BMP-2 Signaling Pathway

    PubMed Central

    Kishi, Yuta; Fujihara, Hisako; Kawaguchi, Koji; Yamada, Hiroyuki; Nakayama, Ryoko; Yamamoto, Nanami; Fujihara, Yuko; Hamada, Yoshiki; Satomura, Kazuhito; Masutani, Mitsuko

    2015-01-01

    Poly(ADP-ribosyl)ation is known to be involved in a variety of cellular processes, such as DNA repair, cell death, telomere regulation, genomic stability and cell differentiation by poly(ADP-ribose) polymerase (PARP). While PARP inhibitors are presently under clinical investigation for cancer therapy, little is known about their side effects. However, PARP involvement in mesenchymal stem cell (MSC) differentiation potentiates MSC-related side effects arising from PARP inhibition. In this study, effects of PARP inhibitors on MSCs were examined. MSCs demonstrated suppressed osteogenic differentiation after 1 µM PJ34 treatment without cytotoxicity, while differentiation of MSCs into chondrocytes or adipocytes was unaffected. PJ34 suppressed mRNA induction of osteogenic markers, such as Runx2, Osterix, Bone Morphogenetic Protein-2, Osteocalcin, bone sialoprotein, and Osteopontin, and protein levels of Bone Morphogenetic Protein-2, Osterix and Osteocalcin. PJ34 treatment also inhibited transcription factor regulators such as Smad1, Smad4, Smad5 and Smad8. Extracellular mineralized matrix formation was also diminished. These results strongly suggest that PARP inhibitors are capable of suppressing osteogenic differentiation and poly(ADP-ribosyl)ation may play a physiological role in this process through regulation of BMP-2 signaling. Therefore, PARP inhibition may potentially attenuate osteogenic metabolism, implicating cautious use of PARP inhibitors for cancer treatments and monitoring of patient bone metabolism levels. PMID:26492236

  15. PARP Inhibitor PJ34 Suppresses Osteogenic Differentiation in Mouse Mesenchymal Stem Cells by Modulating BMP-2 Signaling Pathway.

    PubMed

    Kishi, Yuta; Fujihara, Hisako; Kawaguchi, Koji; Yamada, Hiroyuki; Nakayama, Ryoko; Yamamoto, Nanami; Fujihara, Yuko; Hamada, Yoshiki; Satomura, Kazuhito; Masutani, Mitsuko

    2015-01-01

    Poly(ADP-ribosyl)ation is known to be involved in a variety of cellular processes, such as DNA repair, cell death, telomere regulation, genomic stability and cell differentiation by poly(ADP-ribose) polymerase (PARP). While PARP inhibitors are presently under clinical investigation for cancer therapy, little is known about their side effects. However, PARP involvement in mesenchymal stem cell (MSC) differentiation potentiates MSC-related side effects arising from PARP inhibition. In this study, effects of PARP inhibitors on MSCs were examined. MSCs demonstrated suppressed osteogenic differentiation after 1 µM PJ34 treatment without cytotoxicity, while differentiation of MSCs into chondrocytes or adipocytes was unaffected. PJ34 suppressed mRNA induction of osteogenic markers, such as Runx2, Osterix, Bone Morphogenetic Protein-2, Osteocalcin, bone sialoprotein, and Osteopontin, and protein levels of Bone Morphogenetic Protein-2, Osterix and Osteocalcin. PJ34 treatment also inhibited transcription factor regulators such as Smad1, Smad4, Smad5 and Smad8. Extracellular mineralized matrix formation was also diminished. These results strongly suggest that PARP inhibitors are capable of suppressing osteogenic differentiation and poly(ADP-ribosyl)ation may play a physiological role in this process through regulation of BMP-2 signaling. Therefore, PARP inhibition may potentially attenuate osteogenic metabolism, implicating cautious use of PARP inhibitors for cancer treatments and monitoring of patient bone metabolism levels. PMID:26492236

  16. Osteogenic differentiation of human adipose-derived mesenchymal stem cells on gum tragacanth hydrogel.

    PubMed

    Haeri, Seyed Mohammad Jafar; Sadeghi, Yousef; Salehi, Mohammad; Farahani, Reza Masteri; Mohsen, Nourozian

    2016-05-01

    Currently, natural polymer based hydrogels has attracted great attention of orthopedic surgeons for application in bone tissue engineering. With this aim, osteoinductive capacity of Gum Tragacanth (GT) based hydrogel was compared to collagen hydrogel and tissue culture plate (TCPS). For this purpose, adipose-derived mesenchymal stem cells (AT-MSCs) was cultured on the hydrogels and TCPS and after investigating the biocompatibility of hydrogels using MTT assay, osteoinductivity of hydrogels were evaluated using pan osteogenic markers such as Alizarin red staining, alkaline phosphatase (ALP) activity, calcium content and osteo-related genes. Increasing proliferation trend of AT-MSCs on GT hydrogel demonstrated that TG has no-cytotoxicity and can even be better than the other groups i.e., highest proliferation at day 5. GT hydrogel displayed highest ALP activity and mineralization when compared to the collagen hydrogel and TCPS. Relative gene expression levels have demonstrated that highest expression of Runx2, osteonectin and osteocalcin in the cells cultured GT hydrogel but the expression of collagen type-1 remains constant in hydrogels. Above results demonstrate that GT hydrogel could be an appropriate scaffold for accelerating and supporting the adhesion, proliferation and osteogenic differentiation of stem cells which further can be used for orthopedic applications. PMID:27055599

  17. Vascular smooth muscle cell differentiation to an osteogenic phenotype involves matrix metalloproteinase-2 modulation by homocysteine.

    PubMed

    Liu, Tingjiao; Lin, Jinghan; Ju, Ting; Chu, Lei; Zhang, Liming

    2015-08-01

    Arterial calcification is common in vascular diseases and involves conversion of vascular smooth muscle cells (VSMCs) to an osteoblast phenotype. Clinical studies suggest that the development of atherosclerosis can be promoted by homocysteine (HCY), but the mechanisms remain unclear. Here, we determined whether increases in HCY levels lead to an increase in VSMC calcification and differentiation, and examined the role of an extracellular matrix remodeler, matrix metalloproteinase-2 (MMP-2). Rat VSMCs were exposed to calcification medium in the absence or presence of HCY (10, 100 or 200 μmol/L) or an MMP-2 inhibitor (10(-6) or 10(-5) mol/L). MTT assays were performed to determine the cytotoxicity of the MMP-2 inhibitor in calcification medium containing 200 μmol/L HCY. Calcification was assessed by measurements of calcium deposition and alkaline phosphatase (ALP) activity as well as von Kossa staining. Expression of osteocalcin, bone morphogenetic protein (BMP)-2, and osteopontin, and MMP-2 was determined by immunoblotting. Calcification medium induced osteogenic differentiation of VSMCs. HCY promoted calcification, increased osteocalcin and BMP-2 expression, and decreased expression of osteopontin. MMP-2 expression was increased by HCY in a dose-dependent manner in VSMCs exposed to both control and calcification medium. The MMP-2 inhibitor decreased the calcium content and ALP activity, and attenuated the osteoblastic phenotype of VSMCs. Vascular calcification and osteogenic differentiation of VSMCs were positively regulated by HCY through increased/restored MMP-2 expression, increased expression of calcification proteins, and decreased anti-calcification protein levels. In summary, MMP-2 inhibition may be a protective strategy against VSMC calcification. PMID:25987498

  18. Osteogenic Matrix Cell Sheet Transplantation Enhances Early Tendon Graft to Bone Tunnel Healing in Rabbits

    PubMed Central

    Uematsu, Kota; Akahane, Manabu; Morita, Yusuke; Ogawa, Munehiro; Ueha, Tomoyuki; Shimizu, Takamasa; Kura, Tomohiko; Kawate, Kenji; Tanaka, Yasuhito

    2013-01-01

    The objective of this study was to determine whether osteogenic matrix cell sheets (OMCS) could induce bone formation around grafted tendons, thereby enhancing early stage tendon to bone tunnel healing in skeletally mature male Japanese white rabbits. First, the osteogenic potential of rabbit OMCS was evaluated. Then, the OMCS were transplanted into the interface between the grafted tendon and the bone tunnel created at the tibia. Histological assessments and biomechanical tensile testing were performed after 3 weeks. The rabbit OMCS showed high alkaline phosphatase (ALP) activity, positive staining of ALP, and osteogenic potential when transplanted subcutaneously with beta tricalcium phosphate disks. Newly formed bony walls and positive collagen type I staining were seen around the grafted tendon with OMCS transplantation, whereas such bony walls were thinner or less frequent without OMCS transplantation. Micro-computed tomography images showed significantly higher bone volume in the OMCS transplantation group. The pullout strength was significantly higher with OMCS (0.74 ± 0.23 N/mm2) than without OMCS (0.58 ± 0.15 N/mm2). These results show that OMCS enhance early tendon to bone tunnel healing. This method can be applied to cases requiring early tendon to bone tunnel healing after ligament reconstruction surgery. PMID:24106718

  19. Interleukin 17 inhibits myogenic and promotes osteogenic differentiation of C2C12 myoblasts by activating ERK1,2.

    PubMed

    Kocić, Jelena; Santibañez, Juan F; Krstić, Aleksandra; Mojsilović, Slavko; Dorđević, Ivana Okić; Trivanović, Drenka; Ilić, Vesna; Bugarski, Diana

    2012-04-01

    The present study evaluated the role of interleukin (IL) 17 in multilineage commitment of C2C12 myoblastic cells and investigated associated signaling pathways. The results concerning the effects on cell function showed that IL-17 inhibits the migration of C2C12 cells, while not affecting their proliferation. The data regarding the influence on differentiation demonstrated that IL-17 inhibits myogenic differentiation of C2C12 cells by down-regulating the myogenin mRNA level, myosin heavy chain expression and myotube formation, but promotes their osteogenic differentiation by up-regulating the Runt-related transcription factor 2 mRNA level, cyclooxygenase-2 expression and alkaline phosphatase activity. IL-17 exerted these effects by activating ERK1,2 mitogen activated protein kinase signaling pathway, which in turn regulated the expression of relevant genes and proteins to inhibit myogenic differentiation and induce osteogenic differentiation. Additional analysis showed that the induction of osteogenic differentiation by IL-17 is independent of BMP signaling. The results obtained demonstrate the potential of IL-17 not only to inhibit the myogenic differentiation of C2C12 myoblasts but also to convert their differentiation pathway into that of osteoblast lineage providing new insight into the capacities of IL-17 to modulate the differentiation commitment. PMID:22285818

  20. The Effect of Human Platelet-Rich Plasma on Adipose-Derived Stem Cell Proliferation and Osteogenic Differentiation

    PubMed Central

    Tavakolinejad, Sima; Khosravi, Mohsen; Mashkani, Baratali; Ebrahimzadeh Bideskan, Alireza; Sanjar Mossavi, Nasser; Parizadeh, Seyyed Mohammad Reza; Hamidi Alamdari, Daryoush

    2014-01-01

    Background: The cultured mesenchymal stem cells (MSC) have been used in many clinical trials; however, there are still some concerns about the cultural conditions. One concern is related to the use of FBS as a widely used xenogeneic supplement in the culture system. Human platelet-rich plasma (hPRP) is a candidate replacement for FBS. In this study, the effect of hPRP on MSC proliferation and osteogenic differentiation has been evaluated. Methods: Human adipose-derived stem cells (hADSC) were expanded. Cells from the third passage were characterized by flow cytometric analysis and used for in vitro experiments. Resazurin and alizarin red stains were used for cell proliferation and osteogenic differentiation assays, respectively. Results: Treatment with hPRP resulted in a statistically significant increase in cell proliferation compare to the negative control group (P<0.001). Cell proliferation in the 15% hPRP group was also significantly higher than that in the 10% hPRP group (P<0.05). Additionally, it caused less osteogenic differentiation of the hADSC compared to the FBS (P<0.001), but in comparison to negative control, it caused acceptable mineralization (P<0.001). Conclusion: These findings indicate that hPRP not only improves the proliferation but also it can be a suitable substitution in osteogenic differentiation for clinical purposes. However, the clinical application value of hPRP still needs more investigation. PMID:24842141

  1. The role of reactive oxygen species in mesenchymal stem cell adipogenic and osteogenic differentiation: a review.

    PubMed

    Atashi, Fatemeh; Modarressi, Ali; Pepper, Michael S

    2015-05-15

    Mesenchymal stromal cells (MSCs) are promising candidates for tissue engineering and regenerative medicine. The multipotent stem cell component of MSC isolates is able to differentiate into derivatives of the mesodermal lineage including adipocytes, osteocytes, chondrocytes, and myocytes. Many common pathways have been described in the regulation of adipogenesis and osteogenesis. However, stimulation of osteogenesis appears to suppress adipogenesis and vice-versa. Increasing evidence implicates a tight regulation of these processes by reactive oxygen species (ROS). ROS are short-lived oxygen-containing molecules that display high chemical reactivity toward DNA, RNA, proteins, and lipids. Mitochondrial complexes I and III, and the NADPH oxidase isoform NOX4 are major sources of ROS production during MSC differentiation. ROS are thought to interact with several pathways that affect the transcription machinery required for MSC differentiation including the Wnt, Hedgehog, and FOXO signaling cascades. On the other hand, elevated levels of ROS, defined as oxidative stress, lead to arrest of the MSC cell cycle and apoptosis. Tightly regulated levels of ROS are therefore critical for MSC terminal differentiation, although the precise sources, localization, levels and the exact species of ROS implicated remain to be determined. This review provides a detailed overview of the influence of ROS on adipogenic and osteogenic differentiation in MSCs. PMID:25603196

  2. The Role of Reactive Oxygen Species in Mesenchymal Stem Cell Adipogenic and Osteogenic Differentiation: A Review

    PubMed Central

    Atashi, Fatemeh; Modarressi, Ali

    2015-01-01

    Mesenchymal stromal cells (MSCs) are promising candidates for tissue engineering and regenerative medicine. The multipotent stem cell component of MSC isolates is able to differentiate into derivatives of the mesodermal lineage including adipocytes, osteocytes, chondrocytes, and myocytes. Many common pathways have been described in the regulation of adipogenesis and osteogenesis. However, stimulation of osteogenesis appears to suppress adipogenesis and vice-versa. Increasing evidence implicates a tight regulation of these processes by reactive oxygen species (ROS). ROS are short-lived oxygen-containing molecules that display high chemical reactivity toward DNA, RNA, proteins, and lipids. Mitochondrial complexes I and III, and the NADPH oxidase isoform NOX4 are major sources of ROS production during MSC differentiation. ROS are thought to interact with several pathways that affect the transcription machinery required for MSC differentiation including the Wnt, Hedgehog, and FOXO signaling cascades. On the other hand, elevated levels of ROS, defined as oxidative stress, lead to arrest of the MSC cell cycle and apoptosis. Tightly regulated levels of ROS are therefore critical for MSC terminal differentiation, although the precise sources, localization, levels and the exact species of ROS implicated remain to be determined. This review provides a detailed overview of the influence of ROS on adipogenic and osteogenic differentiation in MSCs. PMID:25603196

  3. On the use of dexamethasone-loaded liposomes to induce the osteogenic differentiation of human mesenchymal stem cells.

    PubMed

    Monteiro, Nelson; Martins, Albino; Ribeiro, Diana; Faria, Susana; Fonseca, Nuno A; Moreira, João N; Reis, Rui L; Neves, Nuno M

    2015-09-01

    Stem cells have received considerable attention by the scientific community because of their potential for tissue engineering and regenerative medicine. The most frequently used method to promote their differentiation is supplementation of the in vitro culture medium with growth/differentiation factors (GDFs). The limitations of that strategy caused by the short half-life of GDFs limit its efficacy in vivo and consequently its clinical use. Thus, the development of new concepts that enable the bioactivity and bioavailability of GDFs to be protected, both in vitro and in vivo, is very relevant. Nanoparticle-based drug delivery systems can be injected, protect the GDFs and enable spatiotemporal release kinetics to be controlled. Liposomes are well-established nanodelivery devices presenting significant advantages, viz. a high load-carrying capacity, relative safety and easy production, and a versatile nature in terms of possible formulations and surface functionalization. The main objective of the present study was to optimize the formulation of liposomes to encapsulate dexamethasone (Dex). Our results showed that the optimized Dex-loaded liposomes do not have any cytotoxic effect on human bone marrow-derived mesenchymal stem cells (hBMSCs). More importantly, they were able to promote an earlier induction of differentiation of hBMSCs into the osteogenic lineage, as demonstrated by the expression of osteoblastic markers, both phenotypically and genotypically. We concluded that Dex-loaded liposomes represent a viable nanoparticle strategy with enhanced safety and efficacy for tissue engineering and regenerative medicine. PMID:24123949

  4. Effects of decellularized matrices derived from periodontal ligament stem cells and SHED on the adhesion, proliferation and osteogenic differentiation of human dental pulp stem cells in vitro.

    PubMed

    Heng, Boon Chin; Zhu, Shaoyue; Xu, Jianguang; Yuan, Changyong; Gong, Ting; Zhang, Chengfei

    2016-04-01

    A major bottleneck to the therapeutic applications of dental pulp stem cells (DPSC) are their limited proliferative capacity ex vivo and tendency to undergo senescence. This may be partly due to the sub-optimal in vitro culture milieu, which could be improved by an appropriate extracellular matrix substratum. This study therefore examined decellularized matrix (DECM) from stem cells derived from human exfoliated deciduous teeth (SHED) and periodontal ligament stem cells (PDLSC), as potential substrata for DPSC culture. Both SHED-DECM and PDLSC-DECM promoted rapid adhesion and spreading of newly-seeded DPSC compared to bare polystyrene (TCPS), with vinculin immunocytochemistry showing expression of more focal adhesions by newly-adherent DPSC cultured on DECM versus TCPS. Culture of DPSC on SHED-DECM and PDLSC-DECM yielded higher proliferation of cell numbers compared to TCPS. The qRT-PCR data showed significantly higher expression of nestin by DPSC cultured on DECM versus the TCPS control. Osteogenic differentiation of DPSC was enhanced by culturing on PDLSC-DECM and SHED-DECM versus TCPS, as demonstrated by alizarin red S staining for mineralized calcium deposition, alkaline phosphatase assay and qRT-PCR analysis of key osteogenic marker expression. Hence, both SHED-DECM and PDLSC-DECM could enhance the ex vivo culture of DPSC under both non-inducing and osteogenic-inducing conditions. PMID:26796232

  5. Macrophages derived from THP-1 promote the osteogenic differentiation of mesenchymal stem cells through the IL-23/IL-23R/β-catenin pathway.

    PubMed

    Tu, Bing; Liu, Shen; Liu, Guangwang; Yan, Wei; Wang, Yugang; Li, Zhiwei; Fan, Cunyi

    2015-11-15

    Abnormal bone formation is a clinically significant dilemma for many conditions in response to injury, inflammation or genetic disease. However, the effects of inflammation on the osteogenic differentiation of mesenchymal stem cells (MSCs) remain unclear. IL-23 secretion from macrophages might contribute to the development of bone formation. Here, we investigated the stimulatory effects of THP-1 macrophage conditioned medium (MΦ CM) on the osteogenic differentiation of human MSCs and the associated signaling pathways. The osteogenic differentiation of MSCs was induced after exposure to osteogenic differentiation medium (OM). MΦ CM significantly increased alkaline phosphate (ALP) activity and calcium mineralization in MSCs. Osteogenic marker genes, including RUNX2, ALP and osteocalcin (OCN), were also up-regulated in MSCs after exposure to MΦ CM. Moreover, western blotting revealed that MΦ CM treatment induced STAT3 and β-catenin activation in MSCs. Furthermore, blockade of IL-23 in MΦ CM not only impaired the osteogenic-promotion effects of macrophage but also decreased the expression of osteogenic maker genes. However, IL-23R silencing suppressed MΦ CM-induced calcium mineralization and osteogenic maker gene expression in MSCs. These data suggest that macrophages derived from THP-1 promote the osteoblastic differentiation of MSCs through the IL-23/IL-23R/β-catenin pathway and macrophages might contribute to the development of bone formation in inflammation. PMID:26477825

  6. Protocols for in vitro Differentiation of Human Mesenchymal Stem Cells into Osteogenic, Chondrogenic and Adipogenic Lineages.

    PubMed

    Ciuffreda, Maria Chiara; Malpasso, Giuseppe; Musarò, Paola; Turco, Valentina; Gnecchi, Massimiliano

    2016-01-01

    Mesenchymal stem cells (MSC) possess high plasticity and the potential to differentiate into several different cell types; this characteristic has implications for cell therapy and reparative biotechnologies. MSC have been originally isolated from the bone marrow (BM-MSC), but they have been found also in other tissues such as adipose tissue, cord blood, synovium, skeletal muscle, and lung. MSC are able to differentiate in vitro and in vivo into several cell types such as bone, osteocytes, chondrocytes, adipocytes, and skeletal myocytes, just to name a few.During the last two decades, an increasing number of studies have proven the therapeutic potential of MSC for the treatment of neurodegenerative diseases, spinal cord and brain injuries, cardiovascular diseases, diabetes mellitus, and diseases of the skeleton. Their immuno-privileged profile allows both autologous and allogeneic use. For all these reasons, the scientific appeal of MSC is constantly on the rise.The identity of MSC is currently based on three main criteria: plastic-adherence capacity, defined epitope profile, and capacity to differentiate in vitro into osteocytes, chondrocytes, and adipocytes. Here, we describe standard protocols for the differentiation of BM-MSC into the osteogenic, chondrogenic, and adipogenic lineages. PMID:27236670

  7. Mechanosensitive TRPM7 mediates shear stress and modulates osteogenic differentiation of mesenchymal stromal cells through Osterix pathway

    PubMed Central

    Liu, Yi-Shiuan; Liu, Yu-An; Huang, Chin-Jing; Yen, Meng-Hua; Tseng, Chien-Tzu; Chien, Shu; Lee, Oscar K.

    2015-01-01

    Microenvironments that modulate fate commitments of mesenchymal stromal cells (MSCs) are composed of chemical and physical cues, but the latter ones are much less investigated. Here we demonstrate that intermittent fluid shear stress (IFSS), a potent and physiologically relevant mechanical stimulus, regulates osteogenic differentiation of MSCs through Transient receptor potential melastatin 7 (TRPM7)-Osterix axis. Immunostaining showed the localization of TRPM7 near or at cell membrane upon IFSS, and calcium imaging analysis demonstrated the transient increase of cytosolic free calcium. Expressions of osteogenic marker genes including Osterix, but not Runx2, were upregulated after three-hour IFSS. Phosphorylation of p38 and Smad1/5 was promoted by IFSS as well. TRPM7 gene knockdown abolished the promotion of bone-related gene expressions and phosphorylation. We illustrate that TRPM7 is mechanosensitive to shear force of 1.2 Pa, which is much lower than 98 Pa pressure loading reported recently, and mediates distinct mechanotransduction pathways. Additionally, our results suggest the differential roles of TRPM7 in endochondral and intramembranous ossification. Together, this study elucidates the mechanotransduction in MSCs fate commitments and displays an efficient mechano-modulation for MSCs osteogenic differentiation. Such findings should be taken into consideration when designing relevant scaffolds and microfluidic devices for osteogenic induction in the future. PMID:26558702

  8. 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

  9. Investigation of low-level laser therapy potentiality on proliferation and differentiation of human osteoblast-like cells in the absence/presence of osteogenic factors

    NASA Astrophysics Data System (ADS)

    Bloise, Nora; Ceccarelli, Gabriele; Minzioni, Paolo; Vercellino, Marco; Benedetti, Laura; De Angelis, Maria Gabriella Cusella; Imbriani, Marcello; Visai, Livia

    2013-12-01

    Several studies have shown that low-level laser irradiation (LLLI) has beneficial effects on bone regeneration. The objective of this study was to examine the in vitro effects of LLLI on proliferation and differentiation of a human osteoblast-like cell line (Saos-2 cell line). Cultured cells were exposed to different doses of LLLI with a semiconductor diode laser (659 nm 10 mW power output). The effects of laser on proliferation were assessed daily up to seven days of culture in cells irradiated once or for three consecutive days with laser doses of 1 or 3 J/cm2. The obtained results showed that laser stimulation enhances the proliferation potential of Saos-2 cells without changing their telomerase pattern or morphological characteristics. The effects on cell differentiation were assessed after three consecutive laser irradiation treatments in the presence or absence of osteo-inductive factors on day 14. Enhanced secretion of proteins specific for differentiation toward bone as well as calcium deposition and alkaline phosphatase activity were observed in irradiated cells cultured in a medium not supplemented with osteogenic factors. Taken together these findings indicate that laser treatment enhances the in vitro proliferation of Saos-2 cells, and also influences their osteogenic maturation, which suggest it is a helpful application for bone tissue regeneration.

  10. Osteogenic differentiation of MC3T3-E1 cells on poly(L-lactide)/Fe3O4 nanofibers with static magnetic field exposure.

    PubMed

    Cai, Qing; Shi, Yuzhou; Shan, Dingying; Jia, Wenkai; Duan, Shun; Deng, Xuliang; Yang, Xiaoping

    2015-10-01

    Proliferation and differentiation of bone-related cells are modulated by many factors such as scaffold design, growth factor, dynamic culture system, and physical simulation. Nanofibrous structure and moderate-intensity (1 mT-1 T) static magnetic field (SMF) have been identified as capable of stimulating proliferation and differentiation of osteoblasts. Herein, magnetic nanofibers were prepared by electrospinning mixture solutions of poly(L-lactide) (PLLA) and ferromagnetic Fe3O4 nanoparticles (NPs). The PLLA/Fe3O4 composite nanofibers demonstrated homogeneous dispersion of Fe3O4 NPs, and their magnetism depended on the contents of Fe3O4 NPs. SMF of 100 mT was applied in the culture of MC3T3-E1 osteoblasts on pure PLLA and PLLA/Fe3O4 composite nanofibers for the purpose of studying the effect of SMF on osteogenic differentiation of osteoblastic cells on magnetic nanofibrous scaffolds. On non-magnetic PLLA nanofibers, the application of external SMF could enhance the proliferation and osteogenic differentiation of MC3T3-E1 cells. In comparison with pure PLLA nanofibers, the incorporation of Fe3O4 NPs could also promote the proliferation and osteogenic differentiation of MC3T3-E1 cells in the absence or presence of external SMF. The marriage of magnetic nanofibers and external SMF was found most effective in accelerating every aspect of biological behaviors of MC3T3-E1 osteoblasts. The findings demonstrated that the magnetic feature of substrate and microenvironment were applicable ways in regulating osteogenesis in bone tissue engineering. PMID:26117751

  11. Proliferation and osteogenic differentiation of human mesenchymal stem cells on zirconia and titanium with different surface topography.

    PubMed

    Hirano, Tomoki; Sasaki, Hodaka; Honma, Shinya; Furuya, Yoshitaka; Miura, Tadashi; Yajima, Yasutomo; Yoshinari, Masao

    2015-01-01

    The purpose of this study was to elucidate behavior of human mesenchymal stem cells (hMSCs) on yttria stabilized tetragonal zirconia polycrystals (TZP) and commercial pure titanium (CpTi) with different surface topography. Mirror-polished (MS), sandblasted with 150-μm alumina (SB150) and SB150 acid-etched (SB150E) were prepared on TZP and CpTi. Proliferation, osteogenic differentiation of hMSCs was evaluated. The scanning electron microscopy showed that micro- and nano-topographies were created on both TZP and CpTi SB150E surfaces. The proliferation ability, ALP activity, expression of Runx2 on the both SB150E specimens was significantly higher than those on the other specimens. These results suggested that creation of micro- and nano-topographies on TZP and CpTi by blast and acid-etching may offer a promising method for enhancing the proliferation and differentiation of hMSCs in clinical application. PMID:26632237

  12. Foxc1 Expression in Early Osteogenic Differentiation Is Regulated by BMP4-SMAD Activity.

    PubMed

    Hopkins, Alexander; Mirzayans, Freda; Berry, Fred

    2016-07-01

    FOXC1 is an important regulator of the initial steps in intramembranous and endochondral ossification processes. As BMP signalling is a key initiator of these processes, we sought to determine whether Foxc1 expression is regulated by such signalling factors. BMP4 treatment of C2C12 cells resulted in an induction in Foxc1 mRNA levels. Chromatin immunoprecipitation assays demonstrated that SMAD proteins interacted with the mouse Foxc1 promoter approximately 300 bp upstream of the transcription start site. This ChIP positive region was cloned into a luciferase reporter and demonstrated to be responsive to BMP4 stimulation. Reduction of Foxc1 levels in C2C12 cells though siRNA impaired BMP4 osteogenic differentiation. In contrast, BMP4 treatment repressed Foxc1 expression in 10T1/2 or D1-ORL mesenchymal cells and MC3T3 preosteoblasts. Finally, siRNA knock-down of Foxc1 in MC3T3 cells resulted in an induction of markers of osteoblast differentiation and an accelerated mineralization. These data indicate that Foxc1 expression is regulated by BMP4 and FOXC1 functions in the commitment of progenitor cells to the osteoblast fate and its expression is reduced when differentiation proceeds. J. Cell. Biochem. 117: 1707-1717, 2016. © 2015 Wiley Periodicals, Inc. PMID:26666591

  13. Osteogenic differentiation of human bone marrow mesenchymal stem cells in hydrogel containing nacre powder.

    PubMed

    Flausse, Alicia; Henrionnet, Christel; Dossot, Manuel; Dumas, Dominique; Hupont, Sébastien; Pinzano, Astrid; Mainard, Didier; Galois, Laurent; Magdalou, Jacques; Lopez, Evelyne; Gillet, Pierre; Rousseau, Marthe

    2013-11-01

    Nacre (or mother of pearl) can facilitate bone cell differentiation and can speed up their mineralization. Here we report on the capability of nacre to induce differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) and the production of extracellular matrix. hBM-MSCs were encapsulated in an alginate hydrogel containing different concentrations of powdered nacre and cultured in the same environment until Day 28. Analysis of osteogenic gene expression, histochemistry, second harmonic generation (SHG) microscopy, and Raman scattering spectroscopy were used to characterize the synthesis of the extracellular matrix. In the presence of nacre powder, a significant increase in matrix synthesis from D21 in comparison with pure alginate was observed. Histochemistry revealed the formation of a new tissue composed of collagen fibers in the presence of nacre (immunostaining and SHG), and hydroxyapatite crystals (Raman) in the alginate beads. These results suggest that nacre is efficient in hBM-MSCs differentiation, extracellular matrix production and mineralization in alginate 3D biomaterials. PMID:23554327

  14. cAMP/PKA signaling inhibits osteogenic differentiation and bone formation in rodent models.

    PubMed

    Siddappa, Ramakrishnaiah; Mulder, Winfried; Steeghs, Ilse; van de Klundert, Christine; Fernandes, Hugo; Liu, Jun; Arends, Roel; van Blitterswijk, Clemens; de Boer, Jan

    2009-08-01

    We previously demonstrated that cAMP-mediated protein kinase A (PKA) activation induces in vitro osteogenesis and in vivo bone formation by human mesenchymal stem cells (hMSCs). To analyze the species-specific response of this phenomenon and to translate our findings into a clinical trial, suitable animal models and cell lines are desirable. In this report, we assessed whether PKA plays a similar proosteogenic role played by two commonly used PKA activators-N6,2'-O-dibutyryl-cAMP (db-cAMP) and 8-bromo cAMP (8b-cAMP)-in a number of model systems. To this end, we treated MC3T3-E1 cells, mouse calvarial osteoblasts, mouse MSCs, and rat MSCs with cAMP. We demonstrate that cAMP inhibits osteogenesis in rodent cell types, evidenced by inhibition of osteogenic markers such as alkaline phosphatase (ALP), osteocalcin (BGLAP), and collagen type 1 (COL1A1). In support of this, ex vivo-cultured mouse calvaria exposed to db-cAMP showed a reduction in bone volume. Interestingly, cAMP even stimulated adipogenic differentiation in rat MSCs. Taken together, our data demonstrate that cAMP inhibits osteogenesis in vitro and bone formation ex vivo in rodent models in contrast to our earlier findings in hMSCs. The species discrepancy in response to various osteogenic signals is a critical need to be tested in clinically relevant models to translate the fundamental findings in lower species level to clinical applications. PMID:19231969

  15. Osteogenic differentiation of human bone marrow mesenchymal stem cells seeded on melt based chitosan scaffolds for bone tissue engineering applications.

    PubMed

    Costa-Pinto, Ana R; Correlo, Vitor M; Sol, Paula C; Bhattacharya, Mrinal; Charbord, Pierre; Delorme, Bruno; Reis, Rui L; Neves, Nuno M

    2009-08-10

    The purpose of this study was to evaluate the growth patterns and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) when seeded onto new biodegradable chitosan/polyester scaffolds. Scaffolds were obtained by melt blending chitosan with poly(butylene succinate) in a proportion of 50% (wt) each and further used to produce a fiber mesh scaffold. hBMSCs were seeded on those structures and cultured for 3 weeks under osteogenic conditions. Cells were able to reduce MTS and demonstrated increasing metabolic rates over time. SEM observations showed cell colonization at the surface as well as within the scaffolds. The presence of mineralized extracellular matrix (ECM) was successfully demonstrated by peaks corresponding to calcium and phosphorus elements detected in the EDS analysis. A further confirmation was obtained when carbonate and phosphate group peaks were identified in Fourier Transformed Infrared (FTIR) spectra. Moreover, by reverse transcriptase (RT)-PCR analysis, it was observed the expression of osteogenic gene markers, namely, Runt related transcription factor 2 (Runx2), type 1 collagen, bone sialoprotein (BSP), and osteocalcin. Chitosan-PBS (Ch-PBS) biodegradable scaffolds support the proliferation and osteogenic differentiation of hBMSCs cultured at their surface in vitro, enabling future in vivo testing for the development of bone tissue engineering therapies. PMID:19621927

  16. Differential osteogenic potential of human adipose-derived stem cells co-cultured with human osteoblasts on polymeric microfiber scaffolds.

    PubMed

    Rozila, Ismail; Azari, Pedram; Munirah, Sha'ban; Wan Safwani, Wan Kamarul Zaman; Gan, Seng Neon; Nur Azurah, Abdul Ghani; Jahendran, Jeevanan; Pingguan-Murphy, Belinda; Chua, Kien Hui

    2016-02-01

    The osteogenic potential of human adipose-derived stem cells (HADSCs) co-cultured with human osteoblasts (HOBs) using selected HADSCs/HOBs ratios of 1:1, 2:1, and 1:2, respectively, is evaluated. The HADSCs/HOBs were seeded on electrospun three-dimensional poly[(R)-3-hydroxybutyric acid] (PHB) blended with bovine-derived hydroxyapatite (BHA). Monocultures of HADSCs and HOBs were used as control groups. The effects of PHB-BHA scaffold on cell proliferation and cell morphology were assessed by AlamarBlue assay and field emission scanning electron microscopy. Cell differentiation, cell mineralization, and osteogenic-related gene expression of co-culture HADSCs/HOBs were examined by alkaline phosphatase (ALP) assay, alizarin Red S assay, and quantitative real time PCR, respectively. The results showed that co-culture of HADSCs/HOBs, 1:1 grown into PHB-BHA promoted better cell adhesion, displayed a significant higher cell proliferation, higher production of ALP, extracellular mineralization and osteogenic-related gene expression of run-related transcription factor, bone sialoprotein, osteopontin, and osteocalcin compared to other co-culture groups. This result also suggests that the use of electrospun PHB-BHA in a co-culture HADSCs/HOBs system may serve as promising approach to facilitate osteogenic differentiation activity of HADSCs through direct cell-to-cell contact with HOBs. PMID:26414782

  17. Celastrol inhibits prostaglandin E2-induced proliferation and osteogenic differentiation of fibroblasts isolated from ankylosing spondylitis hip tissues in vitro

    PubMed Central

    Zou, Yu-Cong; Yang, Xian-Wen; Yuan, Shi-Guo; Zhang, Pei; Li, Yi-Kai

    2016-01-01

    Background Heterotopic ossification on the enthesis, which develops after subsequent inflammation, is one of the most distinctive features in ankylosing spondylitis (AS). Prostaglandin E2 (PGE-2) serves as a key mediator of inflammation and bone remodeling in AS. Celastrol, a well-known Chinese medicinal herb isolated from Tripterygium wilfordii, is widely used in treating inflammatory diseases, including AS. It has been proven that it can inhibit lipopolysac-charide-induced expression of various inflammation mediators, such as PGE-2. However, the mechanism by which celastrol inhibits inflammation-induced bone forming in AS is unclear. Objective To investigate whether celastrol could inhibit isolated AS fibroblast osteogenesis induced by PGE-2. Methods Hip synovial tissues were obtained from six AS patients undergoing total hip replacement in our hospital. Fibroblasts were isolated, primarily cultured, and then treated with PGE-2 for osteogenic induction. Different doses of celastrol and indometacin were added to observe their effects on osteogenic differentiation. Cell proliferation, osteogenic markers, alizarin red staining as well as the activity of alkaline phosphatase were examined in our study. Results Celastrol significantly inhibits cell proliferation of isolated AS fibroblasts and in vitro osteogenic differentiation compared with control groups in a time- and dose-dependent manner. Conclusion Our results demonstrated that celastrol could inhibit isolated AS fibroblast proliferation and in vitro osteogenic differentiation. The interaction of PI3K/AKT signaling and Wnt protein may be involved in the process. Further studies should be performed in vivo and animal models to identify the potential effect of celastrol on the bone metabolism of AS patients. PMID:27022241

  18. Proliferation, osteogenic differentiation, and distribution of rat bone marrow stromal cells in nonwoven fabrics by different culture methods.

    PubMed

    Ichinohe, Norihisa; Takamoto, Tomoaki; Tabata, Yasuhiko

    2008-01-01

    The proliferation, osteogenic differentiation, and distribution patterns of stromal cells from rat bone marrow were investigated in a three-dimensional nonwoven fabric of polyethylene terephthalate fiber by the static, agitated, and stirred culture methods; stirring speeds were 10, 50, and 100 rpm in the stirred culture method. The culture method affected the time profile of proliferation and osteogenic differentiation of cells or their distribution in the fabric. The extent of cell proliferation and osteogenic differentiation became higher in order of the stirred at 100 rpm = the stirred at 50 rpm > the stirred at 10 rpm > the agitated > the static methods. In addition, the cells were more uniformly proliferated in the fabric by the stirred culture method with time than they were proliferated in the fabric by other methods. The alkaline phosphatase (ALP) activity and calcium content were higher for cells cultured by the stirred culture method than those cultured by other methods. The total ALP activity, calcium content, and bone mineral density were higher for every stirred method than those for other methods. However, the distribution uniformity of cells differentiated was low irrespective of the culture method. It is concluded that the extent of proliferation and differentiation of cells or their distribution uniformity in the nonwoven fabrics was influenced by the culture method. PMID:18333809

  19. Differentiations and Functional State of Osteogenic Cells in Conditions of Microgravity

    NASA Astrophysics Data System (ADS)

    Onishchenko, Ganna; Rodionova, Natalia; Markevich, Ganna; Markevich, Ganna

    The space flight factors (space radiation, magnetic fields etc.) affect considerably the state of bone tissue, leading to the development of osteoporosis and osteopenia in the bone skeleton. Many aspects of reactions of bone tissue cells still remain unclear until now. With the use of electron microscopy and autoradiography with 3H-thymidine we studied the samples gathered from the femoral bone epiphyses and metaphyses of rats flown on board American Spacelab -2 and in experiments with modeling of microgravity ("tail suspension" method). In our work the main attention is focused on studying the ultrastructure and metabolism of osteogenetic cells. The degree of differentiation and functional state are evaluated according to the degree of development of organelles for specific biosynthesis: rough endoplasmic reticulum (RER), Golgy complex (GC), as well as the state of mitochondria and cell nucleus. As compared with a control, the population of osteogenetic cells from zones of bone reconstruction shows a decrease in the number of functionally active forms. We can judge of this from the reduction volume of RER, GC, mitochondria in osteoblasts. RER loses architectonics typical for osteoblasts and, as against the control, is represented by short narrow canaliculi distributed throughout the cy-toplasm; some canals disintegrate. GC is slightly pronounced, mitochondria become smaller in size and acquire an optically dark matrix. These phenomena are supposed to be associated with the desorganization of microtubules and microfilaments in the cells under microgravity condi-tions. The number of degrading and apoptotic cells increases in the population of osteoblasts. The dynamics of labeled cells following various intervals after 3H-thymidine injection testifies to a delay in the rates of osteoblasts' differentiation and their transformation to osteocytes in the experiment animals. A lower 3H-glycine uptake by the osteogenic cells and bone matrix as compared with a control is

  20. Lithium chloride attenuates BMP-2 signaling and inhibits osteogenic differentiation through a novel WNT/GSK3- independent mechanism.

    PubMed

    Li, Jingjing; Khavandgar, Zohreh; Lin, Sue-Hwa; Murshed, Monzur

    2011-02-01

    Lithium inhibition of glycogen synthase kinase3 (GSK3) activity has been shown to mimic the canonical WNT signaling. Analogous to WNT, lithium prevents GSK3-mediated phosphorylation of cytosolic transcription factor β-catenin and its subsequent degradation by the proteasome complex. Although stabilization of β-catenin in osteoblasts has been shown to promote bone mass accrual in a mouse model, several studies reported inhibitory effects of lithium supplements on the osteogenic differentiation of cultured mesenchymal stem cells. One possible explanation for these apparent contradictory findings might be that lithium affects the differentiation of osteoblast progenitors through additional signaling events, which independently or in concert with WNT signaling, affect the bone resorption activities in vivo. In the current study, we used murine MC3T3-E1 pre-osteoblasts and a pluripotent mesenchymal cell line C2C12 to investigate lithium effects during the early stages of osteoblast differentiation. We demonstrate here that lithium inhibits BMP-2 signaling to affect osteogenic differentiation in both cell lines. Lithium treatment reduces BMP-2-induced SMAD 1,5,8 phosphorylation in both MC3T3-E1 and C2C12 cells without affecting their dephosphorylation. Additionally, in MC3T3-E1 cells, lithium attenuates BMP-2-induced osteogenic differentiation through GSK3 inhibition; while in C2C12 cells, these negative effects of lithium ions on BMP-2 signaling do not rely on GSK3 inhibition or activation of canonical WNT signaling. Our work suggests the presence of a novel GSK3/WNT-independent mechanism of lithium action during the early stages of osteogenic differentiation. PMID:20932949

  1. TLR4 Activation Promotes Bone Marrow MSC Proliferation and Osteogenic Differentiation via Wnt3a and Wnt5a Signaling

    PubMed Central

    He, Xiaoqing; Wang, Hai; Jin, Tao; Xu, Yongqing; Mei, Liangbin; Yang, Jun

    2016-01-01

    Mesenchymal stem cells (MSCs) from adult bone marrow maintain their self-renewal ability and the ability to differentiate into osteoblast. Thus, adult bone marrow MSCs play a key role in the regeneration of bone tissue. Previous studies indicated that TLR4 is expressed in MSCs and is critical in regulating the fate decision of MSCs. However, the exact functional role and underlying mechanisms of how TLR4 regulate bone marrow MSC proliferation and differentiation are unclear. Here, we found that activated TLR4 by its ligand LPS promoted the proliferation and osteogenic differentiation of MSCs in vitro. TLR4 activation by LPS also increased cytokine IL-6 and IL-1β production in MSCs. In addition, LPS treatment has no effect on inducing cell death of MSCs. Deletion of TLR4 expression in MSCs completely eliminated the effects of LPS on MSC proliferation, osteogenic differentiation and cytokine production. We also found that the mRNA and protein expression of Wnt3a and Wnt5a, two important factors in regulating MSC fate decision, was upregulated in a TLR4-dependent manner. Silencing Wnt3a with specific siRNA remarkably inhibited TLR4-induced MSC proliferation, while Wnt5a specific siRNA treatment significantly antagonized TLR4-induced MSC osteogenic differentiation. These results together suggested that TLR4 regulates bone marrow MSC proliferation and osteogenic differentiation through Wnt3a and Wnt5a signaling. These finding provide new data to understand the role and the molecular mechanisms of TLR4 in regulating bone marrow MSC functions. These data also provide new insight in developing new therapy in bone regeneration using MSCs by modulating TLR4 and Wnt signaling activity. PMID:26930594

  2. The effect of synthetic α-tricalcium phosphate on osteogenic differentiation of rat bone mesenchymal stem cells

    PubMed Central

    Liu, Jinzhong; Zhao, Liang; Ni, Ling; Qiao, Chunyan; Li, Daowei; Sun, Hongchen; Zhang, Zongtao

    2015-01-01

    The reconstruction of large bone defects has been the focus in bone tissue engineering research. By acting as synthetic frameworks for cell growth and tissue formation, biomaterials can play a critical role in bone tissue engineering. Among various biomaterials, calcium phosphate based materials include hydroxyapatite (HA), α-tricalcium phosphate (α-TCP), and β-tricalcium phosphate (β-TCP) are widely used as scaffold materials in bone tissue engineering. However, little is known about the effect of α-TCP alone on the osteogenic differentiation of the BMSCs. To this end, we synthesized α-TCP using a novel co-precipitation method. The synthetic α-TCP was then incubated with rat BMSCs under osteogenic inductive medium culture conditions, followed by the analysis of the mRNA levels of various osteogenesis-related genes, including ALP, Rux2, COL-I, and SP7, using a quantitative RT-PCR method. Following incubation of BMSCs with 20 μg/ml α-TCP, cells reached confluency after 7 days. Additionally, the MTT analysis showed that α-TCP at concentration of 10-20 μg/ml had good biocompatibility with BMSCs, showing no significant inhibition of rat BMSCs proliferation. Furthermore, the synthetic α-TCP (20 μg/ml), when incubated with rat BMSCs in the osteogenic culture medium, increased the mRNA levels of various osteogenesis-related genes, including ALP, Rux2, COL-I, and SP7. Finally, treatment of synthetic α-TCP (20 μg/ml) potentiated calcium nodule formations after incubation with rat BMSCs in osteogenic culture medium for 21 days, as compared with non-treated control. Taken together, the results in the present study suggested that α-TCP alone likely promotes rat BMSCs osteogenic differentiation through up-regulating ALP, Col-I, Runx2, and SP7 gene expression. PMID:26550458

  3. The effect of synthetic α-tricalcium phosphate on osteogenic differentiation of rat bone mesenchymal stem cells.

    PubMed

    Liu, Jinzhong; Zhao, Liang; Ni, Ling; Qiao, Chunyan; Li, Daowei; Sun, Hongchen; Zhang, Zongtao

    2015-01-01

    The reconstruction of large bone defects has been the focus in bone tissue engineering research. By acting as synthetic frameworks for cell growth and tissue formation, biomaterials can play a critical role in bone tissue engineering. Among various biomaterials, calcium phosphate based materials include hydroxyapatite (HA), α-tricalcium phosphate (α-TCP), and β-tricalcium phosphate (β-TCP) are widely used as scaffold materials in bone tissue engineering. However, little is known about the effect of α-TCP alone on the osteogenic differentiation of the BMSCs. To this end, we synthesized α-TCP using a novel co-precipitation method. The synthetic α-TCP was then incubated with rat BMSCs under osteogenic inductive medium culture conditions, followed by the analysis of the mRNA levels of various osteogenesis-related genes, including ALP, Rux2, COL-I, and SP7, using a quantitative RT-PCR method. Following incubation of BMSCs with 20 μg/ml α-TCP, cells reached confluency after 7 days. Additionally, the MTT analysis showed that α-TCP at concentration of 10-20 μg/ml had good biocompatibility with BMSCs, showing no significant inhibition of rat BMSCs proliferation. Furthermore, the synthetic α-TCP (20 μg/ml), when incubated with rat BMSCs in the osteogenic culture medium, increased the mRNA levels of various osteogenesis-related genes, including ALP, Rux2, COL-I, and SP7. Finally, treatment of synthetic α-TCP (20 μg/ml) potentiated calcium nodule formations after incubation with rat BMSCs in osteogenic culture medium for 21 days, as compared with non-treated control. Taken together, the results in the present study suggested that α-TCP alone likely promotes rat BMSCs osteogenic differentiation through up-regulating ALP, Col-I, Runx2, and SP7 gene expression. PMID:26550458

  4. The effect of glow discharge plasma surface modification of polymers on the osteogenic differentiation of committed human mesenchymal stem cells.

    PubMed

    Mwale, Fackson; Wang, Hong Tian; Nelea, Valentin; Luo, Li; Antoniou, John; Wertheimer, Michael R

    2006-04-01

    Little is known of the effect of material surfaces on stem cell differentiation. The present study has addressed the hypothesis that the interaction of mesenchymal stem cells (MSCs) with material surfaces modified by glow discharge plasma is a major regulator of osteogenic differentiation. We found that biaxially oriented polypropylene (BOPP) plasma treated in ammonia significantly reduced up-regulation of expression of osteogenic marker genes, such as alkaline phosphatase (ALP), bone sialoprotein (BSP) and osteocalcin (OC). In contrast, ALP expression was up-regulated when cultured on treated Nylon-6 polyamide (Ny-t) but was substantially reduced when cultured on its pristine counterpart (Ny-p) on day 3. On day 7, ALP expression was down-regulated with MSCs cultured on Ny-t although its expression level was up again on day 14. BSP was expressed weakly on day 3, but was up-regulated when cultured on Ny-t and Ny-p. Its expression reached its maximum on day 14 when cultured on a polystyrene control, while it was cyclically up-regulated on Ny-t. Similarly, there was a slight increase in OC expression when MSCs were cultured on Ny-t and Ny-p on day 3, when compared to control. Thus, the nature of the surface can directly influence MSCs differentiation, ultimately affecting the quality of new tissue formation with BOPP-t suppressing osteogenic differentiation. PMID:16313952

  5. Lithium chloride attenuates the abnormal osteogenic/adipogenic differentiation of bone marrow-derived mesenchymal stem cells obtained from rats with steroid-related osteonecrosis by activating the β-catenin pathway

    PubMed Central

    YU, ZEFENG; FAN, LIHONG; LI, JIA; GE, ZHAOGANG; DANG, XIAOQIAN; WANG, KUNZHENG

    2015-01-01

    Steroid-related osteonecrosis of the femoral head (ONFH) may be a disease that results from the abnormal osteogenic/adipogenic differentiation of bone marrow-derived mesenchymal stem cells (BMMSCs). In the present study, we examined the possible use of lithium in an aim to reverse the abnormal osteogenic/adipogenic differentiation of BMMSCs isolated from rats with steroid-related ONFH (termed ONFH-BMMSCs). BMMSCs obtained from steroid-related ONFH rat femurs were cultured with or without lithium chloride (LiCl). BMMSCs obtained from normal rat femurs were cultured as controls. LiCl significantly increased the expression of osteocalcin and Runx2 in the ONFH-BMMSCs during osteogenic induction. The mineralization of ONFH-BMMSCs following osteogenic induction was also enhanced. Furthermore, LiCl exerted anti-adipogenic effects on the ONFH-BMMSCs by inhibiting the expression of peroxisome proliferator-activated receptor γ (PPARγ) and fatty acid binding protein 4 (Fabp4) during adipogenic induction, and decreasing lipid droplet formation at the end of adipogenic induction. These effects of LiCl on the ONFH-BMMSCs were associated with an increased expression of β-catenin and a decreased expression of phosphorylated GSK-3β at Tyr-216, and these effects were abolished by treatment with quercetin, an antagonist of the β-catenin pathway. The normal osteogenic/adipogenic activity of BMMSCs may be impaired in steroid-related ONFH. However, as demonstrated by our findings, LiCl reduces abnormal adipogenic activity and simultaneously increases the osteogenic differentiation of ONFH-BMMSCs by activating the β-catenin pathway. PMID:26352537

  6. Lithium chloride attenuates the abnormal osteogenic/adipogenic differentiation of bone marrow-derived mesenchymal stem cells obtained from rats with steroid-related osteonecrosis by activating the β-catenin pathway.

    PubMed

    Yu, Zefeng; Fan, Lihong; Li, Jia; Ge, Zhaogang; Dang, Xiaoqian; Wang, Kunzheng

    2015-11-01

    Steroid-related osteonecrosis of the femoral head (ONFH) may be a disease that results from the abnormal osteogenic/adipogenic differentiation of bone marrow-derived mesenchymal stem cells (BMMSCs). In the present study, we examined the possible use of lithium in an aim to reverse the abnormal osteogenic/adipogenic differentiation of BMMSCs isolated from rats with steroid-related ONFH (termed ONFH-BMMSCs). BMMSCs obtained from steroid‑related ONFH rat femurs were cultured with or without lithium chloride (LiCl). BMMSCs obtained from normal rat femurs were cultured as controls. LiCl significantly increased the expression of osteocalcin and Runx2 in the ONFH-BMMSCs during osteogenic induction. The mineralization of ONFH-BMMSCs following osteogenic induction was also enhanced. Furthermore, LiCl exerted anti-adipogenic effects on the ONFH-BMMSCs by inhibiting the expression of peroxisome proliferator-activated receptor γ (PPARγ) and fatty acid binding protein 4 (Fabp4) during adipogenic induction, and decreasing lipid droplet formation at the end of adipogenic induction. These effects of LiCl on the ONFH-BMMSCs were associated with an increased expression of β-catenin and a decreased expression of phosphorylated GSK-3β at Tyr-216, and these effects were abolished by treatment with quercetin, an antagonist of the β-catenin pathway. The normal osteogenic/adipogenic activity of BMMSCs may be impaired in steroid-related ONFH. However, as demonstrated by our findings, LiCl reduces abnormal adipogenic activity and simultaneously increases the osteogenic differentiation of ONFH-BMMSCs by activating the β-catenin pathway. PMID:26352537

  7. MicroRNA-103a-3p controls proliferation and osteogenic differentiation of human adipose tissue-derived stromal cells

    PubMed Central

    Sol Kim, Da; Young Lee, Sun; Hee Lee, Jung; Chan Bae, Yong; Sup Jung, Jin

    2015-01-01

    The elucidation of the molecular mechanisms underlying the differentiation and proliferation of human adipose tissue-derived stromal cells (hADSCs) represents a critical step in the development of hADSCs-based cellular therapies. To examine the role of the microRNA-103a-3p (miR-103a-3p) in hADSCs functions, miR-103a-3p mimics were transfected into hADSCs in order to overexpress miR-103a-3p. Osteogenic differentiation was induced for 14 days in an osetogenic differentiation medium and assessed by using an Alizarin Red S stain. The regulation of the expression of CDK6 (cyclin-dependent kinase 6), a predicted target of miR-103a-3p, was determined by western blot, real-time PCR and luciferase reporter assays. Overexpression of miR-103a-3p inhibited the proliferation and osteogenic differentiation of hADSCs. In addition, it downregulated protein and mRNA levels of predicted target of miR-103a-3p (CDK6 and DICER1). In contrast, inhibition of miR-103a-3p with 2′O methyl antisense RNA increased the proliferation and osteogenic differentiation of hADSCs. The luciferase reporter activity of the construct containing the miR-103a-3p target site within the CDK6 and DICER1 3′-untranslated regions was lower in miR-103a-3p-transfected hADSCs than in control miRNA-transfected hADSCs. RNA interference-mediated downregulation of CDK6 and DICER1 in hADSCs inhibited their proliferation and osteogenic differentiation. The results of the current study indicate that miR-103a-3p regulates the osteogenic differentiation of hADSCs and proliferation of hADSCs by direct targeting of CDK6 and DICER1 partly. These findings further elucidate the molecular mechanisms governing the differentiation and proliferation of hADSCs. PMID:26160438

  8. Could the effect of modeled microgravity on osteogenic differentiation of human mesenchymal stem cells be reversed by regulation of signaling pathways?

    PubMed

    Zheng, Qiang; Huang, Guoping; Yang, Jinfeng; Xu, Yulin; Guo, Chunjuan; Xi, Yongmei; Pan, Zhijun; Wang, Jinfu

    2007-07-01

    Microgravity (MG) results in a reduction in bone formation. Bone formation involves osteogenic differentiation from mesenchymal stem cells (hMSCs) in bone marrow. We modeled MG to determine its effects on osteogenesis of hMSCs and used activators or inhibitors of signaling factors to regulate osteogenic differentiation. Under osteogenic induction, MG reduced osteogenic differentiation of hMSCs and decreased the expression of osteoblast gene markers. The expression of Runx2 was also inhibited, whereas the expression of PPARgamma2 increased. MG also decreased phosphorylation of ERK, but increased phosphorylation of p38MAPK. SB203580, a p38MAPK inhibitor, was able to inhibit the phosphorylation of p38MAPK, but did not reduce the expression of PPARgamma2. Bone morphogenetic protein (BMP) increased the expression of Runx2. Fibroblast growth factor 2 (FGF2) increased the phosphorylation of ERK, but did not significantly increase the expression of osteoblast gene markers. The combination of BMP, FGF2 and SB203580 significantly reversed the effect of MG on osteogenic differentiation of hMSCs. Our results suggest that modeled MG inhibits the osteogenic differentiation and increases the adipogenic differentiation of hMSCs through different signaling pathways. Therefore, the effect of MG on the differentiation of hMSCs could be reversed by the mediation of signaling pathways. PMID:17570829

  9. Development of an early estimation method for predicting later osteogenic differentiation activity of rat mesenchymal stromal cells from their attachment areas

    NASA Astrophysics Data System (ADS)

    Cheng, Kan; Hirose, Motohiro; Wang, Xiupeng; Sogo, Yu; Yamazaki, Atsushi; Ito, Atsuo

    2012-12-01

    Cell morphology has received considerable attention in recent years owing to its possible relationship with cell functions, including proliferation, differentiation, and migration. Recent evidence suggests that extracellular environments can also mediate cell functions, particularly cell adhesion. The aims of this study were to investigate the correlation between osteogenic differentiation activity and the morphology of rat mesenchymal stromal cells (MSCs), and to develop a method of estimating osteogenic differentiation capability of MSCs on biomaterials. We measured the attachment areas of MSCs on substrates with various types of surface after 2 h of seeding, and quantified the amount of osteocalcin secreted from MSCs after 3 weeks of culture under osteogenic differentiation conditions. MSCs with small attachment areas showed a high osteogenic differentiation activity. These findings indicate that cell attachment areas correlate well with the osteogenic differentiation activity of MSCs. They also suggest that the measurement of cell attachment areas is useful for estimating the osteogenic differentiation activity of MSCs and is a practical tool for applications of MSCs in regenerative medicine.

  10. Evaluation of injectable strontium-containing borate bioactive glass cement with enhanced osteogenic capacity in a critical-sized rabbit femoral condyle defect model.

    PubMed

    Zhang, Yadong; Cui, Xu; Zhao, Shichang; Wang, Hui; Rahaman, Mohamed N; Liu, Zhongtang; Huang, Wenhai; Zhang, Changqing

    2015-02-01

    The development of a new generation of injectable bone cements that are bioactive and have enhanced osteogenic capacity for rapid osseointegration is receiving considerable interest. In this study, a novel injectable cement (designated Sr-BBG) composed of strontium-doped borate bioactive glass particles and a chitosan-based bonding phase was prepared and evaluated in vitro and in vivo. The bioactive glass provided the benefits of bioactivity, conversion to hydroxyapatite, and the ability to stimulate osteogenesis, while the chitosan provided a cohesive biocompatible and biodegradable bonding phase. The Sr-BBG cement showed the ability to set in situ (initial setting time = 11.6 ± 1.2 min) and a compressive strength of 19 ± 1 MPa. The Sr-BBG cement enhanced the proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells in vitro when compared to a similar cement (BBG) composed of chitosan-bonded borate bioactive glass particles without Sr. Microcomputed tomography and histology of critical-sized rabbit femoral condyle defects implanted with the cements showed the osteogenic capacity of the Sr-BBG cement. New bone was observed at different distances from the Sr-BBG implants within eight weeks. The bone-implant contact index was significantly higher for the Sr-BBG implant than it was for the BBG implant. Together, the results indicate that this Sr-BBG cement is a promising implant for healing irregularly shaped bone defects using minimally invasive surgery. PMID:25591177

  11. Effects of pH and thermally sensitive hybrid gels on osteogenic differentiation of mesenchymal stem cells.

    PubMed

    Dai, Zhuojun; Shu, Yinglan; Wan, Chao; Wu, Chi

    2015-04-01

    Osteoblastic differentiation of mesenchymal stem cells from bone marrow is an essential step for bone formation. The osteogenesis is normally induced by chemical mediators. Recent laboratory studies have revealed that mechanical properties of an extracellular matrix, typically hydrogels with different modules, also affect the fate of stem cells. The question is how to adjust their mechanical properties inside the body in biomedical applications. In this study, we designed/used a novel extracellular matrix, namely, a hybrid gel made of billions of injectable small thermally and pH-sensitive poly(N-isopropylacrylamide-co-acrylic acid) microgels whose swelling at the body pH and temperature physically jammed them and mesenchymal stem cells together, which enabled us to in situ apply an adjustable mechanical stress on those embedded stem cells. By treating the cell layer with the microgels, we found that an earlier incorporation of the microgels significantly increases the alkaline phosphatase activity, while a later addition of the microgels after the primary calcium deposition enhances the extracellular matrix mineralization in the mesenchymal stem cells cultures accompanied by up-regulation of osteogenic marker genes expression, presumably due to the calcium fixation by the carboxyl groups inside the microgels and the physical contact between the microgels and mesenchymal stem cells layers. These microgels provide an extracellular matrix microenvironment to affect the fate and biological behavior of mesenchymal stem cells, facilitating their potential applications in regenerative therapies. PMID:25361919

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

    PubMed

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

    2007-01-01

    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 x 10(6)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

  13. 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

  14. 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

    2015-12-01

    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. PMID:26482937

  15. Embedded silica nanoparticles in poly(caprolactone) nanofibrous scaffolds enhanced osteogenic potential for bone tissue engineering.

    PubMed

    Ganesh, Nitya; Jayakumar, Rangasamy; Koyakutty, Manzoor; Mony, Ullas; Nair, Shantikumar V

    2012-09-01

    Poly(caprolactone) (PCL) has been frequently considered for bone tissue engineering because of its excellent biocompatibility. A drawback, however, of PCL is its inadequate mechanical strength for bone tissue engineering and its inadequate bioactivity to promote bone tissue regeneration from mesenchymal stem cells. To correct this deficiency, this work investigates the addition of nanoparticles of silica (nSiO(2)) to the scaffold to take advantage of the known bioactivity of silica as an osteogenic material and also to improve the mechanical properties through nanoscale reinforcement of the PCL fibers. The nanocomposite scaffolds and the pristine PCL scaffolds were evaluated physicochemically, mechanically, and biologically in the presence of human mesenchymal stem cells (hMSCs). The results indicated that, when the nanoparticles of size approximately 10 nm (concentrations of 0.5% and 1% w/v) were embedded within, or attached to, the PCL nanofibers, there was a substantial increase in scaffold strength, protein adsorption, and osteogenic differentiation of hMSCs. These nSiO(2) nanoparticles, when directly added to the cells evidently pointed to ingestion of these particles by the cells followed by cell death. The polymer nanofibers appeared to protect the cells by preventing ingestion of the silica nanoparticles, while at the same time adequately exposing them on fiber surfaces for their desired bioactivity. PMID:22725098

  16. Effects of Negative Pressure Wound Therapy on Mesenchymal Stem Cells Proliferation and Osteogenic Differentiation in a Fibrin Matrix

    PubMed Central

    Zhu, Jin; Yu, Aixi; Qi, Baiwen; Li, Zonghuan; Hu, Xiang

    2014-01-01

    Vacuum-assisted closure (VAC) negative pressure wound therapy (NPWT) has been proven to be an effective therapeutic method for the treatment of recalcitrant wounds. However, its role in bone healing remains to be unclear. Here, we investigated the effects of NPWT on rat periosteum-derived mesenchymal stem cells (P-MSCs) proliferation and osteoblastic differentiation in a 3D fibrin matrix. P-MSCs underwent primary culture for three passages before being used to construct cell clots. The fibrin clots were incubated with NPWT under continuous suction at −125 mmHg in a subatmospheric perfusion bioreactor. Clots exposed to atmospheric pressure served as the static control. Compared to the control group, cell proliferation significantly increased in NPWT group after incubation for 3 days. There was no statistical difference in apoptosis rate between two groups. The ALP activity and mineralization of P-MSCs all increased under continuous suction. The expressions of collagen type 1 and transcription factor Cbfa-1 were higher at the 1-, 3-, and 7-day timepoints and the expressions of osteocalcin and integrin β5 were higher at the 3-, and 7-day timepoints in the NPWT group. These results indicate that a short time treatment with NPWT, applied with continuous suction at −125 mmHg, can enhance cellular proliferation of P-MSCs and induce the differentiation toward an osteogenic phenotype. The mechanotransduction molecule integrin β5 was found to be highly expressed after NPWT treatment, which indicates that NPWT may play a positive role in fracture healing through enhance bone formation and decrease bone resorption. PMID:25216182

  17. Glutamine-chitosan modified calcium phosphate nanoparticles for efficient siRNA delivery and osteogenic differentiation

    PubMed Central

    Choi, Bogyu; Cui, Zhong-Kai; Kim, Soyon; Fan, Jiabing; Wu, Benjamin M.

    2015-01-01

    RNA interference (RNAi)-based therapy using small interfering RNA (siRNA) exhibits great potential to treat diseases. Although calcium phosphate (CaP)-based systems are attractive options to deliver nucleic acids due to their good biocompatibility and high affinity with nucleic acids, they are limited by uncontrollable particle formation and inconsistent transfection efficiencies. In this study, we developed a stable CaP nanocarrier system with enhanced intracellular uptake by adding highly cationic, glutamine-conjugated oligochitosan (Gln-OChi). CaP nanoparticles coated with Gln-OChi (CaP/Gln-OChi) significantly enhanced gene transfection and knockdown efficiency in both immortalized cell line (HeLa) and primary mesenchymal stem cells (MSCs) with minimal cytotoxicity. The osteogenic bioactivity of siRNA-loaded CaP/Gln-OChi particles was further confirmed in three-dimensional environments by using photocrosslinkable chitosan hydrogels encapsulating MSCs and particles loaded with siRNA targeting noggin, a bone morphogenetic protein antagonist. These findings suggest that our CaP/Gln-OChi nanocarrier provides an efficient and safe gene delivery system for therapeutic applications. PMID:26413302

  18. Titania nanotube delivery fetal bovine serum for enhancing MC3T3-E1 activity and osteogenic gene expression.

    PubMed

    Peng, Jing; Zhang, Xinming; Li, Zhaoyang; Liu, Yunde; Yang, Xianjin

    2015-11-01

    Titania nanotube (TNT) delivery of fetal bovine serum (FBS) was conducted on titanium (Ti) to enhance bone tissue repair. Scanning electron microscopy (SEM) and energy dispersive spectrometer (EDS) showed FBS increased the tube wall thickness and decreased the tube diameter. Attenuated total reflectance Fourier transform infrared further confirmed that FBS completely covered the TNT and changed the surface composition. Water contact angle tests showed TNT/FBS possessed hydrophilic properties. Compared to original Ti, the TNT/FBS group had more attached osteoblasts after 2h and enhanced filopodia growth at 0.5h. Significantly, more osteoblasts were also observed on TNT/FBS after 7d culturing. FBS was released steadily from TNT; about 70% of FBS had been released at 3d and 90% at 5d, as shown by the bicinchoninic acid method. TNT/FBS also enhanced subsequent osteoblast differentiation and gene expression; the quantum real-time polymerase chain reaction test showed that TNT/FBS up-regulated alkaline phosphatase and osteocalcin gene expression at 7d and 14d. Therefore, TNT/FBS delivered sustained in situ nutrition and enhanced osteoblast activity and osteogenic gene expression. PMID:26249612

  19. Mesenchymal Stem Cells Ageing: Targeting the "Purinome" to Promote Osteogenic Differentiation and Bone Repair.

    PubMed

    Noronha-Matos, J B; Correia-de-Sá, P

    2016-09-01

    Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into bone forming cells. Such ability is compromised in elderly individuals resulting in bone disorders such as osteoporosis, also limiting their clinical usage for cell transplantation and bone tissue engineering strategies. In bone marrow niches, adenine and uracil nucleotides are important local regulators of osteogenic differentiation of MSCs. Nucleotides can be released to the extracellular milieu under both physiological and pathological conditions via (1) membrane cell damage, (2) vesicle exocytosis, (3) ATP-binding cassette transporters, and/or (4) facilitated diffusion through maxi-anion channels, hemichannels or ligand-gated receptor pores. Nucleotides and their derivatives act via adenosine P1 (A1 , A2A , A2B , and A3 ) and nucleotide-sensitive P2 purinoceptors comprising ionotropic P2X and G-protein-coupled P2Y receptors. Purinoceptors activation is terminated by membrane-bound ecto-nucleotidases and other ecto-phosphatases, which rapidly hydrolyse extracellular nucleotides to their respective nucleoside 5'-di- and mono-phosphates, nucleosides and free phosphates, or pyrophosphates. Current knowledge suggests that different players of the "purinome" cascade, namely nucleotide release sites, ecto-nucleotidases and purinoceptors, orchestrate to fine-tuning regulate the activity of MSCs in the bone microenvironment. Increasing studies, using osteoprogenitor cell lines, animal models and, more recently, non-modified MSCs from postmenopausal women, raised the possibility to target chief components of the purinergic signaling pathway to regenerate the ability of aged MSCs to differentiate into functional osteoblasts. This review summarizes the main findings of those studies, prompting for novel therapeutic strategies to control ageing disorders where bone destruction exceeds bone formation, like osteoporosis, rheumatoid arthritis, and fracture mal-union. J. Cell. Physiol. 231: 1852

  20. In Vivo Osteogenic Differentiation of Human Dental Pulp Stem Cells Embedded in an Injectable In Vivo-Forming Hydrogel.

    PubMed

    Jang, Ja Yong; Park, Seung Hun; Park, Ji Hoon; Lee, Bo Keun; Yun, Jeong-Ho; Lee, Bong; Kim, Jae Ho; Min, Byoung Hyun; Kim, Moon Suk

    2016-08-01

    In this study, human dental pulp stem cells (hDPSCs) are examined as a cellular source for bone tissue engineering using an in vivo-forming hydrogel. The hDPSCs are easily harvested in large quantities from extracted teeth. The stemness of harvested hDPSCs indicates their relative tolerance to ex vivo manipulation in culture. The in vitro osteogenic differentiation of hDPSCs is characterized using Alizarin Red S (ARS), von Kossa (VK), and alkaline phosphatase (ALP) staining. The solution of hDPSCs and a methoxy polyethylene glycol-polycaprolactone block copolymer (PC) is easily prepared by simple mixing at room temperature and in no more than 10 s it forms in vivo hydrogels after subcutaneous injection into rats. In vivo osteogenic differentiation of hDPSCs in the in vivo-forming hydrogel is confirmed by micro-computed tomography (CT), histological staining, and gene expression. Micro-CT analysis shows evidence of significant tissue-engineered bone formation in hDPSCs-loaded hydrogel in the presence of osteogenic factors. Differentiated osteoblasts in in vivo-forming hydrogel are identified by ARS and VK staining and are found to exhibit characteristic expression of genes like osteonectin, osteopontin, and osteocalcin. In conclusion, hDPSCs embedded in an in vivo-forming hydrogel may provide benefits as a noninvasive formulation for bone tissue engineering applications. PMID:27074749

  1. Simulated microgravity inhibits osteogenic differentiation of mesenchymal stem cells via depolymerizing F-actin to impede TAZ nuclear translocation

    PubMed Central

    Chen, Zhe; Luo, Qing; Lin, Chuanchuan; Kuang, Dongdong; Song, Guanbin

    2016-01-01

    Microgravity induces observed bone loss in space flight, and reduced osteogenesis of bone mesenchymal stem cells (BMSCs) partly contributes to this phenomenon. Abnormal regulation or functioning of the actin cytoskeleton induced by microgravity may cause the inhibited osteogenesis of BMSCs, but the underlying mechanism remains obscure. In this study, we demonstrated that actin cytoskeletal changes regulate nuclear aggregation of the transcriptional coactivator with PDZ-binding motif (TAZ), which is indispensable for osteogenesis of bone mesenchymal stem cells (BMSCs). Moreover, we utilized a clinostat to model simulated microgravity (SMG) and demonstrated that SMG obviously depolymerized F-actin and hindered TAZ nuclear translocation. Interestingly, stabilizing the actin cytoskeleton induced by Jasplakinolide (Jasp) significantly rescued TAZ nuclear translocation and recovered the osteogenic differentiation of BMSCs in SMG, independently of large tumor suppressor 1(LATS1, an upstream kinase of TAZ). Furthermore, lysophosphatidic acid (LPA) also significantly recovered the osteogenic differentiation of BMSCs in SMG through the F-actin-TAZ pathway. Taken together, we propose that the depolymerized actin cytoskeleton inhibits osteogenic differentiation of BMSCs through impeding nuclear aggregation of TAZ, which provides a novel connection between F-actin cytoskeleton and osteogenesis of BMSCs and has important implications in bone loss caused by microgravity. PMID:27444891

  2. The effect of an alendronate-eluting titanium system to induce osteogenic differentiation in human buccal fat cells (HBFCs)

    NASA Astrophysics Data System (ADS)

    Kim, Sung Eun; Lee, Su-Young; Yun, Young-Pil; Lee, Jae Yong; Park, Kyeongsoon; Lee, Deok-Won; Song, Hae-Ryong

    2012-10-01

    The purpose of this study was to develop alendronate (Aln)-eluting Ti substrates to induce osteogenic differentiation of human buccal fat cells (HBFCs). The surface of pristine Ti was modified by dopamine (DOPA) and then heparin was grafted onto the aminated Ti surfaces to achieve the Aln-eluting Ti system. Aln was subsequently immobilized on the surface of heparinized Ti (Hep-Ti). Pristine Ti and surface-modified-Ti were characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and contact angle. Osteogenic differentiation of HBFCs on the surface of pristine-Ti, Hep-Ti, Aln (1 mg)/Hep-Ti, and Aln (5 mg)/Hep-Ti was demonstrated by alkaline phosphatase (ALP) activity, calcium deposition, and osteocalcin and osteopontin mRNA expression. Successful immobilization of Aln on Hep-Ti was confirmed by XPS and contact angle. Aln/Hep-Ti showed the sustained release for up to 28 days. Additionally, HBFCs cultured on Aln/Hep-Ti substrates showed significantly induced ALP activity, calcium deposition, and osteocalcin and osteopontin mRNA expression. These results suggest that Aln-eluting Ti substrates have a potential effect on osteogenic differentiation of HBFCs and will be a promising material for bone regeneration.

  3. Simulated microgravity inhibits osteogenic differentiation of mesenchymal stem cells via depolymerizing F-actin to impede TAZ nuclear translocation.

    PubMed

    Chen, Zhe; Luo, Qing; Lin, Chuanchuan; Kuang, Dongdong; Song, Guanbin

    2016-01-01

    Microgravity induces observed bone loss in space flight, and reduced osteogenesis of bone mesenchymal stem cells (BMSCs) partly contributes to this phenomenon. Abnormal regulation or functioning of the actin cytoskeleton induced by microgravity may cause the inhibited osteogenesis of BMSCs, but the underlying mechanism remains obscure. In this study, we demonstrated that actin cytoskeletal changes regulate nuclear aggregation of the transcriptional coactivator with PDZ-binding motif (TAZ), which is indispensable for osteogenesis of bone mesenchymal stem cells (BMSCs). Moreover, we utilized a clinostat to model simulated microgravity (SMG) and demonstrated that SMG obviously depolymerized F-actin and hindered TAZ nuclear translocation. Interestingly, stabilizing the actin cytoskeleton induced by Jasplakinolide (Jasp) significantly rescued TAZ nuclear translocation and recovered the osteogenic differentiation of BMSCs in SMG, independently of large tumor suppressor 1(LATS1, an upstream kinase of TAZ). Furthermore, lysophosphatidic acid (LPA) also significantly recovered the osteogenic differentiation of BMSCs in SMG through the F-actin-TAZ pathway. Taken together, we propose that the depolymerized actin cytoskeleton inhibits osteogenic differentiation of BMSCs through impeding nuclear aggregation of TAZ, which provides a novel connection between F-actin cytoskeleton and osteogenesis of BMSCs and has important implications in bone loss caused by microgravity. PMID:27444891

  4. Terminal differentiation of osteogenic cells in the embryonic chick tibia is revealed by a monoclonal antibody against osteocytes.

    PubMed

    Bruder, S P; Caplan, A I

    1990-01-01

    Monoclonal antibodies against the surface of embryonic osteogenic cells have been used to characterize the sequence of transitions involved in the osteoblastic cell lineage. These previous data identified distinct cell stages within the osteogenic lineage, but were incomplete. To further refine and extend these observations, additional monoclonal antibodies were generated against the surface of osteogenic cells by immunizing mice with a heterogeneous population of chick embryonic bone cells. Supernatants from growing hybridoma colonies were immunohistochemically screened against frozen sections of stage 35 (day 9.5) chick tibiae. One cell line, SB-5, which secretes an antibody against the surface of osteogenic cells was successfully cloned, stabilized, and immortalized. Studies on the developmental progression of osteogenesis in the embryonic chick tibia reveal that cells within the lineage stages from Pre-Osteoblast to Secretory Osteoblast were never observed to react with antibody SB-5 at any time. By contrast, strong cell surface immunoreactivity was present on mature osteoblastic cells as they became Osteocytes. Furthermore, in cultures of osteogenic cells derived from embryonic calvaria or tibiae, cells possessing the SB-5 antigen on their surface displayed a morphology remarkably similar to that of Osteocytes found in situ. Double immunofluorescent staining of developing chick tibiae with SB-5 and SB-2, a monoclonal antibody directed against the surface of Secretory Osteoblasts, indicates that these cells proceed through an intermediate lineage step before becoming terminally differentiated Osteocytes. This transitory cell state is characterized by the simultaneous cell surface binding of antibodies SB-2 and SB-5, and is referred to as the Osteocytic Osteoblast stage.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2202356

  5. Effect of enamel matrix derivative and of proline-rich synthetic peptides on the differentiation of human mesenchymal stem cells toward the osteogenic lineage.

    PubMed

    Ramis, Joana Maria; Rubert, Marina; Vondrasek, Jiri; Gayà, Antoni; Lyngstadaas, Staale Petter; Monjo, Marta

    2012-06-01

    With the aim of discovering new molecules for induction of bone formation and biomineralization, combination of bioinformatics and simulation methods were used to design the structure of artificial peptides based on proline-rich domains of enamel matrix proteins. In this study, the effect of such peptides on the differentiation toward the osteogenic lineage of human umbilical cord mesenchymal stem cells (hUCMSCs) was evaluated with or without osteogenic supplements (hydrocortisone, β-glycerol phosphate, and ascorbic acid) and compared to the effect of the commercially available enamel matrix derivative (EMD). It was hypothesized that the differentiation toward the osteogenic lineage of hUCMSCs would be promoted by the treatment with the synthetic peptides when combined with differentiation media, or it could even be directed exclusively by the synthetic peptides. Osteoinductivity was assessed by cell proliferation, bone morphogenetic protein-2 secretion, and gene expression of osteogenic markers after 1, 3, and 14 days of treatment. All peptides were safe with the dosages used, showing lower cell toxicity. P2, P4, and P6 reduced cell proliferation with growing media by 10%-15%. Higher expression of early osteoblast markers was found after 3 days of treatment with EMD in combination with osteogenic supplements, while after 14 days of treatment, cells treated by the different synthetic peptides in combination with osteogenic supplements showed higher osteocalcin mRNA levels. We can conclude that osteogenic differentiation of hUCMSCs is promoted by short-term EMD treatment in combination with osteogenic supplements and by long-term treatment by the synthetic peptides in combination with osteogenic supplements, showing similar results for all the peptide variants analyzed in this study. PMID:22429009

  6. In vitro osteogenic and odontogenic differentiation of human dental pulp stem cells seeded on carboxymethyl cellulose-hydroxyapatite hybrid hydrogel

    PubMed Central

    Teti, Gabriella; Salvatore, Viviana; Focaroli, Stefano; Durante, Sandra; Mazzotti, Antonio; Dicarlo, Manuela; Mattioli-Belmonte, Monica; Orsini, Giovanna

    2015-01-01

    Stem cells from human dental pulp have been considered as an alternative source of adult stem cells in tissue engineering because of their potential to differentiate into multiple cell lineages. Recently, polysaccharide based hydrogels have become especially attractive as matrices for the repair and regeneration of a wide variety of tissues and organs. The incorporation of inorganic minerals as hydroxyapatite nanoparticles can modulate the performance of the scaffolds with potential applications in tissue engineering. The aim of this study was to verify the osteogenic and odontogenic differentiation of dental pulp stem cells (DPSCs) cultured on a carboxymethyl cellulose—hydroxyapatite hybrid hydrogel. Human DPSCs were seeded on carboxymethyl cellulose—hydroxyapatite hybrid hydrogel and on carboxymethyl cellulose hydrogel for 1, 3, 5, 7, 14, and 21 days. Cell viability assay and ultramorphological analysis were carried out to evaluate biocompatibility and cell adhesion. Real Time PCR was carried out to demonstrate the expression of osteogenic and odontogenic markers. Results showed a good adhesion and viability in cells cultured on carboxymethyl cellulose—hydroxyapatite hybrid hydrogel, while a low adhesion and viability was observed in cells cultured on carboxymethyl cellulose hydrogel. Real Time PCR data demonstrated a temporal up-regulation of osteogenic and odontogenic markers in dental pulp stem cells cultured on carboxymethyl cellulose—hydroxyapatite hybrid hydrogel. In conclusion, our in vitro data confirms the ability of DPSCs to differentiate toward osteogenic and odontogenic lineages in presence of a carboxymethyl cellulose—hydroxyapatite hybrid hydrogel. Taken together, our results provide evidence that DPSCs and carboxymethyl cellulose—hydroxyapatite hybrid hydrogel could be considered promising candidates for dental pulp complex and periodontal tissue engineering. PMID:26578970

  7. 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

  8. Promotion of osteoblastic differentiation and osteogenic transcription factor expression on a microgroove titanium surface with immobilized fibronectin or bone sialoprotein II.

    PubMed

    Im, Byung-Jin; Lee, Sang Cheon; Lee, Myung-Hyun; Leesungbok, Richard; Ahn, Su-Jin; Kang, Yoon-Goo; Lee, Do Yun; Yoon, Joon-Ho; Lee, Suk Won

    2016-01-01

    We demonstrate that a composite surface of microgroove titanium (Ti) with immobilized fibronectin (FN) or bone sialoprotein II (BSP2) promotes osteoblastic differentiation and osteogenic transcription factor expression in human bone marrow-derived mesenchymal stem cells (MSCs). Comparisons made between smooth microgrooves, microgrooves with silanization and microgrooves with matrix protein (FN or BSP2)-immobilization Ti surfaces revealed a significant promotion of in vitro osteogenic activity and osteoblastic differentiation at various timelines of culture. An even more significant increase was verified on microgrooves with a matrix protein-immobilization Ti surface in 28 d time-dependent gene expression of the main osteogenic transcription factors, such as ARF4, FRA1, RUNX2, and OSX. As a result, a synergestic effect regarding the promotion of osteogenic transcription factor expression and osteoblastic differentiation in the matrix protein-microgroove Ti composite surface was confirmed. From a multiple regression analysis using various timelines of osteogenic culture as independent variables, day 13 was verified as the most prominent influential timeline for the promotion of osteoblastic differentiation induced by the matrix protein-microgroove Ti composite surface. The FN- or BSP2-microgroove Ti composite surface resulting from silanization can strongly induce the promotion of osteoblastic differentiation in human MSCs. The proposed surface is expected to be useful in the development of a variety of osteogenic biomaterial surfaces. PMID:27327854

  9. Synergistic interaction of platelet derived growth factor (PDGF) with the surface of PLLA/Col/HA and PLLA/HA scaffolds produces rapid osteogenic differentiation.

    PubMed

    Raghavendran, Hanumantha Rao Balaji; Mohan, Saktiswaren; Genasan, Krishnamurithy; Murali, Malliga Raman; Naveen, Sangeetha Vasudevaraj; Talebian, Sepehr; McKean, Robert; Kamarul, Tunku

    2016-03-01

    Scaffolds with structural features similar to the extracellular matrix stimulate rapid osteogenic differentiation in favorable microenvironment and with growth factor supplementation. In this study, the osteogenic potential of electrospun poly-l-lactide/hydroxyapatite/collagen (PLLA/Col/HA, PLLA/HA and PLLA/Col) scaffolds were tested in vitro with the supplementation of platelet derived growth factor-BB (PDGF-BB). Cell attachment and topography, mineralization, extracellular matrix protein localization, and gene expression of the human mesenchymal stromal cells were compared between the fibrous scaffolds PLLA/Col/HA, PLLA/Col, and PLLA/HA. The levels of osteocalcin, calcium, and mineralization were significantly greater in the PLLA/Col/HA and PLLA/HA compared with PLLA/Col. High expression of fibronectin, intracellular adhesion molecule, cadherin, and collagen 1 (Col1) suggests that PLLA/Col/HA and PLLA/HA scaffolds had superior osteoinductivity than PLLA/Col. Additionally, osteopontin, osteocalcin, osterix, Runt-related transcription factor 2 (Runx2), and bone morphogenic protein (BMP2) expression were higher in PLLA/Col/HA and PLLA/HA compared with PLLA/Col. In comparison with PLLA/Col, the PLLA/Col/HA and PLLA/HA scaffolds presented a significant upregulation of the genes Runx2, Col 1, Integrin, osteonectin (ON), bone gamma-carboxyglutamic acid-containing protein (BGALP), osteopontin (OPN), and BMP2. The upregulation of these genes was further increased with PDGF-BB supplementation. These results show that PDGF-BB acts synergistically with PLLA/Col/HA and PLLA/HA to enhance the osteogenic differentiation potential. Therefore, this combination can be used for the rapid expansion of bone marrow stromal cells into bone-forming cells for tissue engineering. PMID:26700235

  10. Synergistic effect of nanomaterials and BMP-2 signalling in inducing osteogenic differentiation of adipose tissue-derived mesenchymal stem cells.

    PubMed

    Lu, ZuFu; Roohani-Esfahani, Seyed-Iman; Li, JiaoJiao; Zreiqat, Hala

    2015-01-01

    The lack of complete understanding in the signalling pathways that control the osteogenic differentiation of mesenchymal stem cells hinders their clinical application in the reconstruction of large bone defects and non-union bone fractures. The aim of this study is to gain insight into the interactions of bone morphogenetic protein-2 (BMP-2) and bone biomimetic scaffolds in directing osteogenic differentiation of adipose tissue-derived mesenchymal stem cells (ASCs) and the underlying signalling pathways involved. We demonstrated that bioactive glass nanoparticles (nBG) incorporated polycaprolactone (PCL) coating on hydroxyapatite/β-tricalcium phosphate (HA/TCP) scaffold exerted a synergistic effect with 3days of BMP-2 treatment in promoting osteogenic gene expression levels (Runx-2, collagen I, osteopontin and bone sialoprotein) and alkaline phosphatase activity in ASCs. Furthermore, we revealed that the synergistic effect was mediated through a mechanism of activating β1-integrin and induction of Wnt-3a autocrine signalling pathways by nBG incorporated scaffold. PMID:25262582

  11. Effects of a hybrid micro/nanorod topography-modified titanium implant on adhesion and osteogenic differentiation in rat bone marrow mesenchymal stem cells

    PubMed Central

    Zhang, Wenjie; Li, Zihui; Huang, Qingfeng; Xu, Ling; Li, Jinhua; Jin, Yuqin; Wang, Guifang; Liu, Xuanyong; Jiang, Xinquan

    2013-01-01

    Background and methods Various methods have been used to modify titanium implant surfaces with the aim of achieving better osseointegration. In this study, we fabricated a clustered nanorod structure on an acid-etched, microstructured titanium plate surface using hydrogen peroxide. We also evaluated biofunctionalization of the hybrid micro/nanorod topography on rat bone marrow mesenchymal stem cells. Scanning electron microscopy and x-ray diffraction were used to investigate the surface topography and phase composition of the modified titanium plate. Rat bone marrow mesenchymal stem cells were cultured and seeded on the plate. The adhesion ability of the cells was then assayed by cell counting at one, 4, and 24 hours after cell seeding, and expression of adhesion-related protein integrin β1 was detected by immunofluorescence. In addition, a polymerase chain reaction assay, alkaline phosphatase and Alizarin Red S staining assays, and osteopontin and osteocalcin immunofluorescence analyses were used to evaluate the osteogenic differentiation behavior of the cells. Results The hybrid micro/nanoscale texture formed on the titanium surface enhanced the initial adhesion activity of the rat bone marrow mesenchymal stem cells. Importantly, the hierarchical structure promoted osteogenic differentiation of these cells. Conclusion This study suggests that a hybrid micro/nanorod topography on a titanium surface fabricated by treatment with hydrogen peroxide followed by acid etching might facilitate osseointegration of a titanium implant in vivo. PMID:23345973

  12. MiR-34a Promotes Osteogenic Differentiation of Human Adipose-Derived Stem Cells via the RBP2/NOTCH1/CYCLIN D1 Coregulatory Network.

    PubMed

    Fan, Cong; Jia, Lingfei; Zheng, Yunfei; Jin, Chanyuan; Liu, Yunsong; Liu, Hao; Zhou, Yongsheng

    2016-08-01

    MiR-34a was demonstrated to be upregulated during the osteogenic differentiation of human adipose-derived stem cells (hASCs). Overexpression of miR-34a significantly increased alkaline phosphatase activity, mineralization capacity, and the expression of osteogenesis-associated genes in hASCs in vitro. Enhanced heterotopic bone formation in vivo was also observed upon overexpression of miR-34a in hASCs. Mechanistic investigations revealed that miR-34a inhibited the expression of retinoblastoma binding protein 2 (RBP2) and reduced the luciferase activity of reporter gene construct comprising putative miR-34a binding sites in the 3' UTR of RBP2. Moreover, miR-34a downregulated the expression of NOTCH1 and CYCLIN D1 and upregulated the expression of RUNX2 by targeting RBP2, NOTCH1, and CYCLIN D1. Taken together, our results suggested that miR-34a promotes the osteogenic differentiation of hASCs via the RBP2/NOTCH1/CYCLIN D1 coregulatory network, indicating that miR-34a-targeted therapy could be a valuable approach to promote bone regeneration. PMID:27453008

  13. MiR-132-3p Regulates the Osteogenic Differentiation of Thoracic Ligamentum Flavum Cells by Inhibiting Multiple Osteogenesis-Related Genes

    PubMed Central

    Qu, Xiaochen; Chen, Zhongqiang; Fan, Dongwei; Sun, Chuiguo; Zeng, Yan

    2016-01-01

    Ossification of the ligamentum flavum (OLF) is a disorder of heterotopic ossification of spinal ligaments and is the main cause of thoracic spinal canal stenosis. Previous studies suggested that miR-132-3p negatively regulates osteoblast differentiation. However, whether miR-132-3p is involved in the process of OLF has not been investigated. In this study, we investigated the effect of miR-132-3p and its target genes forkhead box O1 (FOXO1), growth differentiation factor 5 (GDF5) and SRY-box 6 (SOX6) on the osteogenic differentiation of ligamentum flavum (LF) cells. We demonstrated that miR-132-3p was down-regulated during the osteogenic differentiation of LF cells and negatively regulated the osteoblast differentiation. Further, miR-132-3p targeted FOXO1, GDF5 and SOX6 and down-regulated the protein expression of these genes. Meanwhile, FOXO1, GDF5 and SOX6 were up-regulated after osteogenic differentiation and the down-regulation of endogenous FOXO1, GDF5 or SOX6 suppressed the osteogenic differentiation of LF cells. In addition, we also found FOXO1, GDF5 and SOX6 expression in the ossification front of OLF samples. Overall, these results suggest that miR-132-3p inhibits the osteogenic differentiation of LF cells by targeting FOXO1, GDF5 and SOX6. PMID:27556448

  14. Effects of Tricalcium Silicate Cements on Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells In Vitro

    PubMed Central

    Eid, Ashraf A.; Hussein, Khalid A.; Niu, Li-na; Li, Guo-hua; Watanabe, Ikuya; Al-Shabrawey, Mohamed; Pashley, David H.; Tay, Franklin R.

    2014-01-01

    Tricalcium silicate cements have been successfully employed in the biomedical field as bioactive bone and dentin substitutes, with widely acclaimed osteoactive properties. This research analyzed the effects of different tricalcium silicate cement formulations on the temporal osteoactivity profile of human bone marrow-derived mesenchymal stem cells (hMW-MSCs). These cells were exposed to 4 commercially-available tricalcium silicate cement formulations in osteogenic differentiation medium. After 1, 3, 7 and 10 days, quantitative real time-polymerase chain reaction and Western blotting were performed to detect the expression of target osteogenic markers ALP, RUNX2, OSX, OPN, MSX2, and OCN. After 3, 7, 14 and 21 days, alkaline phosphatase assay was performed to detect changes in intracellular enzyme level. Alizarin Red S assay was performed after 28 days to detect extracellular matrix mineralization. In the presence of tricalcium silicate cements, target osteogenic markers were downregulated at the mRNA and protein levels at all time-points. Intracellular alkaline phosphatase enzyme levels and extracellular mineralization of the experimental groups were not significantly different from the untreated control. Quantitative polymerase chain reaction results showed increases in downregulation of RUNX2, OSX, MSX2 and OCN with increase in time of exposure to the tricalcium silicate cements, while ALP showed peak downregulation at day 7. For Western blotting, OSX, OPN, MSX2 and OCN showed increased downregulation with increased exposure time to the tested cements. Alkaline phosphatase enzyme levels generally declined after day 7. Based on these results, it is concluded that tricalcium silicate cements do not induce osteogenic differentiation of hBM-MSCs in vitro. PMID:24726977

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

    PubMed

    Gong, Kai; Qu, Bo; Liao, Dongfa; Liu, Da; Wang, Cairu; Zhou, Jingsong; Pan, Xianming

    2016-09-01

    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 showed 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

  16. Heparin-induced conformational changes of fibronectin within the extracellular matrix promote hMSC osteogenic differentiation.

    PubMed

    Li, Bojun; Lin, Zhe; Mitsi, Maria; Zhang, Yang; Vogel, Viola

    2015-01-01

    An increasing body of evidence suggests important roles of extracellular matrix (ECM) in regulating stem cell fate. This knowledge can be exploited in tissue engineering applications for the design of ECM scaffolds appropriate to direct stem cell differentiation. By probing the conformation of fibronectin (Fn) using fluorescence resonance energy transfer (FRET), we show here that heparin treatment of the fibroblast-derived ECM scaffolds resulted in more extended conformations of fibrillar Fn in ECM. Since heparin is a highly negatively charged molecule while fibronectin contains segments of positively charged modules, including FnIII13, electrostatic interactions between Fn and heparin might interfere with residual quaternary structure in relaxed fibronectin fibers thereby opening up buried sites. The conformation of modules FnIII12-14 in particular, which contain one of the heparin binding sites as well as binding sites for many growth factors, may be activated by heparin, resulting in alterations in growth factor binding to Fn. Indeed, upregulated osteogenic differentiation was observed when hMSCs were seeded on ECM scaffolds that had been treated with heparin and were subsequently chemically fixed. In contrast, either rigidifying relaxed fibers by fixation alone, or heparin treatment without fixation had no effect. We hypothesize that fibronectin's conformations within the ECM are activated by heparin such as to coordinate with other factors to upregulate hMSC osteogenic differentiation. Thus, the conformational changes of fibronectin within the ECM could serve as a 'converter' to tune hMSC differentiation in extracellular matrices. This knowledge could also be exploited to promote osteogenic stem cell differentiation on biomedical surfaces. PMID:26214191

  17. Boron Nitride Nanotubes Reinforce Tricalcium Phosphate Scaffolds and Promote the Osteogenic Differentiation of Mesenchymal Stem Cells.

    PubMed

    Shuai, Cijun; Gao, Chengde; Feng, Pei; Xiao, Tao; Yu, Kun; Deng, Youwen; Peng, Shuping

    2016-05-01

    Incorporating boron nitride nanotubes (BNNTs) into ceramic matrices is a promising strategy for obtaining multifunctional composites. In this study, the application of BNNTs in reinforcing β-tricalcium phosphate (β-TCP) scaffolds manufactured using laser sintering is demonstrated. BNNTs contribute to the effective inhibition of both grain growth and phase transformation in β-TCP. Moreover, they can strengthen the grain boundaries and boost the fracture mode transition from intergranular to transgranular. BNNTs play an active role in reinforcing β-TCP in terms of load transfer and energy absorption by the synergistic mechanisms of pull-out, peel-off, crack bridging and deflection. With a BNNT content of 4 wt%, the elastic modulus, hardness, compressive strength and fracture toughness of β-TCP increase by 46%, 39%, 109% and 35%, respectively. Umbilical cord mesenchymal stem cells (UC-MSCs) were isolated with high purity, and surface molecule characterization revealed that they were CD90+, CD29+, CD73+, CD31-, CD34- and CD45-. UC-MSCs on BNNTs/β-TCP scaffolds were characterized by more positive Alizarin Red staining as well as up-regulated expression of osteoblast markers, as revealed by quantitative real-time reverse transcriptase polymerase chain reaction analysis and immunofluorescence staining. These results are the first to demonstrate that BNNTs promote the osteogenic differentiation of UC-MSCs, indicating good osteoinductive properties for use in bone scaffolds. This study paves the way for the potential use of a BNNT/β-TCP scaffold in bone repair. PMID:27305816

  18. Isolation, Characterization, Cryopreservation of Human Amniotic Stem Cells and Differentiation to Osteogenic and Adipogenic Cells

    PubMed Central

    Gholizadeh-Ghaleh Aziz, Shiva; Pashaei-Asl, Fatima; Fardyazar, Zahra; Pashaiasl, Maryam

    2016-01-01

    Human stem cells and progenitor cells can be used to treat cancer and replace dysfunctional cells within a tissue or organ. The objective of this study was to identify the appropriate cells type in regenerative medicine and targeted therapy. As an alternative to embryonic and bone marrow stem cells, we examined human amniotic fluid stem cells (hAFSCs), one of the potential source of multipotent stem cells isolated from both cell pellet (using single-stage method), and supernatant of human amniotic fluid. Source of isolation and unique property of the cells emphasize that these cells are one of the promising new tools in therapeutic field. Double sources for isolation and availability of the left over samples in diagnostic laboratory at the same time have less legal and ethical concerns compared with embryonic stem cell studies. Cells were isolated, cultured for 18th passage for 6 months and characterized using qPCR and flow cytometry. Cells showed good proliferative ability in culture condition. The cells successfully differentiated into the adipogenic and osteogenic lineages. Based on these findings, amniotic fluid can be considered as an appropriate and convenient source of human amniotic fluid stem cells. These cells provide potential tools for therapeutic applications in the field of regenerative medicine. To get a better understanding of crosstalk between Oct4/NANOG with osteogenesis and adipogenesis, we used network analysis based on Common Targets algorithm and Common Regulators algorithm as well as subnetwork discovery based on gene set enrichment. Network analysis highlighted the possible role of MIR 302A and MIR let-7g. We demonstrated the high expression of MIR 302A and low expression of MIR let7g in hAFSCs by qPCR. PMID:27434028

  19. Bifunctional bioceramics stimulating osteogenic differentiation of a gingival fibroblast and inhibiting plaque biofilm formation.

    PubMed

    Shen, Ya; Wang, Zhejun; Wang, Jiao; Zhou, Yinghong; Chen, Hui; Wu, Chengtie; Haapasalo, Markus

    2016-04-22

    Gingival recession is a common clinical problem that results in esthetic deficiencies and poor plaque control and predominantly occurs in aged patients. In order to restore the cervical region, ideal biomaterials should possess the ability to stimulate proliferation and osteogenesis/cementogenesis of human gingival fibroblasts (HGF) and have a strong antibiofilm effect. The aim of the present study was to investigate the interactions of HGF and oral multispecies biofilms with Ca, Mg and Si-containing bredigite (BRT, Ca7MgSi4O16) bioceramics. BRT extract induced osteogenic/cementogenic differentiation of HGF and its inhibition of plaque biofilm formation were systematically studied. BRT extract in concentrations lower than <200 mg mL(-1) presented high biocompatibility to HGF cells in 3 days. Ion extracts from BRT also stimulated a series of bone-related gene and protein expressions in HGF cells. Furthermore, BRT extract significantly inhibited oral multispecies plaque biofilm growth on its surface and contributed to over 30% bacterial cell death without additional antibacterial agents in two weeks. A planktonic killing test showed that BRT suppressed 98% plaque bacterial growth compared to blank control in 3 days. The results also revealed that BRT extract has an osteostimulation effect on HGF. The suppression effect on plaque biofilms suggested that BRT might be used as a bioactive material for cervical restoration and that the synergistic effect of bioactive ions, such as Ca, Mg and Si ions, played an important role in the design and construction of bifunctional biomaterials in combination with tissue regeneration and antibiofilm activity. PMID:26806408

  20. 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

  1. Suppression of Evi1 promotes the osteogenic differentiation and inhibits the adipogenic differentiation of bone marrow-derived mesenchymal stem cells in vitro.

    PubMed

    An, Qijun; Wu, Dou; Ma, Yuehong; Zhou, Biao; Liu, Qiang

    2015-12-01

    Osteoporosis (OP) is considered a complex disease with a strong genetic impact, mainly affecting post-menopausal women and is also a common cause of fracture. Elucidating the molecular mechanisms that regulate the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) is crucial to developing treatment strategies to combat OP. In the present study, we found that ectopic viral integration site‑1 (Evi1) was highly expressed during the process of adipogenesis of rat BMSCs. Notably, Evi1 levels markedly increased on day 3 of adipogenic differentiation following the addition of adipogenic induction supplements. In addition, we interfered with the expression of the Evi1 gene in the adipogenesis of BMSCs by supplementing adenoviral plasmids and measured the expression levels of bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN), peroxisome proliferator‑activated receptor γ2 (PPARγ2) and lipoprotein lipase (LPL) by RT-qPCR and western blot analysis. The mRNA and protein levels of osteogenic and adipogenic markers in the BMSCs were up‑ and downregulated, respectively following the silencing of siEvi1. Our experimental results substantiate that the suppression of Evi1 in BMSCs by RNA interference inhibits adipogenic differentiation, while it promotes osteogenic differentiation. The results from our study demonstrated that the Evi1 gene may be targeted as a therapeutic strategy for promoting bone formation. PMID:26497332

  2. Adenovirus-mediated expression of vascular endothelial growth factor-a potentiates bone morphogenetic protein9-induced osteogenic differentiation and bone formation.

    PubMed

    Pi, Chang-Jun; Liang, Kai-Lu; Ke, Zhen-Yong; Chen, Fu; Cheng, Yun; Yin, Liang-Jun; Deng, Zhong-Liang; He, Bai-Cheng; Chen, Liang

    2016-08-01

    Mesenchymal stem cells (MSCs) are suitable seed cells for bone tissue engineering because they can self-renew and undergo differentiation into osteogenic, adipogenic, chondrogenic, or myogenic lineages. Vascular endothelial growth factor-a (VEGF-a), an angiogenic factor, is also involved in osteogenesis and bone repair. However, the effects of VEGF-a on osteogenic MSCs differentiation remain unknown. It was previously reported that bone morphogenetic protein9 (BMP9) is one of the most important osteogenic BMPs. Here, we investigated the effects of VEGF-a on BMP9-induced osteogenesis with mouse embryo fibroblasts (MEFs). We found that endogenous VEGF-a expression was undetectable in MSCs. Adenovirus-mediated expression of VEGF-a in MEFs potentiated BMP9-induced early and late osteogenic markers, including alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). In stem cell implantation assays, VEGF-a augmented BMP9-induced ectopic bone formation. VEGF-a in combination with BMP9 effectively increased the bone volume and osteogenic activity. However, the synergistic effect was efficiently abolished by the phosphoinositide 3-kinase (PI3K)/AKT inhibitor LY294002. These results demonstrated that BMP9 may crosstalk with VEGF-a through the PI3K/AKT signaling pathway to induce osteogenic differentiation in MEFs. Thus, our findings demonstrate the effects of VEGF-a on BMP9-induced bone formation and provide a new potential strategy for treating nonunion fractures, large segmental bony defects, and/or osteoporotic fractures. PMID:27003241

  3. Comparative epigenetic influence of autologous versus fetal bovine serum on mesenchymal stem cells through in vitro osteogenic and adipogenic differentiation.

    PubMed

    Fani, Nesa; Ziadlou, Reihane; Shahhoseini, Maryam; Baghaban Eslaminejad, Mohamadreza

    2016-06-10

    Mesenchymal stem cells (MSCs) derived from bone marrow (BM) represents a useful source of adult stem cells for cell therapy and tissue engineering. MSCs are present at a low frequency in the BM; therefore expansion is necessary before performing clinical studies. Fetal bovine serum (FBS) as a nutritional supplement for in vitro culture of MSCs is a suitable additive for human cell culture, but not regarding subsequent use of these cells for clinical treatment of human patients due to the risk of viral and prion transmission as well as xenogeneic immune responses after transplantation. Recently, autologous serum (AS) has been as a supplement to replace FBS in culture medium. We compared the effect of FBS versus AS on the histone modification pattern of MSCs through in vitro osteogenesis and adipogenesis. Differentiation of stem cells under various serum conditions to a committed state involves global changes in epigenetic patterns that are critically determined by chromatin modifications. Chromatin immunoprecipitation (ChIP) coupled with real-time PCR showed significant changes in the acetylation and methylation patterns in lysine 9 (Lys9) of histone H3 on the regulatory regions of stemness (Nanog, Sox2, Rex1), osteogenic (Runx2, Oc, Sp7) and adipogenic (Ppar-γ, Lpl, adiponectin) marker genes in undifferentiated MSCs, FBS and AS. All epigenetic changes occurred in a serum dependent manner which resulted in higher expression level of stemness genes in undifferentiated MSCs compared to differentiated MSCs and increased expression levels of osteogenic genes in AS compared to FBS. Adipogenic genes showed greater expression in FBS compared to AS. These findings have demonstrated the epigenetic influence of serum culture conditions on differentiation potential of MSCs, which suggest that AS is possibly more efficient serum for osteogenic differentiation of MSCs in cell therapy purposes. PMID:26481420

  4. A genome wide analysis of alternative splicing events during the osteogenic differentiation of human cartilage endplate-derived stem cells.

    PubMed

    Shang, Jin; Wang, Honggang; Fan, Xin; Shangguan, Lei; Liu, Huan

    2016-08-01

    Low back pain is a prevalent disease, which leads to suffering and disabilities in a vast number of individuals. Degenerative disc diseases are usually the underlying causes of low back pain. However, the pathogenesis of degenerative disc diseases is highly complex and difficult to determine. Current therapies for degenerative disc diseases are various. In particular, cell-based therapies have proven to be effective and promising. Our research group has previously isolated and identified the cartilage endplate‑derived stem cells. In addition, alternative splicing is a sophisticated regulatory mechanism, which greatly increases cellular complexity and phenotypic diversity of eukaryotic organisms. The present study continued to investigate alternative splicing events in osteogenic differentiation of cartilage endplate‑derived stem cells. An Affymetrix Human Transcriptome Array 2.0 was used to detect splicing changes between the control and differentiated samples. Additionally, molecular function and pathway analysis were also performed. Following rigorous bioinformatics analysis of the data, 3,802 alternatively spliced genes were identified, and 10 of these were selected for validation by reverse transcription‑polymerase chain reaction. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway analysis also revealed numerous enriched GO terms and signaling pathways. To the best of our knowledge, the present study is the first to investigate alternative splicing mechanisms in osteogenic differentiation of stem cells on a genome‑wide scale. The illumination of molecular mechanisms of stem cell osteogenic differentiation may assist the development novel bioengineered methods to treat degenerative disc diseases. PMID:27278552

  5. Hexosamine-Induced TGF-β Signaling and Osteogenic Differentiation of Dental Pulp Stem Cells Are Dependent on N-Acetylglucosaminyltransferase V

    PubMed Central

    Chen, Yi-Jane; Yao, Chung-Chen; Huang, Chien-Hsun; Chang, Hao-Hueng; Young, Tai-Horng

    2015-01-01

    Glycans of cell surface glycoproteins are involved in the regulation of cell migration, growth, and differentiation. N-acetyl-glucosaminyltransferase V (GnT-V) transfers N-acetyl-d-glucosamine to form β1,6-branched N-glycans, thus playing a crucial role in the biosynthesis of glycoproteins. This study reveals the distinct expression of GnT-V in STRO-1 and CD-146 double-positive dental pulp stem cells (DPSCs). Furthermore, we investigated three types of hexosamines and their N-acetyl derivatives for possible effects on the osteogenic differentiation potential of DPSCs. Our results showed that exogenous d-glucosamine (GlcN), N-acetyl-d-glucosamine (GlcNAc), d-mannosamine (ManN), and acetyl-d-mannosamine (ManNAc) promoted DPSCs' early osteogenic differentiation in the absence of osteogenic supplements, but d-galactosamine (GalN) or N-acetyl-galactosamine (GalNAc) did not. Effects include the increased level of TGF-β receptor type I, activation of TGF-β signaling, and increased mRNA expression of osteogenic differentiation marker genes. The hexosamine-treated DPSCs showed an increased mineralized matrix deposition in the presence of osteogenic supplements. Moreover, the level of TGF-β receptor type I and early osteogenic differentiation were abolished in the DPSCs transfected with siRNA for GnT-V knockdown. These results suggest that GnT-V plays a critical role in the hexosamine-induced activation of TGF-β signaling and subsequent osteogenic differentiation of DPSCs. PMID:26583147

  6. Limited inhibitory effects of non-steroidal antiinflammatory drugs on in vitro osteogenic differentiation in canine cells.

    PubMed

    Oh, Namgil; Sunaga, Takafumi; Yamazaki, Hiroki; Hosoya, Kenji; Takagi, Satoshi; Okumura, Masahiro

    2013-08-01

    Cyclooxygenase (COX)-2 participates essentially in bone healing, demonstrated by COX-2 knockout mice that showed delayed fracture repair. Considerable controversy still exists on inhibitory effects of COX-2 inhibitors on bone healing in clinical cases. To assess stage-dependent effects of short-term treatment of COX-2 inhibitors on osteogenic differentiation, a canine POS osteosarcoma cell line which spontaneously differentiates into osteoblastic cell was exposed to COX-2 inhibitors such as carprofen and meloxicam for 72 hours during three different stages of osteoblast differentiation, including day 0 to 3 (pre-osteoblastic stage), day 4 to 7 (transitional stage) and day 8 to 11 (mature osteoblastic stage). As osteogenic markers, expression of alkaline phosphatase (ALP) was estimated by analysis of mRNA expression, enzymatic activity and ALP staining, and expression of osteocalcin was estimated by analysis of mRNA expression after the drug treatments. Calcified matrix formation was finally observed by von Kossa staining on day 14. Expressions of ALP showed no significant suppression by carprofen and meloxicam during all three stages. However, expressions of osteocalcin mRNA and non-calcified nodule formations were delayed by carprofen and meloxicam during transitional stage. Nevertheless, fully calcified nodule formation was observed in all experimental groups during post-medication period. These results indicate that short-term treatment of carprofen and meloxicam would reversibly suppress the differentiation of osteoblasts. PMID:24059095

  7. Comparison between 8-prenylnarigenin and narigenin concerning their activities on promotion of rat bone marrow stromal cells' osteogenic differentiation in vitro.

    PubMed

    Ming, L G; Ge, B F; Wang, M G; Chen, K M

    2012-12-01

    A number of recent studies have suggested that flavonols (a class of phytochemical with many biological activities), might exert protective effects against post-menopausal bone loss. In the present study, we compared naringenin (NG) and 8-prenylnaringenin (PNG), two major naturally occurring flavonols, on in vitro differentiation of osteoblasts and bone resorbing activity, of rat bone marrow stromal cells (BMSCs). Our results indicated that both compounds, at 10(-6)  m, enhanced BMSCs' differentiation. Then effects of the two compounds at 10(-6)  m on ALP activity, osteocalcin secretion and calcium deposition, were compared over a time course. Numbers and areas of colonies stained for ALP (CFU-F(ALP) ) expression, and mineralized bone nodules, were histochemically analysed after 12 days and 16 days osteogenic induction, respectively. Expression of BMP-2, OPG, OSX, RUNX-2 genes and p38MAPK protein were examined using real-time PCR and western blotting, respectively. The data presented indicate that PNG, significantly enhanced the rat BMSCs' differentiation and mineralization through the BMP-2/p38MAPK/Runx2/Osterix signal pathway, greater than did NG. In conclusion, PNG has a more pronounced ability to enhance osteoblast differentiation and mineralization, than NG. PMID:23106298

  8. The in vitro and in vivo effects of the low molecular weight fucoidan on the bone osteogenic differentiation properties.

    PubMed

    Hwang, Pai-An; Hung, Yu-Lan; Phan, Nam Nhut; Hieu, Bui-Thi-Ngoc; Chang, Po-Ming; Li, Kuan-Lun; Lin, Yen-Chang

    2016-08-01

    Osteoporosis has been reported as a hidden death factor in aged people. So far, prevention and treatment therapies for osteoporosis only slow down the progress but do not treat the disease. Fucoidan has been recognized its roles in anti-tumor, anti-inflammatory, anti-coagulant and antiviral activities. To date, low molecular weight (LMW) fucoidan role in bone loss disease has been not determined yet. Therefore, this study aims to figure out potential effects of LMW fucoidan in osteoporosis in vitro and in vivo. LMW fucoidan was extracted from fresh Sargassum hemiphyllum showing a significant increase in 7F2 cell viability to 150.33 ± 6.50 % relative to normal fucoidan (130.12 ± 5.74 %). The expression of level BMP-2, ALP, osteocalcin significantly increased with 2.28 ± 0.06, 2.18 ± 0.12 and 2.06 ± 0.07 fold, respectively. The RT-PCR assay showed that LMW fucoidan increased mRNA expression of BMP-2, ALP, osteocalcin, COL I, BSP and osteonectin. Furthermore, the bone density and bone ash weight were considerably boosted by the oral administration of 280 mg/kg LMW fucoidan and 100 mg/kg calcium carbonate in C57BL/6J female aged mice. The present finding indicated that LMW fucoidan triggered osteogenic differentiation in vitro, and had an anabolic effect on bone mineralization in vivo. Dietary intake of LMW fucoidan from S. hemiphyllum suggested playing a role in the enhancement of bone loss with increasing age. PMID:26271462

  9. 5-azacytidine improves the osteogenic differentiation potential of aged human adipose-derived mesenchymal stem cells by DNA demethylation.

    PubMed

    Yan, Xueying; Ehnert, Sabrina; Culmes, Mihaela; Bachmann, Anastasia; Seeliger, Claudine; Schyschka, Lilianna; Wang, Zhiyong; Rahmanian-Schwarz, Afshin; Stöckle, Ulrich; De Sousa, Paul A; Pelisek, Jaroslav; Nussler, Andreas K

    2014-01-01

    The therapeutic value of adipose-derived mesenchymal stem cells (Ad-MSCs) for bone regeneration is critically discussed. A possible reason for reduced osteogenic potential may be an age-related deterioration of the Ad-MSCs. In long term in vitro culture, epigenomic changes in DNA methylation are known to cause gene silencing, affecting stem cell growth as well as the differentiation potential. In this study, we observed an age-related decline in proliferation of primary human Ad-MSCs. Decreased Nanog, Oct4 and Lin28A and increased Sox2 gene-expression was accompanied by an impaired osteogenic differentiation potential of Ad-MSCs isolated from old donors (>60 a) as compared to Ad-MSCs isolated from younger donors (<45 a). 5-hydroxymethylcytosine (5 hmC) and 5-methylcytonsine (5 mC) distribution as well as TET gene expression were evaluated to assess the evidence of active DNA demethylation. We observed a decrease of 5 hmC in Ad-MSCs from older donors. Incubation of these cells with 5-Azacytidine induced proliferation and improved the osteogenic differentiation potential in these cells. The increase in AP activity and matrix mineralization was associated with an increased presence of 5 hmC as well as with an increased TET2 and TET3 gene expression. Our data show, for the first time, a decrease of DNA hydroxymethylation in Ad-MSCs which correlates with donor-age and that treatment with 5-Azacytidine provides an approach which could be used to rejuvenate Ad-MSCs from aged donors. PMID:24603866

  10. 5-Azacytidine Improves the Osteogenic Differentiation Potential of Aged Human Adipose-Derived Mesenchymal Stem Cells by DNA Demethylation

    PubMed Central

    Culmes, Mihaela; Bachmann, Anastasia; Seeliger, Claudine; Schyschka, Lilianna; Wang, Zhiyong; Rahmanian-Schwarz, Afshin; Stöckle, Ulrich; De Sousa, Paul A.; Pelisek, Jaroslav; Nussler, Andreas K.

    2014-01-01

    The therapeutic value of adipose-derived mesenchymal stem cells (Ad-MSCs) for bone regeneration is critically discussed. A possible reason for reduced osteogenic potential may be an age-related deterioration of the Ad-MSCs. In long term in vitro culture, epigenomic changes in DNA methylation are known to cause gene silencing, affecting stem cell growth as well as the differentiation potential. In this study, we observed an age-related decline in proliferation of primary human Ad-MSCs. Decreased Nanog, Oct4 and Lin28A and increased Sox2 gene-expression was accompanied by an impaired osteogenic differentiation potential of Ad-MSCs isolated from old donors (>60 a) as compared to Ad-MSCs isolated from younger donors (<45 a). 5-hydroxymethylcytosine (5 hmC) and 5-methylcytonsine (5 mC) distribution as well as TET gene expression were evaluated to assess the evidence of active DNA demethylation. We observed a decrease of 5 hmC in Ad-MSCs from older donors. Incubation of these cells with 5-Azacytidine induced proliferation and improved the osteogenic differentiation potential in these cells. The increase in AP activity and matrix mineralization was associated with an increased presence of 5 hmC as well as with an increased TET2 and TET3 gene expression. Our data show, for the first time, a decrease of DNA hydroxymethylation in Ad-MSCs which correlates with donor-age and that treatment with 5-Azacytidine provides an approach which could be used to rejuvenate Ad-MSCs from aged donors. PMID:24603866

  11. [Effects of isoquercitrin from Craibiodendron yunnanense on osteogenic differentiation of MC3T3-E1 cells].

    PubMed

    Duan, Ai-Zhu; Deng, Xu-Liang; Li, Rong-Tao

    2014-10-01

    Natural products especially flavonoids are being explored for their therapeutic potentials in reducing bone loss and maintaining bone health. The present study is to investigate the effects of isoquercitrin from Craibiodendron yunnanense with different concentrations at 1 x 10(-4), 1 x 10(-5), 1 x 10(-6), 1 x 10(-7) mol x L(-1) on proliferation, differentiation and mineralization of MC3T3-E1. Cell proliferation was assessed by CCK-8 kit at 1, 3, 5 and 7 days of culture. Alkaline phosphatase (ALP) activity were performed qualitatively and quantitatively on day 7, and alizarin red S staining was employed to access the mineralization of cells on day 21. The osteogenic markers ALP, collagen type I (COL 1A1), runt-related transcription factor 2 (Runx-2) and Osterix were detected to analysis early osteogenic differentiation of cells on day 3 by RT-PCR. The results showed that isoquercitrin had a dose-dependent effect on the proliferation, osteogenic differentiation, mineralization and gene expression of MC3T3-E1 in the range from 1 x 10(-7) to 1 x 10(-5) mol x L(-1). At concentrations above 1 x 10(-4) mol x L(-1) isoquercitrin showed cytotoxicity, while 1 x 10(-6) mol x L(-1) is the optimal concentration of isoquercitrin to improve the osteoblastic activity. All these results implied that isoquercitrin might be the major composition of traditional Chinese medicine C. yunnanense to treat bone fractures. PMID:25612450

  12. The inhibitory effect of superparamagnetic iron oxide nanoparticle (Ferucarbotran) on osteogenic differentiation and its signaling mechanism in human mesenchymal stem cells

    SciTech Connect

    Chen, Ying-Chun; Hsiao, Jong-Kai; Liu, Hon-Man; Lai, I-Yin; Yao, Ming; Hsu, Szu-Chun; Ko, Bor-Sheng; Chen, Yao-Chang; Yang, Chung-Shi; Huang, Dong-Ming

    2010-06-01

    Superparamagnetic iron oxide (SPIO) nanoparticles are very useful for monitoring cell trafficking in vivo and distinguish whether cellular regeneration originated from an exogenous cell source, which is a key issue for developing successful stem cell therapies. However, the impact of SPIO labeling on stem cell behavior remains uncertain. Here, we show the inhibitory effect of Ferucarbotran, an ionic SPIO, on osteogenic differentiation and its signaling mechanism in human mesenchymal stem cells. Ferucarbotran caused a dose-dependent inhibition of osteogenic differentiation, abolished the differentiation at high concentration, promoted cell migration, and activated the signaling molecules, {beta}-catenin, a cancer/testis antigen, SSX, and matrix metalloproteinase 2 (MMP2). An iron chelator, desferrioxamine, suppressed all the above Ferucarbotran-induced actions, demonstrating an important role of free iron in the inhibition of osteogenic differentiation that is mediated by the promotion of cell mobilization, involving the activation of a specific signaling pathway.

  13. Effects of Line and Pillar Array Microengineered SiO2 Thin Films on the Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells.

    PubMed

    Carvalho, Angela; Pelaez-Vargas, Alejandro; Hansford, Derek J; Fernandes, Maria H; Monteiro, Fernando J

    2016-02-01

    A primary goal in bone tissue engineering is the design of implants that induce controlled, guided, and rapid healing. The events that normally lead to the integration of an implant into bone and determine the performance of the device occur mainly at the tissue-implant interface. Topographical surface modification of a biomaterial might be an efficient tool for inducing stem cell osteogenic differentiation and replace the use of biochemical stimuli. The main goal of this work was to develop micropatterned bioactive silica thin films to induce the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) only through topographical stimuli. Line and pillar micropatterns were developed by a combination of sol-gel/soft lithography and characterized by scanning electron microscopy, atomic force microscopy, and contact angle measurements. hMSCs were cultured onto the microfabricated thin films and flat control for up to 21 days under basal conditions. The micropatterned groups induced levels of osteogenic differentiation and expression of osteoblast-associated markers higher than those of the flat controls. Via comparison of the micropatterns, the pillars caused a stronger response of the osteogenic differentiation of hMSCs with a higher level of expression of osteoblast-associated markers, ALP activity, and extracellular matrix mineralization after the cells had been cultured for 21 days. These findings suggest that specific microtopographic cues can direct hMSCs toward osteogenic differentiation. PMID:26771563

  14. Notch-Hes pathway mediates the impaired osteogenic differentiation of bone marrow mesenchymal stromal cells from myelodysplastic syndromes patients through the down-regulation of Runx2

    PubMed Central

    Fei, Chengming; Guo, Juan; Zhao, Youshan; Gu, Shucheng; Zhao, Sida; Li, Xiao; Chang, Chunkang

    2015-01-01

    Previous studies have demonstrated that bone marrow mesenchymal stromal cells (BMMSCs) from patients with myelodysplastic syndromes (MDS) display defective proliferative potential and impaired osteogenic differentiation ability. However, the underlying mechanisms are unclear. In the present study, the impaired osteogenic differentiation potential of BMMSCs was found in cases with RARS (83.3%), RCMD (75.0%), RAEB I (44.4%), RAEB II (40%). We also observed that MDS-BMMSCs with impaired osteogenic differentiation potential exhibited accelerate senescence and decreased hematopoietic supporting function. Further, we found that an abnormal activation of Notch-Hes signaling pathway in MDS-BMMSCs. By overexpression of Notch intracellular domain (NICD) in BMMSCs from healthy donors, we confirmed that Notch signaling pathway negatively regulated BMMSCs osteogenesis through inhibition of Runx2 transcriptional activity. Importantly, treatment with DAPT, a γ-secretase inhibitor of Notch signaling reversed the osteogenic differentiation in MDS-BMMSCs. Collectively, we provide evidence that activation of Notch-Hes signaling pathway is involved in the impaired osteogenic differentiation of MDS-BMMSCs and support the concept of a primary BMMSCs defect that might have a contributory effect in MDS pathogenesis. PMID:26692937

  15. Inhibition of miR-222-3p activity promoted osteogenic differentiation of hBMSCs by regulating Smad5-RUNX2 signal axis.

    PubMed

    Yan, Jihong; Guo, Duo; Yang, Shu; Sun, Huaimei; Wu, Bo; Zhou, Deshan

    2016-02-12

    miRNAs are recently found playing important roles in osteogenesis. In this study, we identified that miR-222-3p decreased during osteogenic differentiation of human mesenchymal stem cells (hBMSCs) using Quantitative Real-Time Reverse Transcription PCR (qRT-PCR). Furthermore, we investigated the effect of miR-222-3p on osteogenic differentiation of hBMSCs. Inhibition of miR-222-3p function in hBMSCs using infection of lentiviruses carrying miR-222-3p specific inhibitor promoted expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Whereas, overexpression of miR-222-3p inhibited osteoblast differentiation of hBMSCs in vitro. Moreover, Smad5 and RUNX2, which are the critical transcription factors in osteogenic differentiation, were predicted to be targets of miR-222-3p by bioinformatic analysis. Overexpression of miR-222-3p in hBMSCs significantly suppressed the protein levels of Smad5 and RUNX2, while inhibition of miR-222-3p increased their protein levels. Furthermore, inhibition of miR-222-3p increased phosphorylation of Smad1/5/8, which regulated the expression of osteogenic genes. Our findings suggest that suppression of miR-222-3p activity promoted osteogenic differentiation hBMSCs through regulating Smad5-RUNX2 signaling axis. PMID:26809090

  16. Transcriptomics Comparison between Porcine Adipose and Bone Marrow Mesenchymal Stem Cells during In Vitro Osteogenic and Adipogenic Differentiation

    PubMed Central

    Monaco, Elisa; Bionaz, Massimo; Rodriguez-Zas, Sandra; Hurley, Walter L.; Wheeler, Matthew B.

    2012-01-01

    Bone-marrow mesenchymal stem cells (BMSC) are considered the gold standard for use in tissue regeneration among mesenchymal stem cells (MSC). The abundance and ease of harvest make the adipose-derived stem cells (ASC) an attractive alternative to BMSC. The aim of the present study was to compare the transcriptome of ASC and BMSC, respectively isolated from subcutaneous adipose tissue and femur of 3 adult pigs, during in vitro osteogenic and adipogenic differentiation for up to four weeks. At 0, 2, 7, and 21 days of differentiation RNA was extracted for microarray analysis. A False Discovery Rate ≤0.05 for overall interactions effect and P<0.001 between comparisons were used to determine differentially expressed genes (DEG). Ingenuity Pathway Analysis and DAVID performed the functional analysis of the DEG. Functional analysis of highest expressed genes in MSC and genes more expressed in MSC vs. fully differentiated tissues indicated low immunity and high angiogenic capacity. Only 64 genes were differentially expressed between ASC and BMSC before differentiation. The functional analysis uncovered a potential larger angiogenic, osteogenic, migration, and neurogenic capacity in BMSC and myogenic capacity in ASC. Less than 200 DEG were uncovered between ASC and BMSC during differentiation. Functional analysis also revealed an overall greater lipid metabolism in ASC, while BMSC had a greater cell growth and proliferation. The time course transcriptomic comparison between differentiation types uncovered <500 DEG necessary to determine cell fate. The functional analysis indicated that osteogenesis had a larger cell proliferation and cytoskeleton organization with a crucial role of G-proteins. Adipogenesis was driven by PPAR signaling and had greater angiogenesis, lipid metabolism, migration, and tumorigenesis capacity. Overall the data indicated that the transcriptome of the two MSC is relatively similar across the conditions studied. In addition, functional analysis

  17. Osteochondral differentiation and the emergence of stage-specific osteogenic cell-surface molecules by bone marrow cells in diffusion chambers.

    PubMed

    Bruder, S P; Gazit, D; Passi-Even, L; Bab, I; Caplan, A I

    1990-11-01

    The osteochondral potential and emergence of osteogenic cell-surface molecules by avian marrow cells was evaluated in in vivo diffusion chamber cultures. The chambers were inoculated with unselected marrow cells from young chick tibiae and implanted intraperitoneally into athymic mice. At the light microscopic level, morphologic evidence of de novo bone and cartilage formation, including specific immunostaining by antibody probes, was observed in 14 out of 16 chambers incubated for 20 days or longer. In order to monitor the osteogenic differentiation of the marrow-derived cells, indirect immunofluorescence was performed with monoclonal antibodies against stage-specific cell surface antigens on cells of the embryonic osteogenic lineage. The binding of these and other specific monoclonal antibodies in the developing tissue indicates that the cell surface and extracellular matrix molecules expressed by descendants of marrow-derived mesenchymal progenitor cells are indistinguishable from their in vivo counterparts found in embryonic skeletal structures. Furthermore, the experiments reported here describe the first molecular identification of osteogenic cells by probes which are selective for stage-specific surface antigens on cells of the osteogenic lineage. Importantly, bone formation by these marrow-derived cells appears to occur through a lineage progression which is similar to that observed for embryonic tibial osteoblasts. In summary, these data support the use of diffusion chambers inoculated with avian marrow to study aspects of osteogenic and chondrogenic differentiation. PMID:2268743

  18. 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

  19. 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

  20. Electrospun fibrous scaffolds combined with nanoscale hydroxyapatite induce osteogenic differentiation of human periodontal ligament cells

    PubMed Central

    Wu, Xiaonan; Miao, Leiying; Yao, Yingfang; Wu, Wenlei; Liu, Yu; Chen, Xiaofeng; Sun, Weibin

    2014-01-01

    Periodontal repair is a complex process in which regeneration of alveolar bone is a vital component. The aim of this study was to develop a biodegradable scaffold with good biocompatibility and osteoinductive ability. Two types of composite fibrous scaffolds were produced by electrospinning, ie, type I collagen/poly(ε-caprolactone) (COL/PCL) and type I collagen/poly(ε-caprolactone)/nanoscale hydroxyapatite (COL/PCL/nHA) with an average fiber diameter of about 377 nm. After a simulated body fluid (SBF) immersion test, the COL/PCL/nHA-SBF scaffold developed a rough surface because of the calcium phosphate deposited on the fibers, suggesting that the presence of nHA promoted the mineralization potential of the scaffold. Energy dispersive X-ray spectroscopy clearly showed the calcium and phosphorus content in the COL/PCL/nHA and COL/PCL/nHA-SBF scaffolds, confirming the findings of nHA and calcium phosphate precipitation on scanning electron micrographs. Water contact analysis revealed that nHA could improve the hydrophilic nature of the COL/PCL/nHA-SBF scaffold. The morphology of periodontal ligament cells cultured on COL/PCL-SBF and COL/PCL/nHA-SBF was evaluated by scanning electron microscopy. The results showed that cells adhered to either type of scaffold and were slightly spindle-shaped in the beginning, then extended gradually with stretched filopodia, indicating an ability to fill the fiber pores. A Cell Counting Kit-8 assay showed that both scaffolds supported cell proliferation. However, real-time quantitative polymerase chain reaction analysis showed that expression of the bone-related markers, alkaline phosphatase and osteocalcin, was upregulated only on the COL/PCL/nHA-SBF scaffold, indicating that this scaffold had the ability to induce osteogenic differentiation of periodontal ligament cells. In this study, COL/PCL/nHA-SBF produced by electrospinning followed by biomimetic mineralization had combined electrospun fibers with nHA in it. This scaffold has

  1. Electrospun fibrous scaffolds combined with nanoscale hydroxyapatite induce osteogenic differentiation of human periodontal ligament cells.

    PubMed

    Wu, Xiaonan; Miao, Leiying; Yao, Yingfang; Wu, Wenlei; Liu, Yu; Chen, Xiaofeng; Sun, Weibin

    2014-01-01

    Periodontal repair is a complex process in which regeneration of alveolar bone is a vital component. The aim of this study was to develop a biodegradable scaffold with good biocompatibility and osteoinductive ability. Two types of composite fibrous scaffolds were produced by electrospinning, ie, type I collagen/poly(ε-caprolactone) (COL/PCL) and type I collagen/poly(ε-caprolactone)/nanoscale hydroxyapatite (COL/PCL/nHA) with an average fiber diameter of about 377 nm. After a simulated body fluid (SBF) immersion test, the COL/PCL/nHA-SBF scaffold developed a rough surface because of the calcium phosphate deposited on the fibers, suggesting that the presence of nHA promoted the mineralization potential of the scaffold. Energy dispersive X-ray spectroscopy clearly showed the calcium and phosphorus content in the COL/PCL/nHA and COL/PCL/nHA-SBF scaffolds, confirming the findings of nHA and calcium phosphate precipitation on scanning electron micrographs. Water contact analysis revealed that nHA could improve the hydrophilic nature of the COL/PCL/nHA-SBF scaffold. The morphology of periodontal ligament cells cultured on COL/PCL-SBF and COL/PCL/nHA-SBF was evaluated by scanning electron microscopy. The results showed that cells adhered to either type of scaffold and were slightly spindle-shaped in the beginning, then extended gradually with stretched filopodia, indicating an ability to fill the fiber pores. A Cell Counting Kit-8 assay showed that both scaffolds supported cell proliferation. However, real-time quantitative polymerase chain reaction analysis showed that expression of the bone-related markers, alkaline phosphatase and osteocalcin, was upregulated only on the COL/PCL/nHA-SBF scaffold, indicating that this scaffold had the ability to induce osteogenic differentiation of periodontal ligament cells. In this study, COL/PCL/nHA-SBF produced by electrospinning followed by biomimetic mineralization had combined electrospun fibers with nHA in it. This scaffold has

  2. A Halogen-Containing Stilbene Derivative from the Leaves of Cajanus cajan that Induces Osteogenic Differentiation of Human Mesenchymal Stem Cells.

    PubMed

    Cai, Jia-Zhong; Tang, Rong; Ye, Gui-Fu; Qiu, Sheng-Xiang; Zhang, Nen-Ling; Hu, Ying-Jie; Shen, Xiao-Ling

    2015-01-01

    A new natural halogen-containing stilbene derivative was isolated from the leaves of Cajanus cajan (L.) Millsp. and identified as 3-O-(3-chloro-2-hydroxyl-propanyl)-longistylin A by comprehensive spectroscopic and chemical analysis, and named cajanstilbene H (1). It is the first halogen-containing stilbene derivative found from plants. In human mesenchymal stem cells (hMSC) from bone marrow, 1 did not promote cell proliferation, but distinctly enhanced osteogenic differentiation of hMSC in time- and dose-dependent manners. In six human cancer cell lines, 1 showed a moderate inhibitory effect on cell proliferation, with IC50 values of 21.42-25.85 μmol·L(-1). PMID:26111172

  3. Effect of organic acids on calcium phosphate nucleation and osteogenic differentiation of human mesenchymal stem cells on peptide functionalized nanofibers.

    PubMed

    Barati, Danial; Walters, Joshua D; Shariati, Seyed Ramin Pajoum; Moeinzadeh, Seyedsina; Jabbari, Esmaiel

    2015-05-12

    Carboxylate-rich organic acids play an important role in controlling the growth of apatite crystals and the extent of mineralization in the natural bone. The objective of this work was to investigate the effect of organic acids on calcium phosphate (CaP) nucleation on nanofiber microsheets functionalized with a glutamic acid peptide and osteogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on the CaP-nucleated microsheets. High molecular weight poly(dl-lactide) (DL-PLA) was mixed with low molecular weight L-PLA conjugated with Glu-Glu-Gly-Gly-Cys peptide, and the mixture was electrospun to generate aligned nanofiber microsheets. The nanofiber microsheets were incubated in a modified simulated body fluid (mSBF) supplemented with different organic acids for nucleation and growth of CaP crystals on the nanofibers. Organic acids included citric acid (CA), hydroxycitric acid (HCA), tartaric acid (TART), malic acid (MA), ascorbic acid (AsA), and salicylic acid (SalA). HCA microsheets had the highest CaP content at 240 ± 10% followed by TART and CA with 225 ± 8% and 225 ± 10%, respectively. The Ca/P ratio and percent crystallinity of the nucleated CaP in TART microsheets was closest to that of stoichiometric hydroxyapatite. The extent of CaP nucleation and growth on the nanofiber microsheets depended on the acidic strength and number of hydrogen-bonding hydroxyl groups of the organic acids. Compressive modulus and degradation of the CaP nucleated microsheets were related to percent crystallinity and CaP content. Osteogenic differentiation of hMSCs seeded on the microsheets and cultured in osteogenic medium increased only for those microsheets nucleated with CaP by incubation in CA or AsA-supplemented mSBF. Further, only CA microsheets stimulated bone nodule formation by the seeded hMSCs. PMID:25879768

  4. Osteogenic Differentiation of Human Dental Pulp Stromal Cells on 45S5 Bioglass® Based Scaffolds In Vitro and In Vivo

    PubMed Central

    El-Gendy, Reem; Newby, Phillipa J.; Boccaccini, Aldo R.; Kirkham, Jennifer

    2013-01-01

    The increasing clinical demand for bone substitutes has driven significant progress in cell-based therapies for bone tissue engineering. The underpinning goals for success are to identify the most appropriate cell source and to provide three-dimensional (3D) scaffolds that support cell growth and enhance osteogenic potential. In this study, human dental pulp stromal cells (HDPSCs) were cultured under basal or osteogenic conditions either in monolayers or on 3D Bioglass® scaffolds in vitro for 2 or 4 weeks. Cell–scaffold constructs were also implanted intraperitoneally in nude mice for 8 weeks. Osteogenic potential was assessed using quantitative real-time polymerase chain reaction and histological/immunohistochemical assays. In monolayer culture, osteoinductive conditions enhanced HDPSC expression of osteogenic gene markers (COL1A1, RUNX2, OC, and/or OCN) compared with basal conditions while culture of HDPSCs on 3D scaffolds promoted osteogenic gene expression compared with monolayer culture under both basal and osteogenic conditions. These results were confirmed using histological and immunohistochemical analyses. In vivo implantation of the HDPSC 3D Bioglass constructs showed evidence of sporadic woven bone-like spicules and calcified tissue. In conclusion, this study has demonstrated the potential of using a combination of HDPSCs with 3D 45S5 Bioglass scaffolds to promote bone-like tissue formation in vitro and in vivo, offering a promising approach for clinical bone repair and regeneration. PMID:23046092

  5. Effects of immunosuppressants, FK506 and cyclosporin A, on the osteogenic differentiation of rat mesenchymal stem cells

    PubMed Central

    Byun, Yu-Kyung; Kim, Kyoung-Hwa; Kim, Su-Hwan; Kim, Young-Sung; Koo, Ki-Tae; Kim, Tai-Il; Seol, Yang-Jo; Ku, Young; Rhyu, In-Chul

    2012-01-01

    Purpose The purpose of this study was to investigate the effects of the immunosuppressants FK506 and cyclosporin A (CsA) on the osteogenic differentiation of rat mesenchymal stem cells (MSCs). Methods The effect of FK506 and CsA on rat MSCs was assessed in vitro. The MTT assay was used to determine the deleterious effect of immunosuppressants on stem cell proliferation at 1, 3, and 7 days. Alkaline phosphatase (ALP) activity was analyzed on days 3, 7, and 14. Alizarin red S staining was done on day 21 to check mineralization nodule formation. Real-time polymerase chain reaction (RT-PCR) was also performed to detect the expressions of bone tissue-specific genes on days 1 and 7. Results Cell proliferation was promoted more in the FK506 groups than the control or CsA groups on days 3 and 7. The FK506 groups showed increased ALP activity compared to the other groups during the experimental period. The ALP activity of the CsA groups did not differ from the control group in any of the assessments. Mineralization nodule formation was most prominent in the FK506 groups at 21 days. RT-PCR results of the FK506 groups showed that several bone-related genes-osteopontin, osteonectin, and type I collagen (Col-I)-were expressed more than the control in the beginning, but the intensity of expression decreased over time. Runx2 and Dlx5 gene expression were up-regulated on day 7. The effects of 50 nM CsA on osteonectin and Col-I were similar to those of the FK506 groups, but in the 500 nM CsA group, most of the genes were less expressed compared to the control. Conclusions These results suggest that FK506 enhances the osteoblastic differentiation of rat MSCs. Therefore, FK506 might have a beneficial effect on bone regeneration when immunosuppressants are needed in xenogenic or allogenic stem cell transplantation to treat bone defects. PMID:22803008

  6. Response of MAPK pathway to iron oxide nanoparticles in vitro treatment promotes osteogenic differentiation of hBMSCs.

    PubMed

    Wang, Qiwei; Chen, Bo; Cao, Meng; Sun, Jianfei; Wu, Hao; Zhao, Peng; Xing, Jing; Yang, Yan; Zhang, Xiquan; Ji, Min; Gu, Ning

    2016-04-01

    Iron oxide nanoparticles (IONPs) are generally used in multiple biomedical applications. The tissue repair effect of IONPs had been demonstrated in the previous studies of our group, but the underlying mechanism is unclarified. It is well known that stem cell-based therapies show promising prospect in tissue engineering and regenerative medicine, however, whether IONPs could modulate stem cell fate to promote tissue repair is still unclear. Herein, we found that IONPs could promote osteogenic differentiation of human bone-derived mesenchymal stem cells (hBMSCs) in vitro. To insightfully understand the molecular mechanisms, we performed systematic analyses by use of gene microarray assay and bioinformatics analysis, which revealed that gene expression was widely regulated and classical mitogen-activated protein kinase (MAPK) signal pathway was activated by IONPs treatment. As a result, downstream genes of this pathway were regulated to promote osteogenic differentiation. In summary, the present study elucidates a molecular basis explaining how IONPs effect on hBMSCs, which could have many meaningful impacts for stem cells application in regenerative medicine. PMID:26874888

  7. Calcium phosphate nanoparticles are associated with inorganic phosphate-induced osteogenic differentiation of rat bone marrow stromal cells.

    PubMed

    Chen, Xiao-rong; Bai, Jing; Yuan, Shuai-jun; Yu, Cai-xia; Huang, Jian; Zhang, Tian-lan; Wang, Kui

    2015-08-01

    In the present study, we demonstrated that calcium phosphate (CaP) nanoparticles formed in cell culture media were implicated in the process of high inorganic phosphate (Pi) mediated osteogenic differentiation of rat bone marrow stromal cells (BMSCs). Exposure of BMSCs in vitro to high Pi-containing media reduced alkaline phosphatase (ALP) activity and the expressions of osteoblast-specific genes. The sediments of CaP nanoparticles were observed at the cell surface and some of them were concomitantly found inside cells at high Pi concentration. In addition, treatment the cells with pyrophosphate (PPi), an inhibitor of calcium crystal formation, abrogated the ALP activity induced by high Pi, suggesting the contribution of CaP nanoparticles. Moreover, for isolated CaP nanoparticles, there was a trend of conversion from amorphous calcium phosphate to hydroxyapatite with elevated Pi. The particle size of CaP increased and the surface morphology changed from spherical to irregular due to increased concentrations of serum proteins incorporated into CaP nanoparticles. The study demonstrated that those physicochemical properties of CaP nanoparticles played an important role in modulating BMSCs differentiation. Furthermore, the addition of Pi in the osteogenic media resulted in a dose-dependent increase in matrix mineralization, while treatment of the cells with PPi suppressed Pi-induced calcium deposition. The findings indicated that calcium deposition in the matrix partly came from the spontaneous precipitation of CaP nanoparticles. PMID:26111760

  8. Alginate hydrogel enriched with enamel matrix derivative to target osteogenic cell differentiation in TiO2 scaffolds

    PubMed Central

    Pullisaar, Helen; Verket, Anders; Szoke, Krisztina; Tiainen, Hanna; Haugen, Håvard J; Brinchmann, Jan E; Reseland, Janne E

    2015-01-01

    The purpose of bone tissue engineering is to employ scaffolds, cells, and growth factors to facilitate healing of bone defects. The aim of this study was to assess the viability and osteogenic differentiation of primary human osteoblasts and adipose tissue–derived mesenchymal stem cells from various donors on titanium dioxide (TiO2) scaffolds coated with an alginate hydrogel enriched with enamel matrix derivative. Cells were harvested for quantitative reverse transcription polymerase chain reaction on days 14 and 21, and medium was collected on days 2, 14, and 21 for protein analyses. Neither coating with alginate hydrogel nor alginate hydrogel enriched with enamel matrix derivative induced a cytotoxic response. Enamel matrix derivative–enriched alginate hydrogel significantly increased the expression of osteoblast markers COL1A1, TNFRSF11B, and BGLAP and secretion of osteopontin in human osteoblasts, whereas osteogenic differentiation of human adipose tissue–derived mesenchymal stem cells seemed unaffected by enamel matrix derivative. The alginate hydrogel coating procedure may have potential for local delivery of enamel matrix derivative and other stimulatory factors for use in bone tissue engineering. PMID:26090086

  9. Role of Gender in Burn-Induced Heterotopic Ossification and Mesenchymal Cell Osteogenic Differentiation

    PubMed Central

    Ranganathan, Kavitha; Peterson, Jonathan; Agarwal, Shailesh; Oluwatobi, Eboda; Loder, Shawn; Forsberg, Jonathan A.; Davis, Thomas A.; Wang, Stewart C.; Levi, Benjamin

    2015-01-01

    BACKGROUND Heterotopic ossification (HO) most commonly occurs after burn injury, joint arthroplasty, and trauma. Male gender has been identified as a risk factor for the development of HO. It remains unclear why adult males are more predisposed to this pathology than adult females. In this study, we explore differences in heterotopic ossification between male and female mice using an in vivo burn/tenotomy model. METHODS Our Achilles tenotomy and burn model was used to evaluate the osteogenic potential of tissue-derived mesenchymal stem cells (MSCs) of male and female mice in injured and non-injured mice. Groups consisted of injured male (n=3), injured female (n=3), non-injured male (n=3), and non-injured female (n=3). The osteogenic potential of cells harvested from each group was assessed through RNA and protein levels and quantified using micro-CT scan. Histomorphometry was used to verify micro-CT findings, and immunohistochemistry was used to assess osteogenic signaling at the site of HO. RESULTS MSCs of male mice demonstrated greater osteogenic gene and protein expression than female MSCs (p<.05). Male mice in the burn group formed 35% more bone as compared to female mice in the burn group. This bone formation correlated with increased pSmad and IGF-1 signaling at the HO site in male mice. Differences were also seen between the non-injured male and female groups. CONCLUSIONS We demonstrate that male mice form quantitatively more bone as compared to female mice using our burn/tenotomy model. These findings can be explained at least in part by differences in BMP and IGF-1 signaling. PMID:26017598

  10. Incorporation of Fucoidan in β-Tricalcium phosphate-Chitosan scaffold prompts the differentiation of human bone marrow stromal cells into osteogenic lineage.

    PubMed

    Puvaneswary, Subramaniam; Raghavendran, Hanumantharao Balaji; Talebian, Sepehr; Murali, Malliga Raman; A Mahmod, Suhaeb; Singh, Simmrat; Kamarul, Tunku

    2016-01-01

    In our previous study, we reported the fabrication and characterization of a novel tricalcium phosphate-fucoidan-chitosan (TCP-Fu-Ch) biocomposite scaffold. However, the previous report did not show whether the biocomposite scaffold can exhibit osteogenic differentiation of human bone marrow stromal cells in osteogenic media and normal media supplemented with platelet-derived growth factor (PDGF-BB). On day 15, the release of osteocalcin, was significant in the TCP-Fu-Ch scaffold, when compared with that in the TCP-Ch scaffold, and the level of release was approximately 8 and 6 ng/ml in osteogenic and normal media supplemented with PDGF-BB, respectively. Scanning electron microscopy of the TCP-Fu-Ch scaffold demonstrated mineralization and apatite layer formation on day 14, while the addition of PDGF-BB also improved the osteogenic differentiation of the scaffold. An array of gene expression analysis demonstrated that TCP-Fu-Ch scaffold cultured in osteogenic and normal media supplemented with PDGF-BB showed significant improvement in the expression of collagen 1, Runt-related transcription factor 2, osteonectin, bone gamma-carboxyglutamate protein, alkaline phosphatase, and PPA2, but a decline in the expression of integrin. Altogether, the present study demonstrated that fucoidan-incorporated TCP-Ch scaffold could be used in the differentiation of bone marrow stromal cells and can be a potential candidate for the treatment of bone-related ailments through tissue engineering technology. PMID:27068453

  11. Incorporation of Fucoidan in β-Tricalcium phosphate-Chitosan scaffold prompts the differentiation of human bone marrow stromal cells into osteogenic lineage

    PubMed Central

    Puvaneswary, Subramaniam; Raghavendran, Hanumantharao Balaji; Talebian, Sepehr; Murali, Malliga Raman; A Mahmod, Suhaeb; Singh, Simmrat; Kamarul, Tunku

    2016-01-01

    In our previous study, we reported the fabrication and characterization of a novel tricalcium phosphate-fucoidan-chitosan (TCP-Fu-Ch) biocomposite scaffold. However, the previous report did not show whether the biocomposite scaffold can exhibit osteogenic differentiation of human bone marrow stromal cells in osteogenic media and normal media supplemented with platelet-derived growth factor (PDGF-BB). On day 15, the release of osteocalcin, was significant in the TCP-Fu-Ch scaffold, when compared with that in the TCP-Ch scaffold, and the level of release was approximately 8 and 6 ng/ml in osteogenic and normal media supplemented with PDGF-BB, respectively. Scanning electron microscopy of the TCP-Fu-Ch scaffold demonstrated mineralization and apatite layer formation on day 14, while the addition of PDGF-BB also improved the osteogenic differentiation of the scaffold. An array of gene expression analysis demonstrated that TCP-Fu-Ch scaffold cultured in osteogenic and normal media supplemented with PDGF-BB showed significant improvement in the expression of collagen 1, Runt-related transcription factor 2, osteonectin, bone gamma-carboxyglutamate protein, alkaline phosphatase, and PPA2, but a decline in the expression of integrin. Altogether, the present study demonstrated that fucoidan-incorporated TCP-Ch scaffold could be used in the differentiation of bone marrow stromal cells and can be a potential candidate for the treatment of bone-related ailments through tissue engineering technology. PMID:27068453

  12. Regulation of the osteogenic and adipogenic differentiation of bone marrow-derived stromal cells by extracellular uridine triphosphate: The role of P2Y2 receptor and ERK1/2 signaling

    PubMed Central

    LI, WENKAI; WEI, SHENG; LIU, CHAOXU; SONG, MINGYU; WU, HUA; YANG, YONG

    2016-01-01

    An imbalance in the osteogenesis and adipogenesis of bone marrow-derived stromal cells (BMSCs) is a crucial pathological factor in the development of osteoporosis. Growing evidence suggests that extracellular nucleotide signaling involving the P2 receptors plays a significant role in bone metabolism. The aim of the present study was to investigate the effects of uridine triphosphate (UTP) on the osteogenic and adipogenic differentiation of BMSCs, and to elucidate the underlying mechanisms. The differentiation of the BMSCs was determined by measuring the mRNA and protein expression levels of osteogenic- and adipogenic-related markers, alkaline phosphatase (ALP) staining, alizarin red staining and Oil Red O staining. The effects of UTP on BMSC differentiation were assayed using selective P2Y receptor antagonists, small interfering RNA (siRNA) and an intracellular signaling inhibitor. The incubation of the BMSCs with UTP resulted in a dose-dependent decrease in osteogenesis and an increase in adipogenesis, without affecting cell proliferation. Significantly, siRNA targeting the P2Y2 receptor prevented the effects of UTP, whereas the P2Y6 receptor antagonist (MRS2578) and siRNA targeting the P2Y4 receptor had little effect. The activation of P2Y receptors by UTP transduced to the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. This transduction was prevented by the mitogen-activated protein kinase inhibitor (U0126) and siRNA targeting the P2Y2 receptor. U0126 prevented the effects of UTP on osteogenic- and adipogenic-related gene expression after 24 h of culture, as opposed to 3 to 7 days of culture. Thus, our data suggest that UTP suppresses the osteogenic and enhances the adipogenic differentiation of BMSCs by activating the P2Y2 receptor. The ERK1/2 signaling pathway mediates the early stages of this process. PMID:26531757

  13. Enhanced osteogenic activity and anti-inflammatory properties of Lenti-BMP-2-loaded TiO₂ nanotube layers fabricated by lyophilization following trehalose addition.

    PubMed

    Zhang, Xiaochen; Zhang, Zhiyuan; Shen, Gang; Zhao, Jun

    2016-01-01

    To enhance biocompatibility and osseointegration between titanium implants and surrounding bone tissue, numerous efforts have been made to modify the surface topography and composition of Ti implants. In this paper, Lenti-BMP-2-loaded TiO2 nanotube coatings were fabricated by lyophilization in the presence of trehalose to functionalize the surface. We characterized TiO2 nanotube layers in terms of the following: surface morphology; Lenti-BMP-2 and trehalose release; their ability to induce osteogenesis, proliferation, and anti-inflammation in vitro; and osseointegration in vivo. The anodized TiO2 nanotube surfaces exhibited an amorphous glassy matrix perpendicular to the Ti surface. Both Lenti-BMP-2 and trehalose showed sustained release over the course of 8 days. Results from real-time quantitative polymerase chain reaction studies demonstrated that lyophilized Lenti-BMP-2/TiO2 nanotubes constructed with trehalose (Lyo-Tre-Lenti-BMP-2) significantly promoted osteogenic differentiation of bone marrow stromal cells but not their proliferation. In addition, Lyo-Tre-Lenti-BMP-2 nanotubes effectively inhibited lipopolysaccharide-induced interleukin-1β and tumor necrosis factor-α production. In vivo, the formulation also promoted osseointegration. This study presents a promising new method for surface-modifying biomedical Ti-based implants to simultaneously enhance their osteogenic potential and anti-inflammatory properties, which can better satisfy clinical needs. PMID:26869786

  14. Enhanced osteogenic activity and anti-inflammatory properties of Lenti-BMP-2-loaded TiO2 nanotube layers fabricated by lyophilization following trehalose addition

    PubMed Central

    Zhang, Xiaochen; Zhang, Zhiyuan; Shen, Gang; Zhao, Jun

    2016-01-01

    To enhance biocompatibility and osseointegration between titanium implants and surrounding bone tissue, numerous efforts have been made to modify the surface topography and composition of Ti implants. In this paper, Lenti-BMP-2-loaded TiO2 nanotube coatings were fabricated by lyophilization in the presence of trehalose to functionalize the surface. We characterized TiO2 nanotube layers in terms of the following: surface morphology; Lenti-BMP-2 and trehalose release; their ability to induce osteogenesis, proliferation, and anti-inflammation in vitro; and osseointegration in vivo. The anodized TiO2 nanotube surfaces exhibited an amorphous glassy matrix perpendicular to the Ti surface. Both Lenti-BMP-2 and trehalose showed sustained release over the course of 8 days. Results from real-time quantitative polymerase chain reaction studies demonstrated that lyophilized Lenti-BMP-2/TiO2 nanotubes constructed with trehalose (Lyo-Tre-Lenti-BMP-2) significantly promoted osteogenic differentiation of bone marrow stromal cells but not their proliferation. In addition, Lyo-Tre-Lenti-BMP-2 nanotubes effectively inhibited lipopolysaccharide-induced interleukin-1β and tumor necrosis factor-α production. In vivo, the formulation also promoted osseointegration. This study presents a promising new method for surface-modifying biomedical Ti-based implants to simultaneously enhance their osteogenic potential and anti-inflammatory properties, which can better satisfy clinical needs. PMID:26869786

  15. Early osteoblastic differentiation induced by dexamethasone enhances adenoviral gene delivery to marrow stromal cells.

    PubMed

    Blum, Jeremy S; Parrott, M Brandon; Mikos, Antonios G; Barry, Michael A

    2004-03-01

    We investigated the implications of induced osteogenic differentiation on gene delivery in multipotent rat marrow stromal cells (MSCs). Prior to genetic manipulation cells were cultured with or without osteogenic supplements (5x10(-8) M dexamethasone, 160 microM l-ascorbic acid 2-phosphate, and 10 mM beta-glycerophosphate). Comparison of liposome, retroviral, and adenoviral vectors demonstrated that all three vectors could mediate gene delivery to primary rat MSCs. When these vectors were applied in the absence or presence of osteogenic supplements, we found that MSCs differentiated prior to transduction with adenovirus type 5 vectors produced a 300% increase in transgene expression compared to MSCs that were not exposed to osteogenic supplements. This differentiation effect appeared specific to adenoviral mediated gene delivery, since there was minimal increase in retroviral gene delivery and no increase in liposome gene delivery when MSCs were treated with osteogenic supplements. In addition, we also determined this increase in transgene production to occur at a higher concentration of dexamethasone (5x10(-8) M) in the culture medium of MSCs prior to adenoviral transduction. We found that this increased transgene production could be extended to the osteogenic protein, human bone morphogenetic protein 2 (hBMP-2). When delivered by an adenoviral vector, hBMP-2 transgene production could be increased from 1.4 ng/10(5) cells/3 days to 4.3 ng/10(5) cells/3 days by culture of MSCs with osteogenic supplements prior to transduction. These results indicate that the utility of MSCs as a therapeutic protein delivery mechanism through genetic manipulation can be enhanced by pre-culture of these cells with dexamethasone. PMID:15013104

  16. Silencing tumor necrosis factor-alpha in vitro from small interfering RNA-decorated titanium nanotube array can facilitate osteogenic differentiation of mesenchymal stem cells

    PubMed Central

    Wang, Zhenlin; Hu, Zhiqiang; Zhang, Dawei; Zhuo, Mengchuan; Cheng, Jiwei; Xu, Xingping; Xing, Yongming; Fan, Jie

    2016-01-01

    Titanium implants are known for their bone bonding ability. However, the osseointegration may be severely disturbed in the inflammation environment. In order to enhance osseointegration of the implant in an inflamed environment, the small interfering RNA (siRNA) targeting tumor necrosis factor alpha (TNF-α) was used to functionalize titanium surface for gene silencing. The chitosan–tripolyphosphate–hyaluronate complexes were used to formulate nanoparticles (NPs) with siRNA, which were adsorbed directly by the anodized titanium surface. The surface characterization was analyzed by scanning electron microscope, atomic force microscopy, as well as contact angle measurement. The fluorescence microscope was used to monitor the degradation of the layer. The coculture system was established with mesenchymal stem cells (MSCs) grown directly on functionalized titanium surface and RAW264.7 cells (preactivated by lipopolysaccharide) grown upside in a transwell chamber. The transfection and knockdown efficiency of TNF-α in RAW264.7 cells were determined by fluorescence microscope, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay. The cytoskeleton and osteogenic differentiation of MSCs were also analyzed. Regular vertical aligned nanotubes (~100 nm diameter and ~300 nm length) were generated after anodization of polished titanium. After loading with NPs, the nanotubes were filled and covered by a layer of amorphous particles. The surface topography changed and wettability decreased after covering with NPs. As expected, a burst degradation of the film was observed, which could provide sufficient NPs in the released supernatant and result in transfection and knockdown effects in RAW264.7 cells. The cytoskeleton arrangement of MSCs was elongated and the osteogenic differentiation was also significantly improved on NPs loading surface. In conclusion, the siRNA decorated titanium implant could simultaneously suppress inflammation and improve

  17. TiO2-coated CoCrMo: improving the osteogenic differentiation and adhesion of mesenchymal stem cells in vitro.

    PubMed

    Logan, Niall; Sherif, Anas; Cross, Alison J; Collins, Simon N; Traynor, Alison; Bozec, Laurent; Parkin, Ivan P; Brett, Peter

    2015-03-01

    The current gold standard material for orthopedic applications is titanium (Ti), however, other materials such as cobalt-chromium-molybdenum (CoCrMo) are often preferred due to their wear resistance and mechanical strength. This study investigates if the bioactivity of CoCrMo can be enhanced by coating the surface with titanium oxide (TiO2 ) by atmospheric pressure chemical vapor deposition (CVD), thereby replicating the surface oxide layer found on Ti. CoCrMo, TiO2-coated CoCrMo (CCMT) and Ti substrates were used for this study. Cellular f-actin distribution was shown to be noticeably different between cells on CCMT and CoCrMo after 24 h in osteogenic culture, with cells on CCMT exhibiting greater spread with developed protrusions. Osteogenic differentiation was shown to be enhanced on CCMT compared to CoCrMo, with increased calcium ion content per cell (p < 0.05), greater hydroxyapatite nodule formation (p < 0.05) and reduced type I collagen deposition per cell (p < 0.05). The expression of the focal adhesion protein vinculin was shown to be marginally greater on CCMT compared to CoCrMo, whereas AFM results indicated that CCMT required more force to remove a single cell from the substrate surface compared to CoCrMo (p < 0.0001). These data suggest that CVD TiO2 coatings may have the potential to increase the biocompatibility of CoCrMo implantable devices. PMID:25045159

  18. In Vitro Effects of Low-Intensity Pulsed Ultrasound Stimulation on the Osteogenic Differentiation of Human Alveolar Bone-Derived Mesenchymal Stem Cells for Tooth Tissue Engineering

    PubMed Central

    Lim, KiTaek; Kim, Jangho; Choung, Pill-Hoon; Chung, Jong Hoon

    2013-01-01

    Ultrasound stimulation produces significant multifunctional effects that are directly relevant to alveolar bone formation, which is necessary for periodontal healing and regeneration. We focused to find out effects of specific duty cycles and the percentage of time that ultrasound is being generated over one on/off pulse period, under ultrasound stimulation. Low-intensity pulsed ultrasound ((LIPUS) 1 MHz) with duty cycles of 20% and 50% was used in this study, and human alveolar bone-derived mesenchymal stem cells (hABMSCs) were treated with an intensity of 50 mW/cm2 and exposure time of 10 min/day. hABMSCs exposed at duty cycles of 20% and 50% had similar cell viability (O.D.), which was higher (*P < 0.05) than that of control cells. The alkaline phosphatase (ALP) was significantly enhanced at 1 week with LIPUS treatment in osteogenic cultures as compared to control. Gene expressions showed significantly higher expression levels of CD29, CD44, COL1, and OCN in the hABMSCs under LIPUS treatment when compared to control after two weeks of treatment. The effects were partially controlled by LIPUS treatment, indicating that modulation of osteogenesis in hABMSCs was related to the specific stimulation. Furthermore, mineralized nodule formation was markedly increased after LIPUS treatment than that seen in untreated cells. Through simple staining methods such as Alizarin red and von Kossa staining, calcium deposits generated their highest levels at about 3 weeks. These results suggest that LIPUS could enhance the cell viability and osteogenic differentiation of hABMSCs, and could be part of effective treatment methods for clinical applications. PMID:24195067

  19. Novel nano-composite biomimetic biomaterial allows chondrogenic and osteogenic differentiation of bone marrow concentrate derived cells.

    PubMed

    Grigolo, Brunella; Cavallo, Carola; Desando, Giovanna; Manferdini, Cristina; Lisignoli, Gina; Ferrari, Andrea; Zini, Nicoletta; Facchini, Andrea

    2015-04-01

    In clinical orthopedics suitable materials that induce and restore biological functions together with the right mechanical properties are particularly needed for the regeneration of osteochondral lesions. For this purpose, the ideal scaffold should possess the right properties with respect to degradation, cell binding, cellular uptake, non-immunogenicity, mechanical strength, and flexibility. In addition, it should be easy to handle and serve as a template for chondrocyte and bone cells guiding both cartilage and bone formation. The aim of the present study was to estimate the chondrogenic and osteogenic capability of bone marrow concentrated derived cells seeded onto a novel nano-composite biomimetic material. These properties have been evaluated by means of histological, immunohistochemical and electron microscopy analyses. The data obtained demonstrated that freshly harvested cells obtained from bone marrow were able, once seeded onto the biomaterial, to differentiate either down the chondrogenic and osteogenic pathways as evaluated by the expression and production of specific matrix molecules. These findings support the use, for the repair of osteochondral lesions, of this new nano-composite biomimetic material together with bone marrow derived cells in a "one step" transplantation procedure. PMID:25804305

  20. Calcium-Sensing Receptor-Mediated Osteogenic and Early-Stage Neurogenic Differentiation in Umbilical Cord Matrix Mesenchymal Stem Cells from a Large Animal Model

    PubMed Central

    Martino, Nicola Antonio; Reshkin, Stephan Joel; Ciani, Elena; Dell'Aquila, Maria Elena

    2014-01-01

    Background Umbilical cord matrix mesenchymal stem cells (UCM-MSCs) present a wide range of potential therapeutical applications. The extracellular calcium-sensing receptor (CaSR) regulates physiological and pathological processes. We investigated, in a large animal model, the involvement of CaSR in triggering osteogenic and neurogenic differentiation of two size-sieved UCM-MSC lines, by using AMG641, a novel potent research calcimimetic acting as CaSR agonist. Methodology/Principal Findings Large (>8µm in diameter) and small (<8µm) equine UCM-MSC lines were cultured in medium with high calcium (Ca2+) concentration ([Ca2+]o; 2.87 mM) and dose-response effects of AMG641 (0.01 to 3µM) on cell proliferation were evaluated. Both cell lines were then cultured in osteogenic or neurogenic differentiation medium containing: 1) low [Ca2+]o (0.37 mM); 2) high [Ca2+]o (2.87 mM); 3) AMG641 (0.05, 0.1 or 1 µM) with high [Ca2+]o and 4) the CaSR antagonist NPS2390 (10 mM for 30 min) followed by incubation with AMG641 in high [Ca2+]o. Expression of osteogenic or neurogenic differentiation biomarkers was compared among groups. In both cell lines, AMG641 dose-dependently increased cell proliferation (up to P<0.001). Osteogenic molecular markers expression was differentially regulated by AMG641, with stimulatory (OPN up-regulation) in large or inhibitory (RUNX2 and OPN down-regulation) effects in small cells, respectively. AMG641 significantly increased alkaline phosphatase activity and calcium phosphate deposition in both cell lines. Following treatment with AMG641 during osteogenic differentiation, in both cell lines CaSR expression was inversely related to that of osteogenic markers and inhibition of CaSR by NPS2390 blocked AMG641-dependent responses. Early-stage neurogenic differentiation was promoted/triggered by AMG641 in both cell lines, as Nestin and CaSR mRNA transcription up-regulation were observed. Conclusions/Significance Calcium- and AMG641-induced CaSR stimulation

  1. The osteogenic potential of the marine-derived multi-mineral formula aquamin is enhanced by the presence of vitamin D.

    PubMed

    Widaa, A; Brennan, O; O'Gorman, D M; O'Brien, F J

    2014-05-01

    Bone degenerative diseases are on the increase globally and are often problematic to treat. This has led to a demand to identify supplements that aid bone growth and formation. Aquamin is a natural multi-mineral food supplement, derived from the red algae Lithothamnion species which contains calcium, magnesium and 72 other trace minerals. It has been previously reported to increase bone formation and mineralisation. This study aimed to investigate the 28 day in vitro osteogenic response of Aquamin supplemented with Vitamin D. The osteogenic potential of MC3T3-E1 osteoblast-like cells was analysed in standard osteogenic medium supplemented with Aquamin +/- Vitamin D3, and the controls consisted of osteogenic medium, +/- Vitamin D3. Proliferation of osteoblasts, metabolic activity and cell viability did not differ between Aquamin and the osteogenic control groups. Alkaline phosphatase (ALP) levels and mineralisation were increased by the supplementation of Aquamin, and the addition of Vitamin D3 increased mineralisation for all groups. The combination of Aquamin and Vitamin D3 yielded a significant increase in ALP and mineralisation over Aquamin alone and the standard osteogenic control +/- Vitamin D3. This study demonstrates that Aquamin aids osteogenesis, and that its osteogenic response can be enhanced by combining Aquamin with Vitamin D3. PMID:23873476

  2. MicroRNA expression profiling of human bone marrow mesenchymal stem cells during osteogenic differentiation reveals Osterix regulation by miR-31.

    PubMed

    Baglìo, Serena Rubina; Devescovi, Valentina; Granchi, Donatella; Baldini, Nicola

    2013-09-15

    Osteogenesis is the result of a complex sequence of events that involve the differentiation of mesenchymal stem cells (MSC) into osteoblasts. MSCs are multipotent adult stem cells that can give rise to different cell types of the mesenchymal germ layer. The differentiation fate of MSCs depends on the microenvironmental signals received by these cells and is tightly regulated by multiple pathways that lead to the activation of specific transcription factors. Among the transcription factors involved in osteogenic differentiation Osterix (Sp7) plays a key role and has been shown to be fundamental for bone homeostasis. However, the molecular events governing the expression of this transcription factor are not fully understood. In this study we set out to investigate the changes in the microRNA (miRNA) expression that occur during the osteogenic differentiation of bone marrow-derived MSCs. To this purpose, we analyzed the miRNA expression profile of MSCs deriving from 3 donors during the differentiation and mineralization processes by microarray. 29 miRNAs were significantly and consistently modulated during the osteogenic differentiation and 5 during the mineralization process. Interestingly, most of the differentially expressed miRNAs have been reported to be implicated in stemness maintenance, differentiation and/or oncogenesis. Subsequently, we focused our attention on the regulation of Osterix by miRNAs and demonstrated that one of the miRNAs differentially modulated during osteogenic differentiation, miR-31, controls Osterix expression through association to the 3' untranslated region of this transcription factor. By analyzing miR-31 and Osterix expression levels we found an inverse miRNA-target expression trend during osteogenic differentiation and in osteosarcoma cell lines. Moreover, the inhibition of the microRNA activity led to an increase in the endogenous expression of Osterix. Our results define a miRNA signature characterizing the osteogenic

  3. 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

  4. Inhibition of TGFβ signaling decreases osteogenic differentiation of fibrodysplasia ossificans progressiva fibroblasts in a novel in vitro model of the disease.

    PubMed

    Micha, Dimitra; Voermans, Elise; Eekhoff, Marelise E W; van Essen, Huib W; Zandieh-Doulabi, Behrouz; Netelenbos, Coen; Rustemeyer, Thomas; Sistermans, E A; Pals, Gerard; Bravenboer, Nathalie

    2016-03-01

    Fibrodysplasia ossificans progressiva is a rare genetic disorder characterized by progressive heterotopic ossification. FOP patients develop soft tissue lumps as a result of inflammation-induced flare-ups which leads to the irreversible replacement of skeletal muscle tissue with bone tissue. Classical FOP patients possess a mutation (c.617G>A; R206H) in the ACVR1-encoding gene which leads to dysregulated BMP signaling. Nonetheless, not all FOP patients with this mutation exhibit equal severity in symptom presentation or disease progression which indicates a strong contribution by environmental factors. Given the pro-inflammatory role of TGFβ, we studied the role of TGFβ in the progression of osteogenic differentiation in primary dermal fibroblasts from five classical FOP patients based on a novel method of platelet lysate-based osteogenic transdifferentiation. During the course of transdifferentiation the osteogenic properties of the cells were evaluated by the mRNA expression of Sp7/Osterix, Runx2, Alp, OC and the presence of mineralization. During transdifferentiation the expression of osteoblast markers Runx2 (p<0.05) and Alp were higher in patient cells compared to healthy controls. All cell lines exhibited increase in mineralisation. FOP fibroblasts also expressed higher baseline Sp7/Osterix levels (p<0.05) confirming their higher osteogenic potential. The pharmacological inhibition of TGFβ signaling during osteogenic transdifferentiation resulted in the attenuation of osteogenic transdifferentiation in all cell lines as shown by the decrease in the expression of Runx2 (p<0.05), Alp and mineralization. We suggest that blocking of TGFβ signaling can decrease the osteogenic transdifferentiation of FOP fibroblasts. PMID:26769004

  5. Caffeine inhibits the viability and osteogenic differentiation of rat bone marrow-derived mesenchymal stromal cells

    PubMed Central

    Zhou, Y; Guan, XX; Zhu, ZL; Guo, J; Huang, YC; Hou, WW; Yu, HY

    2010-01-01

    BACKGROUND AND PURPOSE Caffeine is consumed extensively in Europe and North America. As a risk factor for osteoporosis, epidemiological studies have observed that caffeine can decrease bone mineral density, adversely affect calcium absorption and increase the risk of bone fracture. However, the exact mechanisms have not been fully investigated. Here, we examined the effects of caffeine on the viability and osteogenesis of rat bone marrow-derived mesenchymal stromal cells (rBMSCs). EXPERIMENTAL APPROACH Cell viability, apoptosis and necrosis were quantified using thymidine incorporation and flow cytometry. Sequential gene expressions in osteogenic process were measured by real-time PCR. cAMP, alkaline phosphatase and osteocalcin were assessed by immunoassay, spectrophotometry and radioimmunoassay, respectively. Mineralization was determined by calcium deposition. KEY RESULTS After treating BMSCs with high caffeine concentrations (0.1–1 mM), their viability decreased in a concentration-dependent manner. This cell death was primarily due to necrosis and, to a small extent, apoptosis. Genes and protein sequentially expressed in osteogenesis, including Cbfa1/Runx2, collagen I, alkaline phosphatase and its protein, were significantly downregulated except for osteocalcin and its protein. Moreover, caffeine inhibited calcium deposition in a concentration- and time-dependent manner, but increased intracellular cAMP in a concentration-dependent manner. CONCLUSIONS AND IMPLICATIONS By suppressing the commitment of BMSCs to the osteogenic lineage and selectively inhibiting gene expression, caffeine downregulated some important events in osteogenesis and ultimately affected bone mass. PMID:20726981

  6. Activation of the ERK1/2 Signaling Pathway during the Osteogenic Differentiation of Mesenchymal Stem Cells Cultured on Substrates Modified with Various Chemical Groups

    PubMed Central

    He, Jin; Li, Yan-Shu; Wang, Xiu-Mei; Ai, Hong-Jun; Cui, Fu-Zhai

    2013-01-01

    The current study examined the influence of culture substrates modified with the functional groups –OH, –COOH, –NH2, and –CH3 using SAMs technology, in conjunction with TAAB control, on the osteogenic differentiation of rabbit BMSCs. The CCK-8 assay revealed that BMSCs exhibited substrate-dependent cell viability. The cells plated on –NH2- and –OH-modified substrates were well spread and homogeneous, but those on the –COOH- and –CH3-modified substrates showed more rounded phenotype. The mRNA expression of BMSCs revealed that –NH2-modified substrate promoted the mRNA expression and osteogenic differentiation of the BMSCs. The contribution of ERK1/2 signaling pathway to the osteogenic differentiation of BMSCs cultured on the –NH2-modified substrate was investigated in vitro. The –NH2-modified substrate promoted the expression of integrins; the activation of FAK and ERK1/2. Inhibition of ERK1/2 activation by PD98059, a specific inhibitor of the ERK signaling pathway, blocked ERK1/2 activation in a dose-dependent manner, as revealed for expression of Cbfα-1 and ALP. Blockade of ERK1/2 phosphorylation in BMSCs by PD98059 suppressed osteogenic differentiation on chemical surfaces. These findings indicate a potential role for ERK in the osteogenic differentiation of BMSCs on surfaces modified by specific chemical functional groups, indicating that the microenvironment affects the differentiation of BMSCs. This observation has important implications for bone tissue engineering. PMID:24069599

  7. Altered MicroRNA Expression Profile in Exosomes during Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

    PubMed Central

    Zhang, Shui-Jun; Zhao, Chen; Qiu, Bin-Song; Gu, Hai-Feng; Hong, Jian-Fei; Cao, Li; Chen, Yu; Xia, Bing; Bi, Qin; Wang, Ya-Ping

    2014-01-01

    The physiological role of microRNAs (miRNAs) in osteoblast differentiation remains elusive. Exosomal miRNAs isolated from human bone marrow-derived mesenchymal stem cells (BMSCs) culture were profiled using miRNA arrays containing probes for 894 human matured miRNAs. Seventy-nine miRNAs (∼8.84%) could be detected in exosomes isolated from BMSC culture supernatants when normalized to endogenous control genes RNU44. Among them, nine exosomal miRNAs were up regulated and 4 miRNAs were under regulated significantly (Relative fold>2, p<0.05) when compared with the values at 0 day with maximum changes at 1 to 7 days. Five miRNAs (miR-199b, miR-218, miR-148a, miR-135b, and miR-221) were further validated and differentially expressed in the individual exosomal samples from hBMSCs cultured at different time points. Bioinformatic analysis by DIANA-mirPath demonstrated that RNA degradation, mRNA surveillance pathway, Wnt signaling pathway, RNA transport were the most prominent pathways enriched in quantiles with differential exosomal miRNA patterns related to osteogenic differentiation. These data demonstrated exosomal miRNA is a regulator of osteoblast differentiation. PMID:25503309

  8. Expression of OCT-4 and SOX-2 in Bone Marrow-Derived Human Mesenchymal Stem Cells during Osteogenic Differentiation

    PubMed Central

    Matic, Igor; Antunovic, Maja; Brkic, Sime; Josipovic, Pavle; Mihalic, Katarina Caput; Karlak, Ivan; Ivkovic, Alan; Marijanovic, Inga

    2016-01-01

    AIM: Determine the levels of expression of pluripotency genes OCT-4 and SOX-2 before and after osteogenic differentiation of human mesenchymal stem cells (hMSCs). METHODS: Human MSCs were derived from the bone marrow and differentiated into osteoblasts. The analyses were performed on days 0 and 14 of the cell culture. In vitro differentiation was evaluated due to bone markers – alkaline phosphatase (AP) activity and the messenger RNA (mRNA) expression of AP and bone sialoprotein (BSP). The OCT-4 and SOX-2 expression was evaluated at mRNA level by real-time qPCR and at protein level by immunocytochemistry. RESULTS: In vitro cultures on day 14 showed an increase in AP activity and upregulation of AP and BSP gene expression. OCT-4 and SOX-2 in undifferentiated hMSCs on day 0 is detectable and very low compared to tumor cell lines as a positive control. Immunocytochemistry detected OCT-4 in the cell nuclei prior (day 0) and post differentiation (day 14). On the same time points, cultures were negative for SOX-2 protein. CONCLUSION: Messenger RNA for pluripotency markers OCT-4 and SOX-2 isolated from hMSCs was less present, while OCT-4 protein was detected in cell nuclei prior and post differentiation into osteoblast lineage. PMID:27275321

  9. Adipose-derived stem cells undergo spontaneous osteogenic differentiation in vitro when passaged serially or seeded at low density.

    PubMed

    Liu, Y; Zhang, Z; Zhang, C; Deng, W; Lv, Q; Chen, X; Huang, T; Pan, L

    2016-07-01

    Adipose-derived stem cells (ADSCs) are a convenient source of cells for regenerating tissue. Widespread application of ADSCs requires that they propagate efficiently and differentiate in vitro. We investigated the differentiation potential of ADSCs during long-term expansion in vitro and when the cells were seeded at low density. ADSCs were isolated from the inguinal fat pads of 3-week-old male rats, then cultured serially for 12 passages; some ADSCs at passage 3 were seeded at low density. The differentiation potential of ADSCs from passage 3 to passage 12 was assessed by their capacity for adipogenesis and osteogenesis while cultured in specific induction media. Spontaneous osteogenesis of ADSCs at passage 12 and of ADSCs that were seeded at low density was detected by western blotting, alizarin red S staining and measurement of alkaline phosphatase (ALP) activity. We found that with increasing passage number, the adipogenic potential of ADSCs decreased and osteogenic differentiation increased. Alizarin red S staining, bone morphogenetic protein-2 (BMP-2) and runt-related transcription factor 2 (Runx2) expressions, and ALP activity demonstrated that both ADSCs at passage 12 and those that were seeded at low density differentiated into osteoblasts without additional induction factors. PMID:27149413

  10. MiR-708 promotes steroid-induced osteonecrosis of femoral head, suppresses osteogenic differentiation by targeting SMAD3

    PubMed Central

    Hao, Cheng; Yang, Shuhua; Xu, Weihua; Shen, Jacson K.; Ye, Shunan; Liu, Xianzhe; Dong, Zhe; Xiao, Baojun; Feng, Yong

    2016-01-01

    Steroid-induced osteonecrosis of femoral head (ONFH) is a serious complication of glucocorticoid (GC) use. We investigated the differential expression of miRs in the mesenchymal stem cells (MSCs) of patients with ONFH, and aimed to explain the relationship between GC use and the development of MSC dysfunction in ONFH. Cells were collected from bone marrow of patients with ONFH. Samples were assigned to either GCs Group or Control Group at 1:1 matched with control. We then used miRNA microarray analysis and real-time PCR to identify the differentially expressed miRs. We also induced normal MSCs with GCs to verify the differential expression above. Subsequently, we selected some of the miRs for further studies, including miRNA target and pathway prediction, and functional analysis. We discovered that miR-708 was upregulated in ONFH patients and GC-treated MSCs. SMAD3 was identified as a direct target gene of miR-708, and functional analysis demonstrated that miR-708 could markedly suppress osteogenic differentiation and adipogenesis differentiation of MSCs. Inhibition of miR-708 rescued the suppressive effect of GC on osteonecrosis. Therefore, we determined that GC use resulted in overexpression of miR-708 in MSCs, and thus, targeting miR-708 may serve as a novel therapeutic biomarker for the prevention and treatment of ONFH. PMID:26932538

  11. Vibration Induced Osteogenic Commitment of Mesenchymal Stem Cells is Enhanced by Cytoskeletal Remodeling but not Fluid Shear

    PubMed Central

    Uzer, Gunes; Pongkitwitoon, Suphannee; Chan, M Ete; Judex, Stefan

    2013-01-01

    Consistent across studies in humans, animals and cells, the application of vibrations can be anabolic and/or anti-catabolic to bone. The physical mechanisms modulating the vibration-induced response have not been identified. Recently, we developed an in vitro model in which candidate parameters including acceleration magnitude and fluid shear can be controlled independently during vibrations. Here, we hypothesized that vibration induced fluid shear does not modulate mesenchymal stem cell (MSC) proliferation and mineralization and that cell’s sensitivity to vibrations can be promoted via actin stress fiber formation. Adipose derived human MSCs were subjected to vibration frequencies and acceleration magnitudes that induced fluid shear stress ranging from 0.04Pa to 5Pa. Vibrations were applied at magnitudes of 0.15g, 1g, and 2g using frequencies of both 100Hz and 30Hz. After 14d and under low fluid shear conditions associated with 100Hz oscillations, mineralization was greater in all vibrated groups than in controls. Greater levels of fluid shear produced by 30Hz vibrations enhanced mineralization only in the 2g group. Over 3d, vibrations led to the greatest increase in total cell number with the frequency/acceleration combination that induced the smallest level of fluid shear. Acute experiments showed that actin remodeling was necessary for early mechanical up-regulation of RUNX-2 mRNA levels. During osteogenic differentiation, mechanically induced up-regulation of actin remodeling genes including Wiskott-Aldrich syndrome (WAS) protein, a critical regulator of Arp2/3 complex, was related to the magnitude of the applied acceleration but not to fluid shear. These data demonstrate that fluid shear does not regulate vibration induced proliferation and mineralization and that cytoskeletal remodeling activity may play a role in MSC mechanosensitivity. PMID:23870506

  12. Exosomes derived from mineralizing osteoblasts promote ST2 cell osteogenic differentiation by alteration of microRNA expression.

    PubMed

    Cui, Yazhou; Luan, Jing; Li, Haiying; Zhou, Xiaoyan; Han, Jinxiang

    2016-01-01

    Mineralizing osteoblasts (MOBs) can release exosomes, although the functional significance remains unclear. In the present study, we demonstrate that exosomes derived from mineralizing pre-osteoblast MC3T3-E1 cells can promote bone marrow stromal cell (ST2) differentiation to osteoblasts. We reveal that MOB-derived exosomes significantly influence miRNA profiles in recipient ST2 cells, and these changes tend to activate the Wnt signaling pathway by inhibiting Axin1 expression and increasing β-catenin expression. We also suggest that MOB derived-exosomes partly induce the variation in miRNA expression in recipient ST2 cells by exosomal miRNA transfer. These findings suggest an exosome-mediated mode of cell-to-cell communication in the osteogenic microenvironment, and also indicate the potential of MOB exosomes in bone tissue engineering. PMID:26763102

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

    SciTech Connect

    Katsuki, Yuko; Sakamoto, Kei; Minamizato, Tokutaro; Makino, Hatsune; Umezawa, Akihiro; Ikeda, Masa-aki; Perbal, Bernard; Amagasa, Teruo; Yamaguchi, Akira; Katsube, Ken-ichi

    2008-04-11

    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 by Notch/p21 pathway.

  14. Effect of miR-26a-5p on the Wnt/Ca(2+) Pathway and Osteogenic Differentiation of Mouse Adipose-Derived Mesenchymal Stem Cells.

    PubMed

    Li, Shasha; Hu, Chen; Li, Jianwei; Liu, Lei; Jing, Wei; Tang, Wei; Tian, Weidong; Long, Jie

    2016-08-01

    Elucidation of the molecular mechanisms that regulate the differentiation of adipose-derived mesenchymal stem cells into osteogenic cells may lead to new methods for bone tissue engineering. We examined the role of miR-26a-5p in the regulation of osteogenic differentiation of mouse adipose-derived mesenchymal stem cells (mADSCs) by using mimics and inhibitors of this microRNA. Our results showed that over-expression of miR-26a-5p inhibited osteogenesis and that suppression of endogenous miR-26a-5p promoted osteogenesis. Four bioinformatics algorithms indicated that the 3'UTR of Wnt5a was a potential target of miR-26a-5p. We confirmed this prediction by use of dual-luciferase reporter assay and GFP/RFP assay. We also examined the molecular mechanisms by which miR-26a-5p regulates osteogenesis. Fura-2AM and Western blot assays after transfection indicated that miR-26a-5p repressed WNT5A, inhibited calcium flux and protein kinase C, and suppressed osteogenic differentiation of mADSCs. By contrast, miR-26a-5p inhibition activated these signal proteins and promoted osteogenic differentiation. Taken together, our results suggest that up-regulation of miR-26a-5p inhibits osteogenic differentiation of mADSCs by directly targeting the 3'UTR of Wnt5a, thereby down-regulating the Wnt/Ca(2+) signaling pathway. PMID:27040676

  15. Correlation of NADH fluorescence lifetime and oxidative phosphorylation metabolism in the osteogenic differentiation of human mesenchymal stem cell

    NASA Astrophysics Data System (ADS)

    Guo, Han-Wen; Yu, Jia-Sin; Hsu, Shu-Han; Wei, Yau-Huei; Lee, Oscar K.; Dong, Chen-Yuan; Wang, Hsing-Wen

    2015-01-01

    Reduced nicotinamide dinucleotide (NADH) fluorescence lifetime has been broadly used as a metabolic indicator for stem cell imaging. However, the direct relationship between NADH fluorescence lifetime and metabolic pathway and activity remains to be clarified. In this study, we measured the NADH fluorescence lifetime of human mesenchymal stem cells (hMSCs) as well as the metabolic indictors, such as adenosine triphosphate (ATP) level, oxygen consumption, and lactate release, up to 4 weeks under normal osteogenic differentiation and oxidative phosphorylation-attenuated/inhibited differentiation by oligomycin A (OA) treatment. NADH fluorescence lifetime was positively correlated with oxygen consumption and ATP level during energy transformation from glycolysis to oxidative phosphorylation. Under OA treatment, oxidative phosphorylation was attenuated/inhibited (i.e., oxygen consumption remained the same as controls or lower), cells showed attenuated differentiation under glycolysis, and NADH fluorescence lifetime change was not detected. Increased expression of the overall complex proteins was observed in addition to Complex I. We suggested special caution needs to be exercised while interpreting NADH fluorescence lifetime signal in terms of stem cell differentiation.

  16. Porous polylactic acid-silica hybrids: preparation, characterization, and study of mesenchymal stem cell osteogenic differentiation.

    PubMed

    Pandis, Christos; Trujillo, Sara; Matos, Joana; Madeira, Sara; Ródenas-Rochina, Joaquín; Kripotou, Sotiria; Kyritsis, Apostolos; Mano, João F; Gómez Ribelles, José Luis

    2015-02-01

    A novel approach to reinforce polymer porous membranes is presented. In the prepared hybrid materials, the inorganic phase of silica is synthesized in-situ and inside the pores of aminolyzed polylactic acid (PLA) membranes by sol-gel reactions using tetraethylorthosilicate (TEOS) and glycidoxypropyltrimethoxysilane (GPTMS) as precursors. The hybrid materials present a porous structure with a silica layer covering the walls of the pores while GPTMS serves also as coupling agent between the organic and inorganic phase. The adjustment of silica precursors ratio allows the modulation of the thermomechanical properties. Culture of mesenchymal stem cells on these supports in osteogenic medium shows the expression of characteristic osteoblastic markers and the mineralization of the extracellular matrix. PMID:25303745

  17. Metallofullerene nanoparticles promote osteogenic differentiation of bone marrow stromal cells through BMP signaling pathway

    NASA Astrophysics Data System (ADS)

    Yang, Kangning; Cao, Weipeng; Hao, Xiaohong; Xue, Xue; Zhao, Jing; Liu, Juan; Zhao, Yuliang; Meng, Jie; Sun, Baoyun; Zhang, Jinchao; Liang, Xing-Jie

    2013-01-01

    Although endohedral metallofullerenol [Gd@C82(OH)22]n nanoparticles have anti-tumor efficiency and mostly deposit in the bones of mice, how these nanoparticles act in bone marrow stromal cells (MSCs) remains largely unknown. Herein, we observed that [Gd@C82(OH)22]n nanoparticles facilitated the differentiation of MSCs toward osteoblasts, as evidenced by the enhancement of alkaline phosphatase (ALP) activity and mineralized nodule formation upon [Gd@C82(OH)22]n nanoparticle treatment. Mechanistically, the effect of [Gd@C82(OH)22]n nanoparticles on ALP activity was inhibited by the addition of noggin as an inhibitor of the BMP signaling pathway. Moreover, the in vivo results of the ovariectomized rats further indicated that [Gd@C82(OH)22]n nanoparticles effectively improved bone density and prevented osteoporosis.Although endohedral metallofullerenol [Gd@C82(OH)22]n nanoparticles have anti-tumor efficiency and mostly deposit in the bones of mice, how these nanoparticles act in bone marrow stromal cells (MSCs) remains largely unknown. Herein, we observed that [Gd@C82(OH)22]n nanoparticles facilitated the differentiation of MSCs toward osteoblasts, as evidenced by the enhancement of alkaline phosphatase (ALP) activity and mineralized nodule formation upon [Gd@C82(OH)22]n nanoparticle treatment. Mechanistically, the effect of [Gd@C82(OH)22]n nanoparticles on ALP activity was inhibited by the addition of noggin as an inhibitor of the BMP signaling pathway. Moreover, the in vivo results of the ovariectomized rats further indicated that [Gd@C82(OH)22]n nanoparticles effectively improved bone density and prevented osteoporosis. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr33575a

  18. Modulation of osteogenic, adipogenic and myogenic differentiation of mesenchymal stem cells by submicron grooved topography.

    PubMed

    Wang, Peng-Yuan; Li, Wen-Tyng; Yu, Jiashing; Tsai, Wei-Bor

    2012-12-01

    Topographic cues have been recognized crucial on the modulation of cell behavior, and subsequent important for the design of implants, cell-based biomedical devices and tissue-engineered products. Grooved topography direct cells to align anisotropically on the substrates, resulting in an obvious morphological difference compared with the flat and the other topographies. This study aimed at investigating the effects of grooved topography on the differentiation of mesenchymal stem cells (MSCs) into osteoblasts, adipocytes and myoblasts. A series of submicron-grooved polystyrene substrates with equal groove-to-ridge ratio but different width and depth (width/depth (nm): 450/100, 450/350, 900/100, and 900/550) were fabricated based on electron beam lithography and soft lithography techniques. Primary rat MSCs (rMSCs) were cultured on these substrates without induction for differentiation for 6 days, and then subjected to induction for osteogenesis, adipogenesis and myogenesis. While the alignment of rMSCs strongly complied with the direction of the grooves and increased with groove depths, cell attachment on day 1 (~1.5 × 10(4)/cm(2)) and cell proliferation after 6 days of culture (~5 × 10(4)/cm(2)) were not significantly affected by substrate types. Osteogenesis, indicated by alkaline phosphatase activities and calcium deposit, was not significantly modulated by the grooved substrates, compared with the flat control, suggesting that cell alignment may not determine osteoinduction of rMSCs. On the other hand, adipogenesis, indicated by lipid production, was significantly enhanced by the grooved substrates compared with the flat surface (P < 0.001). On the other hand, myogenesis, indicated by desmin and MHC staining, was enhanced by the grooves in a time- and groove size-dependent manner compared with the flat control. The results suggested that grooved topography has an in-depth potential for modulating the commitment of the stem cell lineages, which could benefit

  19. New insights in osteogenic differentiation revealed by mass spectrometric assessment of phosphorylated substrates in murine skin mesenchymal cells

    PubMed Central

    2013-01-01

    Background Bone fractures and loss represent significant costs for the public health system and often affect the patients quality of life, therefore, understanding the molecular basis for bone regeneration is essential. Cytokines, such as IL-6, IL-10 and TNFα, secreted by inflammatory cells at the lesion site, at the very beginning of the repair process, act as chemotactic factors for mesenchymal stem cells, which proliferate and differentiate into osteoblasts through the autocrine and paracrine action of bone morphogenetic proteins (BMPs), mainly BMP-2. Although it is known that BMP-2 binds to ActRI/BMPR and activates the SMAD 1/5/8 downstream effectors, little is known about the intracellular mechanisms participating in osteoblastic differentiation. We assessed differences in the phosphorylation status of different cellular proteins upon BMP-2 osteogenic induction of isolated murine skin mesenchymal stem cells using Triplex Stable Isotope Dimethyl Labeling coupled with LC/MS. Results From 150 μg of starting material, 2,264 proteins were identified and quantified at five different time points, 235 of which are differentially phosphorylated. Kinase motif analysis showed that several substrates display phosphorylation sites for Casein Kinase, p38, CDK and JNK. Gene ontology analysis showed an increase in biological processes related with signaling and differentiation at early time points after BMP2 induction. Moreover, proteins involved in cytoskeleton rearrangement, Wnt and Ras pathways were found to be differentially phosphorylated during all timepoints studied. Conclusions Taken together, these data, allow new insights on the intracellular substrates which are phosphorylated early on during differentiation to BMP2-driven osteoblastic differentiation of skin-derived mesenchymal stem cells. PMID:24148232

  20. Cellular localization of NRF2 determines the self-renewal and osteogenic differentiation potential of human MSCs via the P53–SIRT1 axis

    PubMed Central

    Yoon, D S; Choi, Y; Lee, J W

    2016-01-01

    NRF2 (nuclear factor erythroid-derived 2-like 2) plays an important role in defense against oxidative stress at the cellular level. Recently, the roles of NRF2 in embryonic and adult stem cells have been reported, but its role in maintaining self-renewal and differentiation potential remains unknown. We studied the mechanisms of NRF2 action in mesenchymal stem cells (MSCs) derived from human bone marrow. We found that the cellular localization of NRF2 changed during prolonged cell passage and osteogenic differentiation. Blocking the nuclear import of NRF2 using ochratoxin A (OTA) induced the loss of the self-renewal and osteogenic potential of early-passage (EP) MSCs. Conversely, reinforcing the nuclear import of NRF2 using tert-butylhydroquinone (t-BHQ) improved the self-renewal capacity and maintained the differentiation potential in the osteogenic lineage of EP MSCs. Real-time quantitative PCR and western blot analysis showed that NRF2 positively regulates sirtuin 1 (SIRT1) at the mRNA and protein levels via the negative regulation of p53. The self-renewal and osteogenic potential suppressed in OTA-treated or NRF2-targeting small hairpin RNA (shRNA)-infected EP MSCs were rescued by introducing small interfering RNA (siRNA) targeting p53. t-BHQ treatment in late-passage (LP) MSCs, which lost their self-renewal and osteogenic potential, reversed these effects. In LP MSCs treated with t-BHQ for ∼7 days, the phosphorylation and nuclear localization of NRF2 improved and SIRT1 protein level increased, whereas p53 protein levels decreased. Therefore, our results suggest that NRF2 plays an important role in regulating p53 and SIRT1 to maintain MSC stemness. This study is the first to establish a functional link between NRF2 and SIRT1 expression in the maintenance of MSC self-renewal and differentiation potential. PMID:26866273

  1. The synergistic effect on osteogenic differentiation of human mesenchymal stem cells by diode laser-treated stimulating human umbilical vein endothelial cells

    NASA Astrophysics Data System (ADS)

    Kao, Chia-Tze; Hsu, Tuan-Ti; Huang, Tsui-Hsien; Wu, Yu-Tin; Chen, Yi-Wen; Shie, Ming-You

    2016-02-01

    Angiogenesis plays an important role in determining the biostimulation of bone regeneration, in either new bone or blood vessel formation. Human umbilical cord cells (HUVECs) are important effector cells in angiogenesis and are indispensable for osteogenesis and for their heterogeneity and plasticity. However, there are very few studies about the effects of HUVECs on diode laser-stimulated/regulated osteogenesis. In this study, we used diode laser as a model biostimulation to examine the role of HUVECs on laser-stimulated osteogenesis. Several bone formation-related proteins were also significantly up-regulated by the diode laser stimulation, indicating that HUVECs may participate in diode laser-stimulated osteogenesis. Interestingly, when human mesenchymal stem cells (hMSCs) cultured with HUVECs were diode laser-treated, the osteogenesis differentiation of the hMSCs was significantly promoted, indicating the important role of HUVECs in diode laser-enhanced osteogenesis. Adequately activated HUVECs are vital for the success of diode laser-stimulated hard-tissue regeneration. These findings provided valuable insights into the mechanism of diode laser-stimulated osteogenic differentiation, and a strategy to optimize the evaluation system for the in vitro osteogenesis capacity of laser treatment in periodontal repair.

  2. A novel strontium(II)-modified calcium phosphate bone cement stimulates human-bone-marrow-derived mesenchymal stem cell proliferation and osteogenic differentiation in vitro.

    PubMed

    Schumacher, M; Lode, A; Helth, A; Gelinsky, M

    2013-12-01

    In the present study, the in vitro effects of novel strontium-modified calcium phosphate bone cements (SrCPCs), prepared using two different approaches on human-bone-marrow-derived mesenchymal stem cells (hMSCs), were evaluated. Strontium ions, known to stimulate bone formation and therefore already used in systemic osteoporosis therapy, were incorporated into a hydroxyapatite-forming calcium phosphate bone cement via two simple approaches: incorporation of strontium carbonate crystals and substitution of Ca(2+) by Sr(2+) ions during cement setting. All modified cements released 0.03-0.07 mM Sr(2+) under in vitro conditions, concentrations that were shown not to impair the proliferation or osteogenic differentiation of hMSCs. Furthermore, strontium modification led to a reduced medium acidification and Ca(2+) depletion in comparison to the standard calcium phosphate cement. In indirect and direct cell culture experiments with the novel SrCPCs significantly enhanced cell proliferation and differentiation were observed. In conclusion, the SrCPCs described here could be beneficial for the local treatment of defects, especially in the osteoporotic bone. PMID:23917042

  3. Effects of Culture Substrate Made of Poly(N-isopropylacrylamide-co-acrylic acid) Microgels on Osteogenic Differentiation of Mesenchymal Stem Cells.

    PubMed

    Dai, Zhuojun; Shu, Yinglan; Wan, Chao; Wu, Chi

    2016-01-01

    Poly(N-isopropylacrylamide) (PNIPAM)-based polymers and gels are widely known and studied for their thermoresponsive property. In the biomaterials category, they are regarded as a potential cell culture substrate, not only because of their biocompatibility, but also their special character of allowing controlled detachment of cells via temperature stimulus. Previous research about PNIPAM-based substrates mostly concentrated on their effects in cell adhesion and proliferation. In this study, however, we investigate the influence of the PNIPAM-based substrate on the differentiation capacity of stem cells. Especially, we choose P(NIPAM-AA) microgels as a culture dish coating and mesenchymal stem cells (MSCs) are cultured on top of the microgels. Interestingly, we find that the morphology of MSCs changes remarkably on a microgel-coated surface, from the original spindle form to a more stretched and elongated cell shape. Accompanied by the alternation in morphology, the expression of several osteogenesis-related genes is elevated even without inducing factors. In the presence of full osteogenic medium, MSCs on a microgel substrate show an enhancement in the expression level of osteopontin and alizarin red staining signals, indicating the physical property of substrate has a direct effect on MSCs differentiation. PMID:27618001

  4. Sustained release of TGFbeta3 from PLGA microspheres and its effect on early osteogenic differentiation of human mesenchymal stem cells.

    PubMed

    Moioli, Eduardo K; Hong, Liu; Guardado, Jesse; Clark, Paul A; Mao, Jeremy J

    2006-03-01

    Despite the widespread role of transforming growth factor-beta3 (TGFbeta3) in wound healing and tissue regeneration, its long-term controlled release has not been demonstrated. Here, we report microencapsulation of TGFbeta3 in poly-d-l-lactic-co-glycolic acid (PLGA) microspheres and determine its bioactivity. The release profiles of PLGA-encapsulated TGFbeta3 with 50:50 and 75:25 PLA:PGA ratios differed throughout the experimental period. To compare sterilization modalities of microspheres, bFGF was encapsulated in 50:50 PLGA microspheres and subjected to ethylene oxide (EO) gas, radio-frequency glow discharge (RFGD), or ultraviolet (UV) light. The release of bFGF was significantly attenuated by UV light, but not significantly altered by either EO or RFGD. To verify its bioactivity, TGFbeta3 (1.35 ng/mL) was control-released to the culture of human mesenchymal stem cells (hMSC) under induced osteogenic differentiation. Alkaline phosphatase staining intensity was markedly reduced 1 week after exposing hMSC-derived osteogenic cells to TGFbeta3. This was confirmed by lower alkaline phosphatase activity (2.25 +/- 0.57 mU/mL/ng DNA) than controls (TGFbeta3- free) at 5.8 +/- 0.9 mU/mL/ng DNA (p < 0.05). Control-released TGFbeta3 bioactivity was further confirmed by lack of significant differences in alkaline phosphatase upon direct addition of 1.35 ng/mL TGFbeta3 to cell culture (p > 0.05). These findings provide baseline data for potential uses of microencapsulated TGFbeta3 in wound healing and tissue-engineering applications. PMID:16579687

  5. Differences in the Osteogenic Differentiation Capacity of Omental Adipose-Derived Stem Cells in Obese Patients With and Without Metabolic Syndrome.

    PubMed

    Oliva-Olivera, Wilfredo; Leiva Gea, Antonio; Lhamyani, Said; Coín-Aragüez, Leticia; Alcaide Torres, Juan; Bernal-López, Maria Rosa; García-Luna, Pedro Pablo; Morales Conde, Salvador; Fernández-Veledo, Sonia; El Bekay, Rajaa; Tinahones, Francisco José

    2015-12-01

    Multiple studies have suggested that the reduced differentiation capacity of multipotent adipose tissue-derived mesenchymal stem cells (ASCs) in obese subjects could compromise their use in cell therapy. Our aim was to assess the osteogenic potential of omental ASCs and to examine the status of the isolated CD34(negative)-enriched fraction of omental-derived ASCs from subjects with different metabolic profiles. Omental ASCs from normal-weight subjects and subjects with or without metabolic syndrome were isolated, and the osteogenic potential of omental ASCs was evaluated. Additionally, osteogenic and clonogenic potential, proliferation rate, mRNA expression levels of proteins involved in redox balance, and fibrotic proteins were examined in the CD34(negative)-enriched fraction of omental-derived ASCs. Both the omental ASCs and the CD34(negative)-enriched fraction of omental ASCs from subjects without metabolic syndrome have a greater osteogenic potential than those from subjects with metabolic syndrome. The alkaline phosphatase and osteonectin mRNA were negatively correlated with nicotinamide adenine dinucleotide phosphate oxidase-2 mRNA and the mRNA expression levels of the fibrotic proteins correlated positively with nicotinamide adenine dinucleotide phosphate oxidase-5 mRNA and the homeostasis model assessment. Although the population doubling time was significantly higher in subjects with a body mass index of 25 kg/m(2) or greater, only the CD34(negative)-enriched omental ASC fraction in the subjects with metabolic syndrome had a higher population doubling time than the normal-weight subjects. The osteogenic, clonogenic, fibrotic potential, and proliferation rate observed in vitro suggest that omental ASCs from subjects without metabolic syndrome are more suitable for therapeutic osteogenic applications than those from subjects with metabolic syndrome. PMID:26372179

  6. Bifunctional coating based on carboxymethyl chitosan with stable conjugated alkaline phosphatase for inhibiting bacterial adhesion and promoting osteogenic differentiation on titanium

    NASA Astrophysics Data System (ADS)

    Zheng, Dong; Neoh, Koon Gee; Kang, En-Tang

    2016-01-01

    In this work, alkaline phosphatase (ALP) was covalently immobilized on carboxymethyl chitosan (CMCS)-coated polydopamine (PDA)-functionalized Ti to achieve a bifunctional surface. Our results showed ∼89% reduction in Staphylococcus epidermidis adhesion on this surface compared to that on pristine Ti. The ALP-modified Ti supported cell proliferation, and significantly enhanced cellular ALP activity and calcium deposition of osteoblasts, human mesenchymal stem cells (hMSCs) and human adipose-derived stem cells (hADSCs). The extent of enhancement in the functions of these cells is dependent on the surface density of immobilized ALP. The substrate prepared using an ALP solution of 50 μg/cm2 resulted in 44%, 54% and 129% increase in calcium deposited by osteoblasts, hMSCs and hADSCs, respectively, compared to those cultured on pristine Ti. The ALP-modified substrates also promoted the osteogenic differentiation of hMSCs and hADSCs by up-regulating gene expressions of runt-related transcription factor 2 (RUNX2), osterix (OSX), and osteocalcin (OC) in the two types of stem cells. The surface-immobilized ALP was stable after being subjected to 1 h immersion in 70% ethanol and autoclaving at 121 °C for 20 min. However, the enzymatic bioactivity of the surface-immobilized ALP was reduced by about 50% after these substrates were immersed in phosphate buffered saline (PBS) or PBS containing lysozyme for 14 days.

  7. Functionalization of polycaprolactone scaffolds with hyaluronic acid and β-TCP facilitates migration and osteogenic differentiation of human dental pulp stem cells in vitro.

    PubMed

    Jensen, Jonas; Kraft, David Christian Evar; Lysdahl, Helle; Foldager, Casper Bindzus; Chen, Muwan; Kristiansen, Asger Albæk; Rölfing, Jan Hendrik Duedal; Bünger, Cody Eric

    2015-02-01

    In this study, we sought to assess the osteogenic potential of human dental pulp stem cells (DPSCs) on three different polycaprolactone (PCL) scaffolds. The backbone structure of the scaffolds was manufactured by fused deposition modeling (PCL scaffold). The composition and morphology was functionalized in two of the scaffolds. The first underwent thermal induced phase separation of PCL infused into the pores of the PCL scaffold. This procedure resulted in a highly variable micro- and nanostructured porous (NSP), interconnected, and isotropic tubular morphology (NSP-PCL scaffold). The second scaffold type was functionalized by dip-coating the PCL scaffold with a mixture of hyaluronic acid and β-TCP (HT-PCL scaffold). The scaffolds were cylindrical and measured 5 mm in height and 10 mm in diameter. They were seeded with 1×10(6) human DPSCs, a cell type known to express bone-related markers, differentiate into osteoblasts-like cells, and to produce a mineralized bone-like extracellular matrix. DPSCs were phenotypically characterized by flow cytometry for CD90(+), CD73(+), CD105(+), and CD14(-). DNA, ALP, and Ca(2+) assays and real-time quantitative polymerase chain reaction for genes involved in osteogenic differentiation were analyzed on day 1, 7, 14, and 21. Cell viability and distribution were assessed on day 1, 7, 14, and 21 by fluorescent-, scanning electron-, and confocal microscopy. The results revealed that the DPSCs expressed relevant gene expression consistent with osteogenic differentiation. The NSP-PCL and HT-PCL scaffolds promoted osteogenic differentiation and Ca(2+) deposition after 21 days of cultivation. Different gene expressions associated with mature osteoblasts were upregulated in these two scaffold types, suggesting that the methods in which the scaffolds promote osteogenic differentiation, depends on functionalization approaches. However, only the HT-PCL scaffold was also able to support cell proliferation and cell migration resulting in

  8. Functionalization of Polycaprolactone Scaffolds with Hyaluronic Acid and β-TCP Facilitates Migration and Osteogenic Differentiation of Human Dental Pulp Stem Cells In Vitro

    PubMed Central

    Kraft, David Christian Evar; Lysdahl, Helle; Foldager, Casper Bindzus; Chen, Muwan; Kristiansen, Asger Albæk; Rölfing, Jan Hendrik Duedal; Bünger, Cody Eric

    2015-01-01

    In this study, we sought to assess the osteogenic potential of human dental pulp stem cells (DPSCs) on three different polycaprolactone (PCL) scaffolds. The backbone structure of the scaffolds was manufactured by fused deposition modeling (PCL scaffold). The composition and morphology was functionalized in two of the scaffolds. The first underwent thermal induced phase separation of PCL infused into the pores of the PCL scaffold. This procedure resulted in a highly variable micro- and nanostructured porous (NSP), interconnected, and isotropic tubular morphology (NSP-PCL scaffold). The second scaffold type was functionalized by dip-coating the PCL scaffold with a mixture of hyaluronic acid and β-TCP (HT-PCL scaffold). The scaffolds were cylindrical and measured 5 mm in height and 10 mm in diameter. They were seeded with 1×106 human DPSCs, a cell type known to express bone-related markers, differentiate into osteoblasts-like cells, and to produce a mineralized bone-like extracellular matrix. DPSCs were phenotypically characterized by flow cytometry for CD90+, CD73+, CD105+, and CD14−. DNA, ALP, and Ca2+ assays and real-time quantitative polymerase chain reaction for genes involved in osteogenic differentiation were analyzed on day 1, 7, 14, and 21. Cell viability and distribution were assessed on day 1, 7, 14, and 21 by fluorescent-, scanning electron-, and confocal microscopy. The results revealed that the DPSCs expressed relevant gene expression consistent with osteogenic differentiation. The NSP-PCL and HT-PCL scaffolds promoted osteogenic differentiation and Ca2+ deposition after 21 days of cultivation. Different gene expressions associated with mature osteoblasts were upregulated in these two scaffold types, suggesting that the methods in which the scaffolds promote osteogenic differentiation, depends on functionalization approaches. However, only the HT-PCL scaffold was also able to support cell proliferation and cell migration resulting in even cell

  9. Mussel-inspired alginate gel promoting the osteogenic differentiation of mesenchymal stem cells and anti-infection.

    PubMed

    Zhang, Shiwen; Xu, Kaige; Darabi, Mohammad Ali; Yuan, Quan; Xing, Malcolm

    2016-12-01

    Alginate hydrogels have been used in cell encapsulation for many years but a prevalent issue with pure alginates is that they are unable to provide enough bioactive properties to interact with mammalian cells. This paper discusses the modification of alginate with mussel-inspired dopamine for cell loading and anti-infection. Mouse bone marrow stem cells were immobilized into alginate and alginate-dopamine beads and fibers. Through live-dead and MTT assay, alginates modified by dopamine promoted cell viability and proliferation. In vitro cell differentiation results showed that such an alginate-dopamine gel can promote the osteogenic differentiation of mesenchymal stem cell after PCR and ALP assays. In addition to that, the adhesive prosperities of dopamine allowed for coating the surface of alginate-dopamine gel with silver nanoparticles, which provided the gel with significant antibacterial characteristics. Overall, these results demonstrate that a dopamine-modified alginate gel can be a great tool for cell encapsulation to promote cell proliferation and can be applied to bone regeneration, especially in contaminated bone defects. PMID:27612740

  10. Iota-carrageenan/chitosan/gelatin scaffold for the osteogenic differentiation of adipose-derived MSCs in vitro.

    PubMed

    Li, Junjie; Yang, Boguang; Qian, Yufeng; Wang, Qiyu; Han, Ruijin; Hao, Tong; Shu, Yao; Zhang, Yabin; Yao, Fanglian; Wang, Changyong

    2015-10-01

    In this study, we have developed ι-carrageenan/chitosan/gelatin (CCG) scaffold containing multiple functional groups (-NH2 , -OH, -COOH, and -SO3 H) to resemble the native extracellular matrix (ECM), using the ion-shielding technology and ultrasonic dispersion method. Fourier transform infrared spectroscopy (FTIR) of the CCG scaffolds suggests that the formation of CCG network involves electrostatic interactions between ι-carrageenan (ι-CA) and chitosan/gelatin, and the covalent cross-linking among amino groups of chitosan and/or gelatin. Scanning electron microscopic (SEM) observation reveals that the porous structure of scaffolds can be modulated by the ratio of ι-CA to chitosan/gelatin. The swelling ratio of the hydrogels increases as the ι-CA contents increase. Using differential scanning calorimetry, we found that the double helix structure of ι-CA is only stabilized at low contents of ι-CA in the CCG scaffolds (e.g., 5 wt %). The scaffolds containing 5% ι-CA showed the best protein adsorption capacity (4.46 ± 0.63 μg protein/mg scaffold) and elastic modulus (5.37 ± 1.03 MPa). In addition, the CCG scaffolds exhibit excellent support for adipose-derived mesenchymal stem cells (ADMSCs) attachment and proliferation, and they can improve the osteogenic differentiation and neovascularization capacities of ADMSCs. Overall, we conclude that the CCG may represent an ideal scaffold material for bone tissue engineering. PMID:25449538

  11. miR-223 Regulates Adipogenic and Osteogenic Differentiation of Mesenchymal Stem Cells Through a C/EBPs/miR-223/FGFR2 Regulatory Feedback Loop.

    PubMed

    Guan, Xiaohui; Gao, Yifei; Zhou, Jie; Wang, Jun; Zheng, Fang; Guo, Fei; Chang, Ailing; Li, Xiaoxia; Wang, Baoli

    2015-05-01

    Several miRNAs have recently been identified to regulate adipocyte or osteoblast differentiation or both. In this study, miR-223 was found to be involved in the reciprocal regulation of adipocyte and osteoblast differentiation. miR-223 was induced in primary cultured mouse marrow stromal cell, mesenchymal line C3H10T1/2 and stromal line ST2 after adipogenic treatment. Conversely, it was reduced in preosteoblast MC3T3-E1 after osteogenic treatment. Supplementing miR-223 levels using synthetic miR-223 mimics significantly suppressed the growth of the C3H10T1/2 and ST2 cells and induced the progenitor cells to fully differentiate into adipocytes, along with induction of adipocyte-specific transcription factors peroxisome proliferator-activated receptor γ, CCAAT/enhancer binding protein-α (C/EBPα), and marker genes aP2 and adipsin. By contrast, depletion of the endogenous miR-223 using synthetic miR-223 inhibitor repressed the progenitor cells to differentiate. The effects of miR-223 on adipocyte formation from ST2 cells were also demonstrated by using lentivirus that overexpresses miR-223. Conversely, supplementing miR-223 blocked ST2 to differentiate into osteoblasts. Fibroblast growth factor receptor 2 (Fgfr2), a critical regulator of osteoblast, was shown to be a direct target of miR-223 by using dual luciferase reporter assay. Knockdown of Fgfr2 in C3H10T1/2 downregulated phosphorylation of ERK1/2 and upregulated expression of C/EBPα and dramatically enhanced the differentiation of the cells into adipocytes. Further investigation of mechanisms that control miR-223 expression demonstrated that C/EBPs induced miR-223 expression through binding to the promoter regions of the miR-223. Taken together, our study provides evidences that miR-223 regulates adipocyte and osteoblast differentiation through a novel C/EBPs/miR-223/FGFR2 regulatory feedback loop. PMID:25641499

  12. Human term placenta-derived mesenchymal stromal cells are less prone to osteogenic differentiation than bone marrow-derived mesenchymal stromal cells.

    PubMed

    Pilz, Gregor A; Ulrich, Christine; Ruh, Manuel; Abele, Harald; Schäfer, Richard; Kluba, Torsten; Bühring, Hans-Jörg; Rolauffs, Bernd; Aicher, Wilhelm K

    2011-04-01

    Mesenchymal stromal cells (MSC) can be isolated from different tissues. They are capable of differentiating in vitro, for example, to osteoblasts, chondrocytes, or adipocytes. In contrast to CD34 for hematopoietic stem cells, a distinct MSC-defining antibody is not available. Further, for hematopoietic cells lineage-defining antigens such as CD3 or CD20 are known. In contrast, for MSC-derived cells lineage-associated cell surface markers are far from being established. We therefore investigated expression of cell surface antigens on human term placenta-derived MSC (pMSC) in more detail and correlated expression pattern to the osteogenic differentiation capacity of the MSC. We report that pMSC expressed the typical cell surface antigens at levels comparable to bone marrow-derived MSC (bmMSC), including CD73, CD90, and CD105, but did not express CD11b, CD34, and CD45. Further, CD164, TNAP, and the W5C5 antigens were detected on pMSC, whereas CD349 was not observed. Some pMSC expressed CD146 at low or moderate levels, and their osteogenic differentiation potential was weak. In contrast, bmMSC expressed CD146 at high levels, expression of alkaline phosphatase was significantly higher, and they presented a pronounced osteogenic differentiation potential. We conclude that MSC from different sources differ in their expression of distinct markers, and that this may correlate in part with their lineage determination. Thus, a higher percentage of bmMSC expressed CD146 at prominent levels and such cells may be better suited for bone repair. In contrast, many pMSC expressed CD146 at low or moderate levels. They, therefore, may be suitable for applications in which osteogenic differentiation is undesirable. PMID:21047215

  13. Hyperoside, a flavonoid compound, inhibits proliferation and stimulates osteogenic differentiation of human osteosarcoma cells.

    PubMed

    Zhang, Ning; Ying, Mei-Dan; Wu, Yong-Ping; Zhou, Zhi-Hong; Ye, Zhao-Ming; Li, Hang; Lin, Ding-Sheng

    2014-01-01

    Osteosarcoma, one of the most common malignant bone tumours, is generally considered a differentiation disease caused by genetic and epigenetic disruptions in the terminal differentiation of osteoblasts. Novel therapies based on the non-cytotoxic induction of cell differentiation-responsive pathways could represent a significant advance in treating osteosarcoma; however, effective pharmaceuticals to induce differentiation are lacking. In the present study, we investigated the effect of hyperoside, a flavonoid compound, on the osteoblastic differentiation of U2OS and MG63 osteosarcoma cells in vitro. Our results demonstrated that hyperoside inhibits the proliferation of osteosarcoma cells by inducing G0/G1 arrest in the cell cycle, without causing obvious cell death. Cell migration assay further suggested that hyperoside could inhibit the invasion potential of osteosarcoma cells. Additionally, osteopontin and runt-related transcription factor 2 protein levels and osteocalcin activation were upregulated dramatically in hyperoside-treated osteosarcoma cells, suggesting that hyperoside may stimulates osteoblastic differentiation in osteosarcoma cells. This differentiation was accompanied by the activation of transforming growth factor (TGF)-β and bone morphogenetic protein-2, suggesting that the hyperoside-induced differentiation involves the TGF-β signalling pathway. To our knowledge, this study is the first to evaluate the differentiation effect of hyperoside in osteosarcoma cells and assess the possible potential for hyperoside treatment as a future therapeutic approach for osteosarcoma differentiation therapy. PMID:24983940

  14. Co(II)-mediated effects of plain and plasma immersion ion implanted cobalt-chromium alloys on the osteogenic differentiation of human mesenchymal stem cells.

    PubMed

    Schröck, Kathleen; Lutz, Johanna; Mändl, Stephan; Hacker, Michael C; Kamprad, Manja; Schulz-Siegmund, Michaela

    2015-03-01

    Medical CoCr is one of the main alloys used for metal-on-metal prosthesis in patients with total hip arthroplasty. CoCr surfaces modified by nitrogen plasma immersion ion implantation (PIII) are characterized by improved wear resistance but also showed increased Co(II) ion release under in vitro conditions. For the first time, CoCr modified by nitrogen PIII was evaluated with regard to its effect on the osteogenic differentiation of MSC. The activity of alkaline phosphatase, the expression of the osteogenic genes Runt-related transcription factor 2, osteopontin as well as integrin-binding bone sialoprotein and the production of osteocalcin and hydroxyapatite were determined. The results of our study demonstrate that Co(II) ions released from the alloy affected the osteogenic differentiation of MSC. Distinct differences in differentiation markers were found between pristine and modified alloys for osteocalcin but not for integrin-binding sialoprotein and hydroxyapatite. Interestingly, osteopontin was upregulated in naive and differentiated MSC by Co(II) ions and modified CoCr, likely through the induction of a cellular hypoxic response. The findings of this study contribute to a better understanding of possible risk factors with regard to a clinical applicability of surface modified CoCr implant materials. PMID:25469667

  15. Promotion of osteogenic differentiation of stem cells and increase of bone-bonding ability in vivo using urease-treated titanium coated with calcium phosphate and gelatin

    NASA Astrophysics Data System (ADS)

    Huang, Zhong-Ming; Qi, Yi-Ying; Du, Shao-Hua; Feng, Gang; Unuma, Hidero; Yan, Wei-Qi

    2013-10-01

    Because of its excellent biocompatibility and low allergenicity, titanium has been widely used for bone replacement and tissue engineering. To produce a desirable composite with enhanced bone response and mechanical strength, in this study bioactive calcium phosphate (CaP) and gelatin composites were coated onto titanium (Ti) via a novel urease technique. The cellular responses to the CaP/gelatin/Ti (CaP/gel/Ti) and bone bonding ability were evaluated with proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) on CaP/gel/Ti and CaP/Ti in vitro. The results showed that the optical density values, alkaline phosphatase expression and genes expression of MSCs on CaP/gel/Ti were similar to those on CaP/Ti, yet significantly higher than those on pure Ti (p < 0.05). CaP/gel/Ti and CaP/Ti rods (2 mm in diameter, 10 mm in length) were also implanted into femoral shaft of rabbits and pure Ti rods served as control (n = 10). Histological examination, scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) measurements were performed at 4 and 8 weeks after the operation. The histological and SEM observations demonstrated clearly that more new bone formed on the surface of CaP/gel/Ti than in the other two groups at each time point. The CaP/gel/Ti bonded to the surrounding bone directly with no intervening soft tissue layer. An interfacial layer, containing Ti, Ca and P, was found to form at the interface between bone and the implant on all three groups by EDS analysis. However, the content of Ca, P in the surface of CaP/gel/Ti implants was more than in the other two groups at each time point. The CaP/gel/Ti modified by the urease method was not only beneficial for MSCs proliferation and osteogenic differentiation, but also favorable for bone bonding ability on Ti implants in vivo, suggesting that Ti functionalized with CaP and gelatin might have a great potential in clinical joint replacement or dental implants.

  16. Glucose oxidase facilitates osteogenic differentiation and mineralization of embryonic stem cells through the activation of Nrf2 and ERK signal transduction pathways.

    PubMed

    Sim, Hyun-Jaung; Kim, Jae-Hwan; Kook, Sung-Ho; Lee, Seung-Youp; Lee, Jeong-Chae

    2016-08-01

    Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/heme oxygenase-1 (HO-1) signal is known to play important roles in controlling bone homeostasis. This study examined how oxidative stress affects the mineralization of embryonic stem (ES) cells by exposing them to glucose oxidase (GO), which continuously generates H2O2 at low concentrations. The roles of Nrf2/HO-1 and mitogen-activated protein kinases on osteogenesis in GO-exposed ES cells were also investigated. GO treatment at relatively low concentrations did not change the viability of ES cells, whereas it enhanced osteogenic differentiation and mineralization in the cells. GO treatment (1 mU/ml) augmented the induction of runt-related transcription factor 2 (Runx2), Nrf2, and HO-1 in ES cells. GO-mediated acceleration of Runx2 expression and mineralization was inhibited either by Nrf2 knockdown or by treating with 5 μM PD98059, an inhibitor of phospho-extracellular signal-regulated kinase (p-ERK). The GO-stimulated mineralization was also suppressed by treating the cells with reduced glutathione or catalase, but not by superoxide dismutase or N-acetyl-cysteine. Collectively, our results demonstrate that a mild oxidative stress activates Nrf2/HO-1 signaling and an ERK-mediated pathway, and facilitates the mineralization of ES cells with a corresponding increase in Runx2. PMID:27431005

  17. The phytoestrogen genistein enhances osteogenesis and represses adipogenic differentiation of human primary bone marrow stromal cells.

    PubMed

    Heim, M; Frank, O; Kampmann, G; Sochocky, N; Pennimpede, T; Fuchs, P; Hunziker, W; Weber, P; Martin, I; Bendik, I

    2004-02-01

    In the present study, we investigated the role of the phytoestrogen genistein and 17beta-estradiol in human bone marrow stromal cells, undergoing induced osteogenic or adipogenic differentiation. Profiling of estrogen receptors (ERs)-alpha, -beta1, -beta2, -beta3, -beta4, -beta5, and aromatase mRNAs revealed lineage-dependent expression patterns. During osteogenic differentiation, the osteoblast-determining core binding factor-alpha1 showed a progressive increase, whereas the adipogenic regulator peroxisome proliferator-activated receptor gamma (PPARgamma) was sequentially decreased. This temporal regulation of lineage-determining marker genes was strongly enhanced by genistein during the early osteogenic phase. Moreover, genistein increased alkaline phosphatase mRNA levels and activity, the osteoprotegerin:receptor activator of nuclear factor-kappaB ligand gene expression ratio, and the expression of TGFbeta1. During adipogenic differentiation, down-regulation in the mRNA levels of PPARgamma and CCAAT/enhancer-binding protein-alpha at d 3 and decreased lipoprotein lipase and adipsin mRNA levels at d 21 were observed after genistein treatment. This led to a lower number of adipocytes and a reduction in the size of their lipid droplets. At d 3 of adipogenesis, TGFbeta1 was strongly up-regulated by genistein in an ER-dependent manner. Blocking the TGFbeta1 pathway abolished the effects of genistein on PPARgamma protein levels and led to a reduction in the proliferation rate of precursor cells. Overall, genistein enhanced the commitment and differentiation of bone marrow stromal cells to the osteoblast lineage but did not influence the late osteogenic maturation markers. Adipogenic differentiation and maturation, on the other hand, were reduced by genistein (and 17beta-estradiol) via an ER-dependent mechanism involving autocrine or paracrine TGFbeta1 signaling. PMID:14605006

  18. A Comparative Analysis of the In Vitro Effects of Pulsed Electromagnetic Field Treatment on Osteogenic Differentiation of Two Different Mesenchymal Cell Lineages

    PubMed Central

    Ceccarelli, Gabriele; Bloise, Nora; Mantelli, Melissa; Gastaldi, Giulia; Fassina, Lorenzo; De Angelis, Maria Gabriella Cusella; Ferrari, Davide; Imbriani, Marcello

    2013-01-01

    Abstract Human mesenchymal stem cells (MSCs) are a promising candidate cell type for regenerative medicine and tissue engineering applications. Exposure of MSCs to physical stimuli favors early and rapid activation of the tissue repair process. In this study we investigated the in vitro effects of pulsed electromagnetic field (PEMF) treatment on the proliferation and osteogenic differentiation of bone marrow MSCs (BM-MSCs) and adipose-tissue MSCs (ASCs), to assess if both types of MSCs could be indifferently used in combination with PEMF exposure for bone tissue healing. We compared the cell viability, cell matrix distribution, and calcified matrix production in unstimulated and PEMF-stimulated (magnetic field: 2 mT, amplitude: 5 mV) mesenchymal cell lineages. After PEMF exposure, in comparison with ASCs, BM-MSCs showed an increase in cell proliferation (p<0.05) and an enhanced deposition of extracellular matrix components such as decorin, fibronectin, osteocalcin, osteonectin, osteopontin, and type-I and -III collagens (p<0.05). Calcium deposition was 1.5-fold greater in BM-MSC–derived osteoblasts (p<0.05). The immunofluorescence related to the deposition of bone matrix proteins and calcium showed their colocalization to the cell-rich areas for both types of MSC-derived osteoblast. Alkaline phosphatase activity increased nearly 2-fold (p<0.001) and its protein content was 1.2-fold higher in osteoblasts derived from BM-MSCs. The quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis revealed up-regulated transcription specific for bone sialoprotein, osteopontin, osteonectin, and Runx2, but at a higher level for cells differentiated from BM-MSCs. All together these results suggest that PEMF promotion of bone extracellular matrix deposition is more efficient in osteoblasts differentiated from BM-MSCs. PMID:23914335

  19. Hydrogels functionalized with N-cadherin mimetic peptide enhance osteogenesis of hMSCs by emulating the osteogenic niche.

    PubMed

    Zhu, Meiling; Lin, Sien; Sun, Yuxin; Feng, Qian; Li, Gang; Bian, Liming

    2016-01-01

    N-cadherin is considered to be the key factor in directing cell-cell interactions during mesenchymal condensation, which is essential to osteogenesis. In this study, hyaluronic acid (HA) hydrogels are biofunctionalized with an N-cadherin mimetic peptide to mimic the pro-osteogenic niche in the endosteal space to promote the osteogenesis of human mesenchymal stem cells (hMSCs). Results show that the conjugation of the N-cadherin peptide in the HA hydrogels enhances the expression of the osteogenic marker genes in the seeded hMSCs. Furthermore, the biofunctionalized HA hydrogels promote the alkaline phosphatase activity, type I collagen deposition, and matrix mineralization by the seeded hMSCs under both in vitro and in vivo condition. We postulate that the biofunctionalized hydrogels emulates the N-cadherin-mediated homotypic cell-cell adhesion among MSCs and the "orthotypic" interaction between the osteoblasts and MSCs. These findings demonstrate that the biofunctionalized HA hydrogels provide a supportive niche microenvironment for the osteogenesis of hMSCs. PMID:26580785

  20. Osteogenic differentiation of human mesenchymal stem cells in collagen matrices: effect of uniaxial cyclic tensile strain on bone morphogenetic protein (BMP-2) mRNA expression.

    PubMed

    Sumanasinghe, Ruwan D; Bernacki, Susan H; Loboa, Elizabeth G

    2006-12-01

    Human mesenchymal stem cells (hMSCs) differentiate down an osteogenic pathway with appropriate mechanical and/or chemical stimuli. This study describes the successful culture of hMSCs in 3D collagen matrices under mechanical strain. Bone marrow-derived hMSCs were seeded in linear 3D type I collagen matrices and subjected to 0%, 10%, or 12% uniaxial cyclic tensile strain at 1 Hz for 4 h/day for 7 or 14 days. Cell viability studies indicated that hMSCs remained viable throughout the culture period irrespective of the applied strain level. Real-time RT-PCR studies indicated a significant increase in BMP-2 mRNA expression levels in hMSCs strained at 10% compared to the same day unstrained controls after both 7 and 14 days. An increase in BMP-2 was also observed in hMSCs subjected to 12% strain, but the increase was significant only in the 14-day sample. This is the first report of the culture of bone marrow-derived hMSCs in 3D collagen matrices under cyclic strain, and the first demonstration that strain alone can induce osteogenic differentiation without the addition of osteogenic supplements. Induction of bone differentiation in 3D culture is a critical step in the creation of bioengineered bone constructs. PMID:17518682

  1. Improvement of Distribution and Osteogenic Differentiation of Human Mesenchymal Stem Cells by Hyaluronic Acid and β-Tricalcium Phosphate-Coated Polymeric Scaffold In Vitro.

    PubMed

    Chen, Muwan; Le, Dang Q S; Kjems, Jørgen; Bünger, Cody; Lysdahl, Helle

    2015-01-01

    Bone tissue engineering requires a well-designed scaffold that can be biodegradable, biocompatible, and support the stem cells to osteogenic differentiation. Porous polycaprolactone (PCL) scaffold prepared by fused deposition modeling is an attractive biomaterial that has been used in clinic. However, PCL scaffolds lack biological function and osteoinductivity. In this study, we functionalized the PCL scaffolds by embedding them with a matrix of hyaluronic acid/β-tricalcium phosphate (HA/TCP). Human mesenchymal stem cells (MSCs) were cultured on scaffolds with and without coating to investigate proliferation and osteogenic differentiation. The DNA amount was significantly higher in the HA/TCP-coated scaffold on day 21. At the gene expression level, HA/TCP coating significantly increased the expression of ALP and COLI on day 4. These data correlated with the ALP activity peaking on day 7 in the HA/TCP-coated scaffold. Scanning electron microscope and histological analysis revealed that the cell matrix and calcium deposition were distributed more uniformly in the coated scaffolds compared to scaffolds without coating. In conclusion, the HA/TCP coating improved cellular proliferation, osteogenic differentiation, and uniform distribution of the cellular matrix in vitro. The HA/TCP-PCL scaffold holds great promise to accommodate human bone marrow-derived MSCs for bone reconstruction purposes, which warrants future in vivo studies. PMID:26487981

  2. Improvement of Distribution and Osteogenic Differentiation of Human Mesenchymal Stem Cells by Hyaluronic Acid and β-Tricalcium Phosphate-Coated Polymeric Scaffold In Vitro

    PubMed Central

    Chen, Muwan; Le, Dang Q.S.; Kjems, Jørgen; Bünger, Cody; Lysdahl, Helle

    2015-01-01

    Abstract Bone tissue engineering requires a well-designed scaffold that can be biodegradable, biocompatible, and support the stem cells to osteogenic differentiation. Porous polycaprolactone (PCL) scaffold prepared by fused deposition modeling is an attractive biomaterial that has been used in clinic. However, PCL scaffolds lack biological function and osteoinductivity. In this study, we functionalized the PCL scaffolds by embedding them with a matrix of hyaluronic acid/β-tricalcium phosphate (HA/TCP). Human mesenchymal stem cells (MSCs) were cultured on scaffolds with and without coating to investigate proliferation and osteogenic differentiation. The DNA amount was significantly higher in the HA/TCP-coated scaffold on day 21. At the gene expression level, HA/TCP coating significantly increased the expression of ALP and COLI on day 4. These data correlated with the ALP activity peaking on day 7 in the HA/TCP-coated scaffold. Scanning electron microscope and histological analysis revealed that the cell matrix and calcium deposition were distributed more uniformly in the coated scaffolds compared to scaffolds without coating. In conclusion, the HA/TCP coating improved cellular proliferation, osteogenic differentiation, and uniform distribution of the cellular matrix in vitro. The HA/TCP-PCL scaffold holds great promise to accommodate human bone marrow-derived MSCs for bone reconstruction purposes, which warrants future in vivo studies. PMID:26487981

  3. Validation of Housekeeping Genes to Study Human Gingival Stem Cells and Their In Vitro Osteogenic Differentiation Using Real-Time RT-qPCR

    PubMed Central

    Taïhi, Ihsène; Nassif, Ali; Berbar, Tsouria; Isaac, Juliane; Berdal, Ariane; Gogly, Bruno; Fournier, Benjamin Philippe

    2016-01-01

    Gingival stem cells (GSCs) are recently isolated multipotent cells. Their osteogenic capacity has been validated in vitro and may be transferred to human cell therapy for maxillary large bone defects, as they share a neural crest cell origin with jaw bone cells. RT-qPCR is a widely used technique to study gene expression and may help us to follow osteoblast differentiation of GSCs. For accurate results, the choice of reliable housekeeping genes (HKGs) is crucial. The aim of this study was to select the most reliable HKGs for GSCs study and their osteogenic differentiation (dGSCs). The analysis was performed with ten selected HKGs using four algorithms: ΔCt comparative method, GeNorm, BestKeeper, and NormFinder. This study demonstrated that three HKGs, SDHA, ACTB, and B2M, were the most stable to study GSC, whereas TBP, SDHA, and ALAS1 were the most reliable to study dGSCs. The comparison to stem cells of mesenchymal origin (ASCs) showed that SDHA/HPRT1 were the most appropriate for ASCs study. The choice of suitable HKGs for GSCs is important as it gave access to an accurate analysis of osteogenic differentiation. It will allow further study of this interesting stem cells source for future human therapy. PMID:26880978

  4. Evaluation of a thiolated chitosan scaffold for local delivery of BMP-2 for osteogenic differentiation and ectopic bone formation.

    PubMed

    Bae, In-Ho; Jeong, Byung-Chul; Kook, Min-Suk; Kim, Sun-Hun; Koh, Jeong-Tae

    2013-01-01

    Thiolated chitosan (Thio-CS) is a well-established pharmaceutical excipient for drug delivery. However, its use as a scaffold for bone formation has not been investigated. The aim of this study was to evaluate the potential of Thio-CS in bone morphogenetic protein-2 (BMP-2) delivery and bone formation. In vitro study showed that BMP-2 interacted with the Thio-CS and did not affect the swelling behavior. The release kinetics of BMP-2 from the Thio-CS was slightly delayed (70%) within 7 days compared with that from collagen gel (Col-gel, 85%), which is widely used in BMP-2 delivery. The BMP-2 released from Thio-CS increased osteoblastic cell differentiation but did not show any cytotoxicity until 21 days. Analysis of the in vivo ectopic bone formation at 4 weeks of posttransplantation showed that use of Thio-CS for BMP-2 delivery induced more bone formation to a greater extent (1.8 fold) than that of Col-gel. However, bone mineral density in both bones was equivalent, regardless of Thio-CS or Col-gel carrier. Taken together, Thio-CS system might be useful for delivering osteogenic protein BMP-2 and present a promising bone regeneration strategy. PMID:24024213

  5. A Multicompartment Holder for Spinner Flasks Improves Expansion and Osteogenic Differentiation of Mesenchymal Stem Cells in Three-Dimensional Scaffolds

    PubMed Central

    Teixeira, Graciosa Q.; Barrias, Cristina C.; Lourenço, Ana H.

    2014-01-01

    In the tissue engineering field dynamic culture systems, such as spinner flasks, are widely used due to their ability to improve mass transfer in suspension cell cultures. However, this culture system is often unsuitable to culture cells in three-dimensional (3D) scaffolds. To address this drawback, we designed a multicompartment holder for 3D cell culture, easily adaptable to spinner flasks. Here, the device was tested with human mesenchymal stem cells (MSCs) seeded in 3D porous chitosan scaffolds that were maintained in spinner flasks under dynamic conditions (50 rpm). Standard static culture conditions were used as control. The dynamic conditions were shown to significantly increase MSCs proliferation over 1 week (approximately 6-fold) and to improve cell distribution within the scaffold. Moreover, they also promoted osteogenic differentiation of MSCs, inducing an earlier peak in alkaline phosphatase (ALP) activity, and a more homogenous ALP staining and matrix mineralization in the whole scaffolds, but particularly in the center. Overall, this study shows a new multicompartment holder to culture 3D scaffolds that can broaden the application of spinner flasks. PMID:24650268

  6. Degradation of IF1 controls energy metabolism during osteogenic differentiation of stem cells

    PubMed Central

    Sánchez-Aragó, María; García-Bermúdez, Javier; Martínez-Reyes, Inmaculada; Santacatterina, Fulvio; Cuezva, José M

    2013-01-01

    Differentiation of human mesenchymal stem cells (hMSCs) requires the rewiring of energy metabolism. Herein, we demonstrate that the ATPase inhibitory factor 1 (IF1) is expressed in hMSCs and in prostate and colon stem cells but is not expressed in the differentiated cells. IF1 inhibits oxidative phosphorylation and regulates the activity of aerobic glycolysis in hMSCs. Silencing of IF1 in hMSCs mimics the metabolic changes observed in osteocytes and accelerates cellular differentiation. Activation of IF1 degradation acts as the switch that regulates energy metabolism during differentiation. We conclude that IF1 is a stemness marker important for maintaining the quiescence state. PMID:23722655

  7. Periostin: A Downstream Mediator of EphB4-Induced Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

    PubMed Central

    Zhang, Fei; Zhang, Zehua; Sun, Dong; Dong, Shiwu; Xu, Jianzhong; Dai, Fei

    2016-01-01

    Erythropoietin-producing hepatocyte B4 (EphB4) has been reported to be a key molecular switch in the regulation of bone homeostasis, but the underlying mechanism remains poorly understood. In this study, we investigated the role of EphB4 in regulating the expression of periostin (POSTN) within bone marrow-derived mesenchymal stem cells (MSCs) and assessed its effect and molecular mechanism of osteogenic induction in vitro. Treatment with ephrinB2-FC significantly increased the expression of POSTN in MSCs, and the inhibition of EphB4 could abrogate this effect. In addition, osteogenic markers were upregulated especially in MSCs overexpressing EphB4. To elucidate the underlying mechanism of cross talk between EphB4 and the Wnt pathway, we detected the change in protein expression of phosphorylated-glycogen synthase kinase 3β-serine 9 (p-GSK-3β-Ser9) and β-catenin, as well as the osteogenic markers Runx2 and COL1. The results showed that GSK-3β activation and osteogenic marker expression levels were downregulated by ephrinB2-FC treatment, but these effects were inhibited by blocking integrin αvβ3 in MSCs. Our findings demonstrate that EphB4 can promote osteogenic differentiation of MSCs via upregulation of POSTN expression. It not only helps to reveal the interaction mechanism between EphB4 and Wnt pathway but also brings a better understanding of EphB4/ephrinB2 signaling in bone homeostasis. PMID:26788070

  8. Long noncoding RNA related to periodontitis interacts with miR-182 to upregulate osteogenic differentiation in periodontal mesenchymal stem cells of periodontitis patients.

    PubMed

    Wang, L; Wu, F; Song, Y; Li, X; Wu, Q; Duan, Y; Jin, Z

    2016-01-01

    Periodontitis impairs the osteogenic differentiation of human periodontal mesenchymal stem cells (hPDLSCs), but the underlying molecular mechanisms are still poorly understood. Long noncoding RNAs (lncRNAs) have been demonstrated to have significant roles under both physiologic and pathological conditions. In this study, we performed comprehensive lncRNA profiling by lncRNA microarray analysis and identified a novel lncRNA, osteogenesis impairment-related lncRNA of PDLSCs from periodontitis patients (lncRNA-POIR), the expression of which was significantly decreased in PDLSCs from periodontitis patients (pPDLSCs) and was upregulated by osteogenic induction. To study the functions of lncRNA-POIR, we prepared cells with overexpression and knockdown of lncRNA-POIR and found that lncRNA-POIR positively regulated osteogenic differentiation of hPDLSCs and pPDLSCs both in vitro and in vivo. Using quantitative real-time PCRs (qPCRs) and luciferase reporter assays, we demonstrated that lncRNA-POIR may act as a competing endogenous RNA (ceRNA) for miR-182, leading to derepression of its target gene, FoxO1. In this process, lncRNA-POIR and miR-182 suppress each other and form a network to regulate FoxO1. FoxO1 increased bone formation of pPDLSCs by competing with TCF-4 for β-catenin and inhibiting the canonical Wnt pathway. Finally, inflammation increases miR-182 expression through the nuclear factor-κB pathway, and the miR-182 overexpression in the inflammatory microenvironment resulted in an imbalance in the lncRNA-POIR-miR-182 regulatory network. In conclusion, our results provide novel evidence that this lncRNA-miRNA (microRNA) regulatory network has a significant role in osteogenic differentiation of pPDLSCs and that it has potential as a therapeutic target in mesenchymal stem cells during inflammation. PMID:27512949

  9. 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. PMID:23897753

  10. The parathyroid hormone-related protein is secreted during the osteogenic differentiation of human dental follicle cells and inhibits the alkaline phosphatase activity and the expression of DLX3.

    PubMed

    Klingelhöffer, C; Reck, A; Ettl, T; Morsczeck, C

    2016-08-01

    The dental follicle is involved in tooth eruption and it expresses a great amount of the parathyroid hormone-related protein (PTHrP). PTHrP as an extracellular protein is required for a multitude of different regulations of enchondral bone development and differentiation of bone precursor cells and of the development of craniofacial tissues. The dental follicle contains also precursor cells (DFCs) of the periodontium. Isolated DFCs differentiate into periodontal ligament cells, alveolar osteoblast and cementoblasts. However, the role of PTHrP during the human periodontal development remains elusive. Our study evaluated the influence of PTHrP on the osteogenic differentiation of DFCs under in vitro conditions for the first time. The PTHrP protein was highly secreted after 4days of the induction of the osteogenic differentiation of DFCs with dexamethasone (2160.5pg/ml±345.7SD. in osteogenic differentiation medium vs. 315.7pg/ml±156.2SD. in standard cell culture medium; Student's t Test: p<0.05 (n=3)). We showed that the supplementation of the osteogenic differentiation medium with PTHrP inhibited the alkaline phosphatase activity and the expression of the transcription factor DLX3, but the depletion of PTHrP did not support the differentiation of DFCs. Previous studies have shown that Indian Hedgehog (IHH) induces PTHrP and that PTHrP, in turn, inhibits IHH via a negative feedback loop. We showed that SUFU (Suppressor Of Fused Homolog) was not regulated during the osteogenic differentiation in DFCs. So, neither the hedgehog signaling pathway induced PTHrP nor PTHrP suppressed the hedgehog signaling pathway during the osteogenic differentiation in DFCs. In conclusion, our results suggest that PTHrP regulates independently of the hedgehog signaling pathway the osteogenic differentiated in DFCs. PMID:27368119

  11. Inhibition of Ape1 Redox Activity Promotes Odonto/osteogenic Differentiation of Dental Papilla Cells

    PubMed Central

    Chen, Tian; Liu, Zhi; Sun, Wenhua; Li, Jingyu; Liang, Yan; Yang, Xianrui; Xu, Yang; Yu, Mei; Tian, Weidong; Chen, Guoqing; Bai, Ding

    2015-01-01

    Dentinogenesis is the formation of dentin, a substance that forms the majority of teeth, and this process is performed by odontoblasts. Dental papilla cells (DPCs), as the progenitor cells of odontoblasts, undergo the odontogenic differentiation regulated by multiple cytokines and paracrine signal molecules. Ape1 is a perfect paradigm of the function complexity of a biological macromolecule with two major functional regions for DNA repair and redox regulation, respectively. To date, it remains unclear whether Ape1 can regulate the dentinogenesis in DPCs. In the present study, we firstly examed the spatio-temporal expression of Ape1 during tooth germ developmental process, and found the Ape1 expression was initially high and then gradually reduced along with the tooth development. Secondly, the osteo/odontogenic differentiation capacity of DPCs was up-regulated when treated with either Ape1-shRNA or E3330 (a specific inhibitor of the Ape1 redox function), respectively. Moreover, we found that the canonical Wnt signaling pathway was activated in this process, and E3330 reinforced-osteo/odontogenic differentiation capacity was suppressed by Dickkopf1 (DKK1), a potent antagonist of canonical Wnt signaling pathway. Taken together, we for the first time showed that inhibition of Ape1 redox regulation could promote the osteo/odontogenic differentiation capacity of DPCs via canonical Wnt signaling pathway. PMID:26639148

  12. Inhibition of Ape1 Redox Activity Promotes Odonto/osteogenic Differentiation of Dental Papilla Cells.

    PubMed

    Chen, Tian; Liu, Zhi; Sun, Wenhua; Li, Jingyu; Liang, Yan; Yang, Xianrui; Xu, Yang; Yu, Mei; Tian, Weidong; Chen, Guoqing; Bai, Ding

    2015-01-01

    Dentinogenesis is the formation of dentin, a substance that forms the majority of teeth, and this process is performed by odontoblasts. Dental papilla cells (DPCs), as the progenitor cells of odontoblasts, undergo the odontogenic differentiation regulated by multiple cytokines and paracrine signal molecules. Ape1 is a perfect paradigm of the function complexity of a biological macromolecule with two major functional regions for DNA repair and redox regulation, respectively. To date, it remains unclear whether Ape1 can regulate the dentinogenesis in DPCs. In the present study, we firstly examed the spatio-temporal expression of Ape1 during tooth germ developmental process, and found the Ape1 expression was initially high and then gradually reduced along with the tooth development. Secondly, the osteo/odontogenic differentiation capacity of DPCs was up-regulated when treated with either Ape1-shRNA or E3330 (a specific inhibitor of the Ape1 redox function), respectively. Moreover, we found that the canonical Wnt signaling pathway was activated in this process, and E3330 reinforced-osteo/odontogenic differentiation capacity was suppressed by Dickkopf1 (DKK1), a potent antagonist of canonical Wnt signaling pathway. Taken together, we for the first time showed that inhibition of Ape1 redox regulation could promote the osteo/odontogenic differentiation capacity of DPCs via canonical Wnt signaling pathway. PMID:26639148

  13. Osteogenic Differentiation of Human Mesenchymal Stem Cells in Freeze-Gelled Chitosan/Nano β-Tricalcium Phosphate Porous Scaffolds Crosslinked with Genipin

    PubMed Central

    Siddiqui, Nadeem; Pramanik, Krishna; Jabbari, Esmaiel

    2015-01-01

    The objective of this work was to investigate material properties and osteogenic differentiation of human mesenchymal stem cells (hMSCs) in genipin (GN) crosslinked chitosan/nano β-tricalcium phosphate (CS/nano β-TCP) scaffolds, and compare the results with tripolyphosphate (TPP) crosslinked scaffolds. Porous crosslinked CS/nano β-TCP scaffolds were produced by freeze-gelation using GN (CBG scaffold) and TPP (CBT scaffold) as crosslinkers. The prepared CBT and CBG scaffolds were characterized with respect to porosity, pore size, water content, wettability, compressive strength, mass loss, and osteogenic differentiation of hMSCs. All scaffolds displayed interconnected honeycomb-like microstructures. There was a significant difference between the average pore size, porosity, contact angle, and percent swelling of CBT and CBG scaffolds. The average pore size of CBG scaffolds was higher than CBT, the porosity of CBG was lower than CBT, the water contact angle of CBG was higher than CBT, and the percent swelling of CBG was lower than CBT. At a given crosslinker concentration, there was not a significant difference in compressive modulus and mass loss of CBG and CBT scaffolds. Metabolic activity of hMSCs seeded in CBG scaffolds was slightly higher than CBT. Furthermore, CBG scaffolds displayed slightly higher extent of mineralization after 21 days incubation in osteogenic medium compared to CBT. PMID:26046270

  14. Evaluation of an in vivo heterotopic model of osteogenic differentiation of equine bone marrow and muscle mesenchymal stem cells in fibrin glue scaffold.

    PubMed

    McDuffee, Laurie A; Esparza Gonzalez, Blanca P; Nino-Fong, Rodolfo; Aburto, Enrique

    2014-02-01

    Autologous mesenchymal stem cells (MSCs) have been used as a potential cell-based therapy in various animal and human diseases. Their differentiation capacity makes them useful as a novel strategy in the treatment of tissue injury in which the healing process is compromised or delayed. In horses, bone healing is slow, taking a minimum of 6-12 months. The osteogenic capacity of equine bone marrow and muscle MSCs mixed with fibrin glue or phosphate-buffered saline (PBS) as a scaffold is assessed. Bone production by the following groups was compared: Group 1, bone marrow (BM) MSCs in fibrin glue; Group 2, muscle (M) MSCs in fibrin glue; Group 3, BM MSCs in PBS; Group 4, M MSCs in PBS and as a control; Group 5, fibrin glue without cells. BM and M MSCs underwent osteogenic stimulation for 48 h prior to being injected intramuscularly into nude mice. After 4 weeks, the mice were killed and muscle samples were collected and evaluated for bone formation and mineralization by using radiology, histochemistry and immunohistochemistry. Positive bone formation and mineralization were confirmed in Group 1 in nude mice based on calcium deposition and the presence of osteocalcin and collagen type I; in addition, a radiopaque area was observed on radiographs. However, no evidence of mineralization or bone formation was observed in Groups 2-5. In this animal model, equine BM MSCs mixed with fibrin glue showed better osteogenic differentiation capacity compared with BM MSCs in PBS and M MSCs in either carrier. PMID:24258028

  15. Osteogenic differentiation of human mesenchymal stem cells in 3-D Zr-Si organi