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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. DNA N6-methyladenine demethylase ALKBH1 enhances osteogenic differentiation of human MSCs

    PubMed Central

    Zhou, Chenchen; Liu, Yuting; Li, Xiaobing; Zou, Jing; Zou, Shujuan

    2016-01-01

    ALKBH1 was recently discovered as a demethylase for DNA N6-methyladenine (N6-mA), a new epigenetic modification, and interacts with the core transcriptional pluripotency network of embryonic stem cells. However, the role of ALKBH1 and DNA N6-mA in regulating osteogenic differentiation is largely unknown. In this study, we demonstrated that the expression of ALKBH1 in human mesenchymal stem cells (MSCs) was upregulated during osteogenic induction. Knockdown of ALKBH1 increased the genomic DNA N6-mA levels and significantly reduced the expression of osteogenic-related genes, alkaline phosphatase activity, and mineralization. ALKBH1-depleted MSCs also exhibited a restricted capacity for bone formation in vivo. By contrast, the ectopic overexpression of ALKBH1 enhanced osteoblastic differentiation. Mechanically, we found that the depletion of ALKBH1 resulted in the accumulation of N6-mA on the promoter region of ATF4, which subsequently silenced ATF4 transcription. In addition, restoring the expression of ATP by adenovirus-mediated transduction successfully rescued osteogenic differentiation. Taken together, our results demonstrate that ALKBH1 is indispensable for the osteogenic differentiation of MSCs and indicate that DNA N6-mA modifications area new mechanism for the epigenetic regulation of stem cell differentiation. PMID:27785372

  4. Simvastatin enhances Rho/actin/cell rigidity pathway contributing to mesenchymal stem cells' osteogenic differentiation.

    PubMed

    Tai, I-Chun; Wang, Yao-Hsien; Chen, Chung-Hwan; Chuang, Shu-Chun; Chang, Je-Ken; Ho, Mei-Ling

    2015-01-01

    Recent studies have indicated that statins induce osteogenic differentiation both in vitro and in vivo. The molecular mechanism of statin-stimulated osteogenesis is unknown. Activation of RhoA signaling increases cytoskeletal tension, which plays a crucial role in the osteogenic differentiation of mesenchymal stem cells. We thus hypothesized that RhoA signaling is involved in simvastatin-induced osteogenesis in bone marrow mesenchymal stem cells. We found that although treatment with simvastatin shifts localization of RhoA protein from the membrane to the cytosol, the treatment still activates RhoA dose-dependently because it reduces the association with RhoGDIα. Simvastatin also increased the expression of osteogenic proteins, density of actin filament, the number of focal adhesions, and cellular tension. Furthermore, disrupting actin cytoskeleton or decreasing cell rigidity by using chemical agents reduced simvastatin-induced osteogenic differentiation. In vivo study also confirms that density of actin filament is increased in simvastatin-induced ectopic bone formation. Our study is the first to demonstrate that maintaining intact actin cytoskeletons and enhancing cell rigidity are crucial in simvastatin-induced osteogenesis. The results suggested that simvastatin, which is an osteoinductive factor and acts by increasing actin filament organization and cell rigidity combined with osteoconductive biomaterials, may benefit stem-cell-based bone regeneration. PMID:26451103

  5. 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.

  6. Simvastatin enhances Rho/actin/cell rigidity pathway contributing to mesenchymal stem cells’ osteogenic differentiation

    PubMed Central

    Tai, I-Chun; Wang, Yao-Hsien; Chen, Chung-Hwan; Chuang, Shu-Chun; Chang, Je-Ken; Ho, Mei-Ling

    2015-01-01

    Recent studies have indicated that statins induce osteogenic differentiation both in vitro and in vivo. The molecular mechanism of statin-stimulated osteogenesis is unknown. Activation of RhoA signaling increases cytoskeletal tension, which plays a crucial role in the osteogenic differentiation of mesenchymal stem cells. We thus hypothesized that RhoA signaling is involved in simvastatin-induced osteogenesis in bone marrow mesenchymal stem cells. We found that although treatment with simvastatin shifts localization of RhoA protein from the membrane to the cytosol, the treatment still activates RhoA dose-dependently because it reduces the association with RhoGDIα. Simvastatin also increased the expression of osteogenic proteins, density of actin filament, the number of focal adhesions, and cellular tension. Furthermore, disrupting actin cytoskeleton or decreasing cell rigidity by using chemical agents reduced simvastatin-induced osteogenic differentiation. In vivo study also confirms that density of actin filament is increased in simvastatin-induced ectopic bone formation. Our study is the first to demonstrate that maintaining intact actin cytoskeletons and enhancing cell rigidity are crucial in simvastatin-induced osteogenesis. The results suggested that simvastatin, which is an osteoinductive factor and acts by increasing actin filament organization and cell rigidity combined with osteoconductive biomaterials, may benefit stem-cell-based bone regeneration. PMID:26451103

  7. Simvastatin enhances Rho/actin/cell rigidity pathway contributing to mesenchymal stem cells' osteogenic differentiation.

    PubMed

    Tai, I-Chun; Wang, Yao-Hsien; Chen, Chung-Hwan; Chuang, Shu-Chun; Chang, Je-Ken; Ho, Mei-Ling

    2015-01-01

    Recent studies have indicated that statins induce osteogenic differentiation both in vitro and in vivo. The molecular mechanism of statin-stimulated osteogenesis is unknown. Activation of RhoA signaling increases cytoskeletal tension, which plays a crucial role in the osteogenic differentiation of mesenchymal stem cells. We thus hypothesized that RhoA signaling is involved in simvastatin-induced osteogenesis in bone marrow mesenchymal stem cells. We found that although treatment with simvastatin shifts localization of RhoA protein from the membrane to the cytosol, the treatment still activates RhoA dose-dependently because it reduces the association with RhoGDIα. Simvastatin also increased the expression of osteogenic proteins, density of actin filament, the number of focal adhesions, and cellular tension. Furthermore, disrupting actin cytoskeleton or decreasing cell rigidity by using chemical agents reduced simvastatin-induced osteogenic differentiation. In vivo study also confirms that density of actin filament is increased in simvastatin-induced ectopic bone formation. Our study is the first to demonstrate that maintaining intact actin cytoskeletons and enhancing cell rigidity are crucial in simvastatin-induced osteogenesis. The results suggested that simvastatin, which is an osteoinductive factor and acts by increasing actin filament organization and cell rigidity combined with osteoconductive biomaterials, may benefit stem-cell-based bone regeneration.

  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.

  10. 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

  11. The Enhancement of Human Embryonic Stem Cell Osteogenic Differentiation with Nano-Fibrous Scaffolding

    PubMed Central

    Smith, Laura A.; Liu, Xiaohua; Hu, Jiang; Ma, Peter X.

    2010-01-01

    Human embryonic stem cells (hESC) hold great promise as a cell source for tissue engineering since they possess the ability to differentiate into any cell type within the body. However, much work must still be done to control the differentiation of the hESC to the desired lineage. In this study, we examined the effects of the nano-fibrous (NF) architecture in both two dimensional (2-D) poly(L-lactic acid) (PLLA) thin matrices and 3-D PLLA scaffolds in vitro to assess their affect on the osteogenic differentiation of hESC in vitro compared to more traditional solid films and solid-walled (SW) scaffolds. In 2-D culture, hESC on NF thin matrices were found to express collagen type 1, Runx2, and osteocalcin mRNA of higher levels than the hESC on the solid films after 1 week of culture and increased mineralization was observed on the NF matrices compared to the solid films after 3 weeks of culture. After 6 weeks of 3-D culture, the hESC on the NF scaffolds expressed signifcantly more osteocalcin mRNA compared to these on the SW scaffolds. The data indicates that the NF architecture enhances the osteogenic differentiation of the hESC compared to more traditional scaffolding architecture. PMID:20430439

  12. Novel Polypyrrole-Coated Polylactide Scaffolds Enhance Adipose Stem Cell Proliferation and Early Osteogenic Differentiation

    PubMed Central

    Pelto, Jani; Björninen, Miina; Pälli, Aliisa; Talvitie, Elina; Hyttinen, Jari; Mannerström, Bettina; Suuronen Seppanen, Riitta; Kellomäki, Minna; Miettinen, Susanna; Haimi, Suvi

    2013-01-01

    An electrically conductive polypyrrole (PPy) doped with a bioactive agent is an emerging functional biomaterial for tissue engineering. We therefore used chondroitin sulfate (CS)-doped PPy coating to modify initially electrically insulating polylactide resulting in novel osteogenic scaffolds. In situ chemical oxidative polymerization was used to obtain electrically conductive PPy coating on poly-96L/4D-lactide (PLA) nonwoven scaffolds. The coated scaffolds were characterized and their electrical conductivity was evaluated in hydrolysis. The ability of the coated and conductive scaffolds to enhance proliferation and osteogenic differentiation of human adipose stem cells (hASCs) under electrical stimulation (ES) in three-dimensional (3D) geometry was compared to the noncoated PLA scaffolds. Electrical conductivity of PPy-coated PLA scaffolds (PLA-PPy) was evident at the beginning of hydrolysis, but decreased during the first week of incubation due to de-doping. PLA-PPy scaffolds enhanced hASC proliferation significantly compared to the plain PLA scaffolds at 7 and 14 days. Furthermore, the alkaline phosphatase (ALP) activity of the hASCs was generally higher in PLA-PPy seeded scaffolds, but due to patient variation, no statistical significance could be determined. ES did not have a significant effect on hASCs. This study highlights the potential of novel PPy-coated PLA scaffolds in bone tissue engineering. PMID:23126228

  13. Platelet-rich concentrate in serum free medium enhances osteogenic differentiation of bone marrow-derived human mesenchymal stromal cells.

    PubMed

    Samuel, Shani; Ahmad, Raja Elina; Ramasamy, Thamil Selvee; Karunanithi, Puvanan; Naveen, Sangeetha Vasudevaraj; Murali, Malliga Raman; Abbas, Azlina A; Kamarul, Tunku

    2016-01-01

    Previous studies have shown that platelet concentrates used in conjunction with appropriate growth media enhance osteogenic differentiation of human mesenchymal stromal cells (hMSCs). However, their potential in inducing osteogenesis of hMSCs when cultured in serum free medium has not been explored. Furthermore, the resulting osteogenic molecular signatures of the hMSCs have not been compared to standard osteogenic medium. We studied the effect of infrequent supplementation (8-day interval) of 15% non-activated platelet-rich concentrate (PRC) in serum free medium on hMSCs proliferation and differentiation throughout a course of 24 days, and compared the effect with those cultured in a standard osteogenic medium (OM). Cell proliferation was analyzed by alamar blue assay. Gene expression of osteogenic markers (Runx2, Collagen1, Alkaline Phosphatase, Bone morphogenetic protein 2, Osteopontin, Osteocalcin, Osteonectin) were analyzed using Q-PCR. Immunocytochemical staining for osteocalcin, osteopontin and transcription factor Runx2 were done at 8, 16 and 24 days. Biochemical assays for the expression of ALP and osteocalcin were also performed at these time-points. Osteogenic differentiation was further confirmed qualitatively by Alizarin Red S staining that was quantified using cetylpyridinium chloride. Results showed that PRC supplemented in serum free medium enhanced hMSC proliferation, which peaked at day 16. The temporal pattern of gene expression of hMSCs under the influence of PRC was comparable to that of the osteogenic media, but at a greater extent at specific time points. Immunocytochemical staining revealed stronger staining for Runx2 in the PRC-treated group compared to OM, while the staining for Osteocalcin and Osteopontin were comparable in both groups. ALP activity and Osteocalcin/DNA level were higher in the PRC group. Cells in the PRC group had similar level of bone mineralization as those cultured in OM, as reflected by the intensity of Alizarin red

  14. Platelet-rich concentrate in serum free medium enhances osteogenic differentiation of bone marrow-derived human mesenchymal stromal cells

    PubMed Central

    Ramasamy, Thamil Selvee; Karunanithi, Puvanan; Naveen, Sangeetha Vasudevaraj; Murali, Malliga Raman; Abbas, Azlina A.; Kamarul, Tunku

    2016-01-01

    Previous studies have shown that platelet concentrates used in conjunction with appropriate growth media enhance osteogenic differentiation of human mesenchymal stromal cells (hMSCs). However, their potential in inducing osteogenesis of hMSCs when cultured in serum free medium has not been explored. Furthermore, the resulting osteogenic molecular signatures of the hMSCs have not been compared to standard osteogenic medium. We studied the effect of infrequent supplementation (8-day interval) of 15% non-activated platelet-rich concentrate (PRC) in serum free medium on hMSCs proliferation and differentiation throughout a course of 24 days, and compared the effect with those cultured in a standard osteogenic medium (OM). Cell proliferation was analyzed by alamar blue assay. Gene expression of osteogenic markers (Runx2, Collagen1, Alkaline Phosphatase, Bone morphogenetic protein 2, Osteopontin, Osteocalcin, Osteonectin) were analyzed using Q-PCR. Immunocytochemical staining for osteocalcin, osteopontin and transcription factor Runx2 were done at 8, 16 and 24 days. Biochemical assays for the expression of ALP and osteocalcin were also performed at these time-points. Osteogenic differentiation was further confirmed qualitatively by Alizarin Red S staining that was quantified using cetylpyridinium chloride. Results showed that PRC supplemented in serum free medium enhanced hMSC proliferation, which peaked at day 16. The temporal pattern of gene expression of hMSCs under the influence of PRC was comparable to that of the osteogenic media, but at a greater extent at specific time points. Immunocytochemical staining revealed stronger staining for Runx2 in the PRC-treated group compared to OM, while the staining for Osteocalcin and Osteopontin were comparable in both groups. ALP activity and Osteocalcin/DNA level were higher in the PRC group. Cells in the PRC group had similar level of bone mineralization as those cultured in OM, as reflected by the intensity of Alizarin red

  15. Platelet-rich concentrate in serum free medium enhances osteogenic differentiation of bone marrow-derived human mesenchymal stromal cells

    PubMed Central

    Ramasamy, Thamil Selvee; Karunanithi, Puvanan; Naveen, Sangeetha Vasudevaraj; Murali, Malliga Raman; Abbas, Azlina A.; Kamarul, Tunku

    2016-01-01

    Previous studies have shown that platelet concentrates used in conjunction with appropriate growth media enhance osteogenic differentiation of human mesenchymal stromal cells (hMSCs). However, their potential in inducing osteogenesis of hMSCs when cultured in serum free medium has not been explored. Furthermore, the resulting osteogenic molecular signatures of the hMSCs have not been compared to standard osteogenic medium. We studied the effect of infrequent supplementation (8-day interval) of 15% non-activated platelet-rich concentrate (PRC) in serum free medium on hMSCs proliferation and differentiation throughout a course of 24 days, and compared the effect with those cultured in a standard osteogenic medium (OM). Cell proliferation was analyzed by alamar blue assay. Gene expression of osteogenic markers (Runx2, Collagen1, Alkaline Phosphatase, Bone morphogenetic protein 2, Osteopontin, Osteocalcin, Osteonectin) were analyzed using Q-PCR. Immunocytochemical staining for osteocalcin, osteopontin and transcription factor Runx2 were done at 8, 16 and 24 days. Biochemical assays for the expression of ALP and osteocalcin were also performed at these time-points. Osteogenic differentiation was further confirmed qualitatively by Alizarin Red S staining that was quantified using cetylpyridinium chloride. Results showed that PRC supplemented in serum free medium enhanced hMSC proliferation, which peaked at day 16. The temporal pattern of gene expression of hMSCs under the influence of PRC was comparable to that of the osteogenic media, but at a greater extent at specific time points. Immunocytochemical staining revealed stronger staining for Runx2 in the PRC-treated group compared to OM, while the staining for Osteocalcin and Osteopontin were comparable in both groups. ALP activity and Osteocalcin/DNA level were higher in the PRC group. Cells in the PRC group had similar level of bone mineralization as those cultured in OM, as reflected by the intensity of Alizarin red

  16. Morinda citrifolia leaves enhance osteogenic differentiation and mineralization of human periodontal ligament cells.

    PubMed

    Boonanantanasarn, Kanitsak; Janebodin, Kajohnkiart; Suppakpatana, Prapan; Arayapisit, Tawepong; Rodsutthi, Jit-aree; Chunhabundit, Panjit; Boonanuntanasarn, Surintorn; Sripairojthikoon, Wanida

    2012-01-01

    This present study investigated the potential of Morinda citrifolia leaf aqueous extract to induce osteogenic differentiation and matrix mineralization of human periodontal ligament (hPDL) cells. Human periodontal ligament cells were cultured in complete medium, ascorbic acid with β-glycerophosphate, or Morinda citrifolia leaf aqueous extract. Morinda citrifolia leaf aqueous extract significantly increased alkaline phosphatase activity compared to culturing in complete medium or ascorbic acid with β-glycerophosphate. Matrixcontaining mineralized nodules were formed only when the cells were cultured in the presence of Morinda citrifolia leaf aqueous extract. These nodules showed positive alizarin red S staining and were rich in calcium and phosphorus according to energy dispersive X-ray analysis. In conclusion, Morinda citrifolia leaf extract promoted osteogenic differentiation and matrix mineralization in human periodontal ligament cells, a clear indication of the therapeutic potential of Morinda citrifolia leaves in bone and periodontal tissue regeneration.

  17. 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.

  18. Peptide decorated nano-hydroxyapatite with enhanced bioactivity and osteogenic differentiation via polydopamine coating.

    PubMed

    Sun, Yuhua; Deng, Yi; Ye, Ziyou; Liang, Shanshan; Tang, Zhihui; Wei, Shicheng

    2013-11-01

    To be better used as implant materials in bone graft substitutes, bioactivity and osteogenesis of nano-hydroxyapatite (nano-HA) need to be further enhanced. Inspired by adhesive proteins in mussels, here we developed a novel bone forming peptide decorated nano-HA material. In this study, nano-HA was coated by one-step pH-induced polymerization of dopamine, and then the peptide was grafted onto polydopamine (pDA) coated nano-HA (HA-pDA) through catechol chemistry. Our results demonstrated that the peptide-conjugated nano-HA crystals could induce the adhesion and proliferation of MG-63 cells. Moreover, the highly alkaline phosphatase activity of the functionalized nano-HA indicated that the grafted peptide could maintain its biological activity after immobilization onto the surface of HA-pDA, especially at the concentration of 100μg/mL. These modified nano-HA crystals with better bioactivity and osteogenic differentiation hold great potential to be applied as bioactive materials in bone repairing, bone regeneration and bio-implant coating applications.

  19. Sphingosine-1-phosphate-enhanced Wnt5a promotes osteogenic differentiation in C3H10T1/2 cells.

    PubMed

    Hashimoto, Yoko; Kobayashi, Mari; Matsuzaki, Etsuko; Higashi, Katsumasa; Takahashi-Yanaga, Fumi; Takano, Aiko; Hirata, Masato; Nishimura, Fusanori

    2016-10-01

    In this study, we investigated the involvement of Wnt signaling in sphingosine-1-phosphate (S1P)-enhanced osteogenic differentiation of C3H10T1/2 pluripotent stem cells. We found that S1P enhanced the expression of Wnt5a and low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6) during osteogenic differentiation. Wnt5a-neutralizing antibody inhibited S1P-enhanced expression of LRP5/6 and alkaline phosphatase, which are essential for osteogenic differentiation. Conversely, S1P did not affect endogenous canonical Wnt signaling. Taken together, S1P-enhanced Wnt5a promotes LRP5/6 expression, resulting in the trigger of osteogenic differentiation of C3H10T1/2 cells. These findings suggest a potential beneficial role for S1P in bone regeneration. PMID:27486054

  20. Sphingosine-1-phosphate-enhanced Wnt5a promotes osteogenic differentiation in C3H10T1/2 cells.

    PubMed

    Hashimoto, Yoko; Kobayashi, Mari; Matsuzaki, Etsuko; Higashi, Katsumasa; Takahashi-Yanaga, Fumi; Takano, Aiko; Hirata, Masato; Nishimura, Fusanori

    2016-10-01

    In this study, we investigated the involvement of Wnt signaling in sphingosine-1-phosphate (S1P)-enhanced osteogenic differentiation of C3H10T1/2 pluripotent stem cells. We found that S1P enhanced the expression of Wnt5a and low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6) during osteogenic differentiation. Wnt5a-neutralizing antibody inhibited S1P-enhanced expression of LRP5/6 and alkaline phosphatase, which are essential for osteogenic differentiation. Conversely, S1P did not affect endogenous canonical Wnt signaling. Taken together, S1P-enhanced Wnt5a promotes LRP5/6 expression, resulting in the trigger of osteogenic differentiation of C3H10T1/2 cells. These findings suggest a potential beneficial role for S1P in bone regeneration.

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

    PubMed

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

    2014-05-01

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

  2. Silicate-substituted calcium phosphate with enhanced strut porosity stimulates osteogenic differentiation of human mesenchymal stem cells.

    PubMed

    De Godoy, Roberta Ferro; Hutchens, Stacy; Campion, Charlie; Blunn, Gordon

    2015-01-01

    While many synthetic ceramic bone graft substitutes (BGSs) have osteoconductive properties (e.g. provide a physical scaffold for osteointegration of surrounding bone tissue), certain BGSs are osteostimulative in that they actively upregulate mesenchymal stem cell proliferation and stimulate differentiation into osteoblast-like cells. The osteostimulative properties of silicate-substituted calcium phosphate with enhanced porosity (SiCaP EP) were evaluated in vitro with STRO-1+ immunoselected human bone marrow derived mesenchymal stem cells (HBMSCs). Osteostimulative materials (SiCaP) and Bioglass 45S5 (Bioglass) were also assessed as positive controls along with non-silicate substituted hydroxyapatite as a negative control. HBMSCs were also assessed on Thermanox discs cultured in basal and osteogenic media to determine when osteogenic differentiation could be significantly detected with this in vitro cell system. HBMSC viability and necrosis, total DNA content, alkaline phosphatase (ALP) expression, and osteocalcin expression were evaluated after 7, 14, 21, and 28 days. It was demonstrated that SiCaP EP is osteostimulative based on its propensity to support STRO-1+ HBMSC proliferation and ability to promote the differentiation of HBMSCs down the osteoblastic lineage from ALP-expressing, matrix-producing osteoblasts to Osteocalcin-producing pre-osteocytes without the presence of external osteogenic factors. SiCaP EP permitted greater HBMSC attachment as well as ALP and Osteocalcin expression than Bioglass which may be attributed to its microstructure and chemistry.

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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.

  10. Delivery of siRNA via cationic Sterosomes to enhance osteogenic differentiation of mesenchymal stem cells.

    PubMed

    Cui, Zhong-Kai; Fan, Jiabing; Kim, Soyon; Bezouglaia, Olga; Fartash, Armita; Wu, Benjamin M; Aghaloo, Tara; Lee, Min

    2015-11-10

    Noggin is a specific antagonist of bone morphogenetic proteins (BMPs) that can prevent the interaction of BMPs with their receptors. RNA interfering molecules have been used to downregulate noggin expression and thereby stimulate BMP signaling and osteogenesis. Cationic liposomes are considered one of the most efficient non-viral systems for gene delivery. In the past decade, non-phospholipid liposomes (Sterosomes) formulated with single-chain amphiphiles and high content of sterols have been developed. In particular, Sterosomes composed of stearylamine (SA) and cholesterol (Chol) display distinct properties compared with traditional phospholipid liposomes, including increased positive surface charges and enhanced particle stability. Herein, we report SA/Chol Sterosome and small interfering RNA (siRNA) complexes that significantly enhanced cellular uptake and gene knockdown efficiencies in adipose derived mesenchymal stem cells with minimal cytotoxicity compared with commercially available lipofectamine 2000. Furthermore, we confirmed osteogenic efficacy of these Sterosomes loaded with noggin siRNA in in vitro two- and three-dimensional settings as well as in a mouse calvarial defect model. The delivery of siRNA via novel SA/Chol Sterosomes presents a powerful method for efficient gene knockdown. These distinct nanoparticles may present a promising alternative approach for gene delivery.

  11. 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

  12. Humanized culture of periosteal progenitors in allogeneic serum enhances osteogenic differentiation and in vivo bone formation.

    PubMed

    Roberts, Scott J; Owen, Helen C; Tam, Wai Long; Solie, Lien; Van Cromphaut, Sophie J; Van den Berghe, Greet; Luyten, Frank P

    2014-02-01

    The translation of stem cell-based regenerative solutions from the laboratory to the clinic is often hindered by the culture conditions used to expand cell populations. Although fetal bovine serum (FBS) is widely used, regulatory bodies and safety concerns encourage alternative, xeno-free culturing practices. In an attempt to apply this approach to a bone-forming combination product of human periosteal progenitors (human periosteum derived cells) on a clinically used calcium phosphate carrier, FBS was substituted for human allogeneic serum (hAS) during cell expansion. It was found that cell proliferation was increased in hAS along with an apparent commitment to the osteogenic lineage, indicated by enhanced Runx2 expression, as well as alkaline phosphatase activity and matrix mineralization. Following analysis of signaling pathways, it was found that interferon-mediated signaling was downregulated, whereas JAK-STAT signaling was upregulated. STAT3 phosphorylation was enhanced in hAS-cultured human periosteum derived cells, inhibition of which ablated the proliferative effect of hAS. Furthermore, following in vivo implantation of hAS-cultured cells on NuOss scaffolds, enhanced bone formation was observed compared with FBS (71% increase, p < .001). Interestingly, the de novo-formed bone appeared to have a higher ratio of immature regions to mature regions, indicating that after 8 weeks implantation, tissue-formation processes were continuing. Integration of the implant with the environment appeared to be altered, with a decrease in calcium phosphate grain size and surface area, indicative of accelerated resorption. This study highlights the advantages of using humanized culture conditions for the expansion of human periosteal progenitors intended for bone regeneration.

  13. 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

  14. 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

  15. Boron enhances odontogenic and osteogenic differentiation of human tooth germ stem cells (hTGSCs) in vitro.

    PubMed

    Taşlı, Pakize Neslihan; Doğan, Ayşegül; Demirci, Selami; Şahin, Fikrettin

    2013-06-01

    Stem cell technology has been a great hope for the treatment of many common problems such as Parkinson's disease, Alzheimer's disease, diabetes, cancer, and tissue regeneration. Therefore, the main challenge in hard tissue engineering is to make a successful combination of stem cells and efficient inductors in the concept of stem cell differentiation into odontogenic and osteogenic cell types. Although some boron derivatives have been reported to promote bone and teeth growth in vivo, the molecular mechanism of bone formation has not been elucidated yet. Different concentrations of sodium pentaborate pentahydrate (NaB) were prepared for the analysis of cell toxicity and differentiation evaluations. The odontogenic, osteogenic differentiation and biomineralization of human tooth germ stem cells (hTGSCs) were evaluated by analyzing the mRNA expression levels, odontogenic and osteogenic protein expressions, alkaline phosphatase (ALP) activity, mineralization, and calcium deposits. The NaB-treated group displayed the highest ALP activity and expression of osteo- and odontogenic-related genes and proteins compared to the other groups and baseline. In the current study, increased in vitro odontogenic and osteogenic differentiation capacity of hTGSCs by NaB application has been shown for the first time. The study offers considerable promise for the development of new scaffold systems combined with NaB in both functional bone and tooth tissue engineering.

  16. Hydroxyapatite Nanoparticle Reinforced Peptide Amphiphile Nanomatrix Enhances the Osteogenic Differentiation of Mesenchymal Stem Cells by Compositional Ratios

    PubMed Central

    Vines, Jeremy B.; Lim, Dong-Jin; Anderson, Joel M.; Jun, Ho-Wook

    2012-01-01

    In the field of bone tissue engineering, there is a need for materials that mimic the native bone extracellular matrix (ECM). This need is met through the creation of biphasic composites intended to mimic both the organic and inorganic facets of the native bone ECM. However, few studies have created composites with organic ECM analogous components capable of directing cellular behaviors and many are not fabricated in the nanoscale. Furthermore, few attempts have been made at investigating how variations of organic and inorganic components affect the osteogenic differentiation of human mesenchymal stem cells (hMSCs). To address these issues, biphasic nanomatrix composites consisting of hydroxyapatite nanoparticles (HANPs) embedded within peptide amphiphile (PA) nanofibers tailored with the RGDS cellular adhesion motif (PA-RGDS) were created at various HANP to PA-RGDS ratios. Fabrication of these biphasic nanomatrix composites were confirmed via scanning electron microscope (SEM) and transmission electron microscope (TEM). The long-term cellularity and osteogenic differentiation of hMSCs in response to the different compositional ratios was then assessed by quantifying the timed expression of genes indicative of osteogenic differentiation, alkaline phosphatase activity, and DNA content over time. Decreased cellularity and the expression of genes over time correlated with increasing compositional ratios between HANP and PA-RGDS. The highest HANP to PA-RGDS ratio (66% HANP) exhibited the greatest improvement to the osteogenic differentiation of hMSCs. Overall, these results demonstrate that the compositional ratio of biphasic nanomatrix composites plays an important role in influencing the osteogenic differentiation of hMSCs. Based on the observations presented within this study, these biphasic nanomatrix composites show promise for future usage in bone tissue engineering applications. PMID:22842043

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. Apoptosis repressor with caspase recruitment domain enhances survival and promotes osteogenic differentiation of human osteoblast cells under Zoledronate treatment

    PubMed Central

    Hu, Longwei; Han, Jing; Yang, Xi; Wang, Yang; Pan, Hongya; Xu, Liqun

    2016-01-01

    Zoledronate is one of the most potent nitrogen-containing bisphosphonates which has been demonstrated to result in osteoblast apoptosis and impact osteogenic differentiation in vitro. This effect of Zoledronate on osteoblasts may partially explain bisphosphonate-associated osteonecrosis of the jaw, a serious complication associated with treatment with bisphosphonates. Apoptosis repressor with caspase recruitment domain (ARC) is a multifunctional inhibitor of apoptosis that is physiologically expressed predominantly in post-mitotic cells such as cardiomyocytes, neurons and skeletal muscle cells. However, its effect on human osteoblasts remains unclear. The current study aimed to investigate the effects of ARC on human osteoblasts under the treatment of high concentrations of Zoledronate. ARC-overexpressed human osteoblasts were established and were exposed to Zoledronate with different concentrations (0, 1 and 5 µM) in vitro. Cell numbers were detected using the MTT assay, and flow cytometry was used to identity cell apoptosis. Alkaline phosphatase staining, quantitative analysis and ectopic osteogenesis in nude mice were used to evaluate the osteogenic differentiation of ARC-overexpressed osteoblasts. It was observed that ARC is able to reverse the inhibitory effect of Zoldronate on osteoblasts. ARC is additionally able to promote osteogenic differentiation of osteoblasts and inhibit their apoptosis. These observations suggest a critical role for ARC in the regulation of human osteoblasts under Zoledronate treatment. PMID:27573706

  8. 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

  9. TM-25659 enhances osteogenic differentiation and suppresses adipogenic differentiation by modulating the transcriptional co-activator TAZ

    PubMed Central

    Jang, EJ; Jeong, H; Kang, JO; Kim, NJ; Kim, MS; Choi, SH; Yoo, SE; Hong, JH; Bae, MA; Hwang, ES

    2012-01-01

    BACKGROUND AND PURPOSE The transcriptional co-activator with PDZ-binding motif (TAZ) is characterized as a transcriptional modulator of mesenchymal stem cell differentiation into osteoblasts and adipocytes. Moreover, increased TAZ activity in the nucleus enhances osteoblast differentiation and suppresses adipocyte development by interacting with runt-related transcription factor 2 (RUNX2) and PPARγ, respectively. Therefore, it would be of interest to identify low MW compounds that modulate nuclear TAZ activity. EXPERIMENTAL APPROACH High-throughput screening was performed using a library of low MW compounds in order to identify TAZ modulators that enhance nuclear TAZ localization. The effects and molecular mechanisms of a TAZ modulator have been characterized in osteoblast and adipocyte differentiation. KEY RESULTS We identified 2-butyl-5-methyl-6-(pyridine-3-yl)-3-[2′-(1H-tetrazole-5-yl)-biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridine] (TM-25659) as a TAZ modulator. TM-25659 enhanced nuclear TAZ localization in a dose-dependent manner and attenuated PPARγ-mediated adipocyte differentiation by facilitating PPARγ suppression activity of TAZ. In addition, TAZ-induced RUNX2 activity activation was further increased in osteoblasts, causing increased osteoblast differentiation. Accordingly, TM-25659 suppressed bone loss in vivo and decreased weight gain in an obesity model. After oral administration, TM-25659 had a favourable pharmacokinetic profile. CONCLUSION AND IMPLICATIONS TM-25659 stimulated nuclear TAZ localization and thus caused TAZ to suppress PPARγ-dependent adipogenesis and enhance RUNX2-induced osteoblast differentiation in vitro and in vivo. Our data suggest that TM-25659 could be beneficial in the control of obesity and bone loss. PMID:21913895

  10. 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

  11. 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

  12. Demethylation of IGFBP5 by Histone Demethylase KDM6B Promotes Mesenchymal Stem Cell-Mediated Periodontal Tissue Regeneration by Enhancing Osteogenic Differentiation and Anti-Inflammation Potentials.

    PubMed

    Liu, Dayong; Wang, Yuejun; Jia, Zhi; Wang, Liping; Wang, Jinsong; Yang, Dongmei; Song, Jianqiu; Wang, Songlin; Fan, Zhipeng

    2015-08-01

    Mesenchymal stem cell (MSC)-mediated periodontal tissue regeneration is considered a promising method for periodontitis treatment. The molecular mechanism underlying directed differentiation and anti-inflammatory actions remains unclear, thus limiting potential MSC application. We previously found that insulin-like growth factor binding protein 5 (IGFBP5) is highly expressed in dental tissue-derived MSCs compared with in non-dental tissue-derived MSCs. IGFBP5 is mainly involved in regulating biological activity of insulin-like growth factors, and its functions in human MSCs and tissue regeneration are unclear. In this study, we performed gain- and loss-of-function assays to test whether IGFBP5 could regulate the osteogenic differentiation and anti-inflammatory potential in MSCs. We found that IGFBP5 expression was upregulated upon osteogenic induction, and that IGFBP5 enhanced osteogenic differentiation in MSCs. We further showed that IGFBP5 prompted the anti-inflammation effect of MSCs via negative regulation of NFκB signaling. Depletion of the histone demethylase lysine (K)-specific demethylase 6B (KDM6B) downregulated IGFBP5 expression by increasing histone K27 methylation in the IGFBP5 promoter. Moreover, IGFBP5 expression in periodontal tissues was downregulated in individuals with periodontitis compared with in healthy people, and IGFBP5 enhanced MSC-mediated periodontal tissue regeneration and alleviated local inflammation in a swine model of periodontitis. In conclusion, our present results reveal a new function for IGFBP5, provide insight into the mechanism underlying the directed differentiation and anti-inflammation capacities of MSCs, and identify a potential target mediator for improving tissue regeneration.

  13. Design of biomimetic and bioactive cold plasma-modified nanostructured scaffolds for enhanced osteogenic differentiation of bone marrow-derived mesenchymal stem cells.

    PubMed

    Wang, Mian; Cheng, Xiaoqian; Zhu, Wei; Holmes, Benjamin; Keidar, Michael; Zhang, Lijie Grace

    2014-03-01

    The objective of this study was to design a biomimetic and bioactive tissue-engineered bone construct via a cold atmospheric plasma (CAP) treatment for directed osteogenic differentiation of human bone morrow mesenchymal stem cells (MSCs). Porous nanocrystalline hydroxyapatite/chitosan scaffolds were fabricated via a lyophilization procedure. The nanostructured bone scaffolds were then treated with CAP to create a more favorable surface for cell attachment, proliferation, and differentiation. The CAP-modified scaffolds were characterized via scanning electron microscope, Raman spectrometer, contact angle analyzer, and white light interferometer. In addition, optimal CAP treatment conditions were determined. Our in vitro study shows that MSC adhesion and infiltration were significantly enhanced on CAP modified scaffolds. More importantly, it was demonstrated that CAP-modified nanostructured bone constructs can greatly promote total protein, collagen synthesis, and calcium deposition after 3 weeks of culture, thus making them a promising implantable scaffold for bone regeneration. Moreover, the fibronectin and vitronection adsorption experiments by enzyme-linked immunosorbent assay demonstrated that more adhesion-mediated protein adsorption on the CAP-treated scaffolds. Since the initial specific protein absorption on scaffold surfaces can lead to further recruitment as well as activation of favorable cell functions, it is suggested that our enhanced stem cell growth and osteogenic function may be related to more protein adsorption resulting from surface roughness and wettability modification. The CAP modification method used in this study provides a quick one-step process for cell-favorable tissue-engineered scaffold architecture remodeling and surface property alteration.

  14. 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.

  15. Biocompatibility and enhanced osteogenic differentiation of human mesenchymal stem cells in response to surface engineered poly(D,L-lactic-co-glycolic acid) microparticles.

    PubMed

    Rogers, Catherine M; Deehan, David J; Knuth, Callie A; Rose, Felicity R A J; Shakesheff, Kevin M; Oldershaw, Rachel A

    2014-11-01

    Tissue engineering strategies can be applied to enhancing osseous integration of soft tissue grafts during ligament reconstruction. Ligament rupture results in a hemarthrosis, an acute intra-articular bleed rich in osteogenic human mesenchymal stem cells (hMSCs). With the aim of identifying an appropriate biomaterial with which to combine hemarthrosis fluid-derived hMSCs (HF-hMSCs) for therapeutic application, this work has investigated the biocompatibility of microparticles manufactured from two forms of poly(D,L-lactic-co-glycolic acid) (PLGA), one synthesized with equal monomeric ratios of lactic acid to glycolic acid (PLGA 50:50) and the other with a higher proportion of lactic acid (PLGA 85:15) which confers a longer biodegradation time. The surfaces of both types of microparticles were functionalized by plasma polymerization with allylamine to increase hydrophilicity and promote cell attachment. HF-hMSCs attached to and spread along the surface of both forms of PLGA microparticle. The osteogenic response of HF-hMSCs was enhanced when cultured with PLGA compared with control cultures differentiated on tissue culture plastic and this was independent of the type of polymer used. We have demonstrated that surface engineered PLGA microparticles are an appropriate biomaterial for combining with HF-hMSCs and the selection of PLGA is relevant only when considering the biodegradation time for each biomedical application.

  16. Osteogenic differentiation of mesenchymal stem cells in defined protein beads.

    PubMed

    Lund, Amanda W; Bush, Jeff A; Plopper, George E; Stegemann, Jan P

    2008-10-01

    There is a need to develop improved methods for directing and maintaining the differentiation of human mesenchymal stem cells (hMSC) for regenerative medicine. Here, we present a method for embedding cells in defined protein microenvironments for the directed osteogenic differentiation of hMSC. Composite matrices of collagen I and agarose were produced by emulsification and simultaneous polymerization in the presence of hMSC to produce 30-150 mum diameter hydrogel "beads." The proliferation, morphology, osteogenic gene expression, and calcium deposition of hMSC in bead environments were compared to other two- and three-dimensional culture environments over 14-21 days in culture. Cells embedded within 40% collagen beads exhibited equivalent proliferation rates to those in gel disks, but showed upregulation of bone sialoprotein and increased calcium deposition over 2D controls. Osteocalcin gene expression was not changed in 3D beads and disks, while collagen type I gene expression was downregulated relative to cells in 2D culture. The hydrogel bead format allows controlled cell differentiation and is a cell delivery vehicle that may also enhance vascular invasion and host incorporation. Our results indicate that the application of such beads can be used to promote the osteogenic phenotype in hMSC, which is an important step toward using them in bone repair applications.

  17. 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

  18. 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.

  19. The regulatory landscape of osteogenic differentiation.

    PubMed

    Håkelien, Anne-Mari; Bryne, Jan Christian; Harstad, Kristine G; Lorenz, Susanne; Paulsen, Jonas; Sun, Jinchang; Mikkelsen, Tarjei S; Myklebost, Ola; Meza-Zepeda, Leonardo A

    2014-10-01

    Differentiation of osteoblasts from mesenchymal stem cells (MSCs) is an integral part of bone development and homeostasis, and may when improperly regulated cause disease such as bone cancer or osteoporosis. Using unbiased high-throughput methods we here characterize the landscape of global changes in gene expression, histone modifications, and DNA methylation upon differentiation of human MSCs to the osteogenic lineage. Furthermore, we provide a first genome-wide characterization of DNA binding sites of the bone master regulatory transcription factor Runt-related transcription factor 2 (RUNX2) in human osteoblasts, revealing target genes associated with regulation of proliferation, migration, apoptosis, and with a significant overlap with p53 regulated genes. These findings expand on emerging evidence of a role for RUNX2 in cancer, including bone metastases, and the p53 regulatory network. We further demonstrate that RUNX2 binds to distant regulatory elements, promoters, and with high frequency to gene 3' ends. Finally, we identify TEAD2 and GTF2I as novel regulators of osteogenesis. PMID:24898411

  20. 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

  1. 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

  2. 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

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

    PubMed

    Logan, Niall; Brett, Peter

    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

  4. 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

  5. Heparin affects human bone marrow stromal cell fate: Promoting osteogenic and reducing adipogenic differentiation and conversion.

    PubMed

    Simann, Meike; Schneider, Verena; Le Blanc, Solange; Dotterweich, Julia; Zehe, Viola; Krug, Melanie; Jakob, Franz; Schilling, Tatjana; Schütze, Norbert

    2015-09-01

    Heparins are broadly used for the prevention and treatment of thrombosis and embolism. Yet, osteoporosis is considered to be a severe side effect in up to one third of all patients on long-term treatment. However, the mechanisms underlying this clinical problem are only partially understood. To investigate if heparin affects differentiation of skeletal precursors, we examined the effects of heparin on the osteogenic and adipogenic lineage commitment and differentiation of primary human bone marrow stromal cells (hBMSCs). Due to the known inverse relationship between adipogenesis and osteogenesis and the capacity of pre-differentiated cells to convert into the respective other lineage, we also determined heparin effects on osteogenic conversion and adipogenic differentiation/conversion. Interestingly, heparin did not only significantly increase mRNA expression and enzyme activity of the osteogenic marker alkaline phosphatase (ALP), but it also promoted mineralization during osteogenic differentiation and conversion. Furthermore, the mRNA expression of the osteogenic marker bone morphogenic protein 4 (BMP4) was enhanced. In addition, heparin administration partly prevented adipogenic differentiation and conversion demonstrated by reduced lipid droplet formation along with a decreased expression of adipogenic markers. Moreover, luciferase reporter assays, inhibitor experiments and gene expression analyses revealed that heparin had putative permissive effects on osteogenic signaling via the BMP pathway and reduced the mRNA expression of the Wnt pathway inhibitors dickkopf 1 (DKK1) and sclerostin (SOST). Taken together, our data show a rather supportive than inhibitory effect of heparin on osteogenic hBMSC differentiation and conversion in vitro. Further studies will have to investigate the net effects of heparin administration on bone formation versus bone resorption in vivo to unravel the molecular mechanisms of heparin-associated osteoporosis and reconcile

  6. Shear stress induces osteogenic differentiation of human mesenchymal stem cells

    PubMed Central

    Yourek, Gregory; McCormick, Susan M; Mao, Jeremy J; Reilly, Gwendolen C

    2014-01-01

    Aim To determine whether fluid flow-induced shear stress affects the differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs) into osteogenic cells. Materials & methods hMSCs cultured with or without osteogenic differentiation medium were exposed to fluid flow-induced shear stress and analyzed for alkaline phosphatase activity and expression of osteogenic genes. Results Immediately following shear stress, alkaline phosphatase activity in osteogenic medium was significantly increased. At days 4 and 8 of culture the mRNA expression of bone morphogenetic protein-2 and osteopontin was significantly higher in hMSCs subjected to shear stress than those cultured in static conditions. However, hMSCs cultured in osteogenic differentiation medium were less responsive in gene expression of alkaline phosphatase and bone morphogenetic protein-2. Conclusion These data demonstrate that shear stress stimulates hMSCs towards an osteoblastic phenotype in the absence of chemical induction, suggesting that certain mechanical stresses may serve as an alternative to chemical stimulation of stem cell differentiation. PMID:20868327

  7. 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

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

    PubMed

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

    2016-03-01

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

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. Polarization of an electroactive functional film on titanium for inducing osteogenic differentiation

    PubMed Central

    Zhou, Zhengnan; Li, Weiping; He, Tianrui; Qian, Lei; Tan, Guoxin; Ning, Chengyun

    2016-01-01

    To enhance the surface bioactivity of titanium (Ti) prostheses, an electroactive polyvinylidene fluoride (PVDF) film was prepared on a Ti substrate to provide a mimetic of the electrical microenvironment, which facilitated the performance of cell functions. The results of cell proliferation and differentiation assays indicated that polarization of the PVDF-Ti (PTi) altered its surface charge, thus inducing adhesion, proliferation and osteogenic differentiation of cells. The polarized PVDF-Ti (PPTi) may therefore find applications in bone regeneration. PMID:27762318

  16. 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.

  17. 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

  18. 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

  19. 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

  20. Osteogenic differentiation strategies for adipose-derived mesenchymal stem cells.

    PubMed

    Kroeze, Robert Jan; Knippenberg, Marlene; Helder, Marco N

    2011-01-01

    Adipose stem cell preparations, either obtained as a freshly isolated so-called stromal vascular fraction (SVF) or as cells cultured to homogeneity and then referred to as adipose stem cells (ASCs), have found widespread use in a broad variety of studies on tissue engineering and regenerative medicine applications, including bone repair.For newcomers within the field, but also for established research laboratories having up to 10 years of expertise in this research area, it may be convenient to strive for, and use consensus protocols (1) for studying the osteogenic differentiation potential of ASC preparations in vitro, and (2) for osteogenic induction regimes for in vivo implementation. To assist in achieving this goal, this chapter describes various step-by-step osteogenic differentiation protocols for adipose-derived stem cell populations (SVF as well as ASCs) currently applied within our laboratory, with particular emphasis on protocols aimed at intra-operative use. The protocols describe the use of inducing compounds, including the bone morphogenetic proteins (BMPs), 1,25-dihydroxyvitamin-D3, and polyamines, as well as methods and parameters for evaluating the level of differentiation achieved.We would appreciate receiving feedback on the protocols described; this will facilitate the development of consensus protocols, which in turn will allow better comparison of data sets generated by different research groups. This continuing standardization, which might be reported on at international meetings like those of IFATS ( http://www.IFATS.org ), might be of benefit for the whole ASC research community.

  1. Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation

    PubMed Central

    Hwang, Jun-Ha; Byun, Mi Ran; Kim, A. Rum; Kim, Kyung Min; Cho, Hang Jun; Lee, Yo Han; Kim, Juwon; Jeong, Mi Gyeong; Hwang, Eun Sook; Hong, Jeong-Ho

    2015-01-01

    Mesenchymal stem cell (MSC) differentiation is regulated by the extracellular matrix (ECM) through activation of intracellular signaling mediators. The stiffness of the ECM was shown to be an important regulatory factor for MSC differentiation, and transcriptional coactivator with PDZ-binding motif (TAZ) was identified as an effector protein for MSC differentiation. However, the detailed underlying mechanism regarding the role of ECM stiffness and TAZ in MSC differentiation is not yet fully understood. In this report, we showed that ECM stiffness regulates MSC fate through ERK or JNK activation. Specifically, a stiff hydrogel matrix stimulates osteogenic differentiation concomitant with increased nuclear localization of TAZ, but inhibits adipogenic differentiation. ERK and JNK activity was significantly increased in cells cultured on a stiff hydrogel. TAZ activation was induced by ERK or JNK activation on a stiff hydrogel because exposure to an ERK or JNK inhibitor significantly decreased the nuclear localization of TAZ, indicating that ECM stiffness-induced ERK or JNK activation is important for TAZ-driven osteogenic differentiation. Taken together, these results suggest that ECM stiffness regulates MSC differentiation through ERK or JNK activation. PMID:26262877

  2. Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation.

    PubMed

    Hwang, Jun-Ha; Byun, Mi Ran; Kim, A Rum; Kim, Kyung Min; Cho, Hang Jun; Lee, Yo Han; Kim, Juwon; Jeong, Mi Gyeong; Hwang, Eun Sook; Hong, Jeong-Ho

    2015-01-01

    Mesenchymal stem cell (MSC) differentiation is regulated by the extracellular matrix (ECM) through activation of intracellular signaling mediators. The stiffness of the ECM was shown to be an important regulatory factor for MSC differentiation, and transcriptional coactivator with PDZ-binding motif (TAZ) was identified as an effector protein for MSC differentiation. However, the detailed underlying mechanism regarding the role of ECM stiffness and TAZ in MSC differentiation is not yet fully understood. In this report, we showed that ECM stiffness regulates MSC fate through ERK or JNK activation. Specifically, a stiff hydrogel matrix stimulates osteogenic differentiation concomitant with increased nuclear localization of TAZ, but inhibits adipogenic differentiation. ERK and JNK activity was significantly increased in cells cultured on a stiff hydrogel. TAZ activation was induced by ERK or JNK activation on a stiff hydrogel because exposure to an ERK or JNK inhibitor significantly decreased the nuclear localization of TAZ, indicating that ECM stiffness-induced ERK or JNK activation is important for TAZ-driven osteogenic differentiation. Taken together, these results suggest that ECM stiffness regulates MSC differentiation through ERK or JNK activation.

  3. 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.

  4. 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

  5. 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

  6. Housekeeping gene stability influences the quantification of osteogenic markers during stem cell differentiation to the osteogenic lineage

    PubMed Central

    Posada, Olga M.; Gallego-Perez, Daniel; Higuita-Castro, Natalia; Sarassa, Carlos; Hansford, Derek J.; Agudelo-Florez, Piedad; López, Luis E.

    2010-01-01

    Real-time reverse transcription PCR (RT-qPCR) relies on a housekeeping or normalizer gene whose expression remains constant throughout the experiment. RT-qPCR is commonly used for characterization of human bone marrow mesenchymal stem cells (hBMSCs). However, to the best of our knowledge, there are no studies validating the expression stability of the genes used as normalizers during hBMSCs differentiation. This work aimed to study the stability of the housekeeping genes β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ribosomal protein L13A (RPL13A) during the osteogenic differentiation of hBMSCs. Their stability was evaluated via RT-qPCR in 14 and 20 day differentiation assays to the osteogenic lineage. Different normalization strategies were evaluated to quantify the osteogenic markers collagen type I, bone sialoprotein and osteonectin. Cell differentiation was confirmed via alizarin red staining. The results demonstrated up-regulation of β-actin with maximum fold changes (MFC) of 4.38. GAPDH and RPL13A were not regulated by osteogenic media after 14 days and presented average fold changes lower than 2 in 20 day cultures. RPL13A (MFC < 2) had a greater stability when normalizing as a function of culture time compared with GAPDH (MFC ≤ 2.2), which resulted in expression patterns of the osteogenic markers more consistent with the observed differentiation process. The results suggest that β-actin regulation could be associated with the morphological changes characteristic of hBMSCs osteogenic differentiation, and provide evidence for the superior performance of RPL13A as a normalizer gene in osteogenic differentiation studies of hBMSCs. This work highlights the importance of validating the normalizer genes used for stem cells characterization via RT-qPCR. PMID:20396946

  7. Housekeeping gene stability influences the quantification of osteogenic markers during stem cell differentiation to the osteogenic lineage.

    PubMed

    Quiroz, Felipe Garcia; Posada, Olga M; Gallego-Perez, Daniel; Higuita-Castro, Natalia; Sarassa, Carlos; Hansford, Derek J; Agudelo-Florez, Piedad; López, Luis E

    2010-04-01

    Real-time reverse transcription PCR (RT-qPCR) relies on a housekeeping or normalizer gene whose expression remains constant throughout the experiment. RT-qPCR is commonly used for characterization of human bone marrow mesenchymal stem cells (hBMSCs). However, to the best of our knowledge, there are no studies validating the expression stability of the genes used as normalizers during hBMSCs differentiation. This work aimed to study the stability of the housekeeping genes beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ribosomal protein L13A (RPL13A) during the osteogenic differentiation of hBMSCs. Their stability was evaluated via RT-qPCR in 14 and 20 day differentiation assays to the osteogenic lineage. Different normalization strategies were evaluated to quantify the osteogenic markers collagen type I, bone sialoprotein and osteonectin. Cell differentiation was confirmed via alizarin red staining. The results demonstrated up-regulation of beta-actin with maximum fold changes (MFC) of 4.38. GAPDH and RPL13A were not regulated by osteogenic media after 14 days and presented average fold changes lower than 2 in 20 day cultures. RPL13A (MFC < 2) had a greater stability when normalizing as a function of culture time compared with GAPDH (MFC osteogenic markers more consistent with the observed differentiation process. The results suggest that beta-actin regulation could be associated with the morphological changes characteristic of hBMSCs osteogenic differentiation, and provide evidence for the superior performance of RPL13A as a normalizer gene in osteogenic differentiation studies of hBMSCs. This work highlights the importance of validating the normalizer genes used for stem cells characterization via RT-qPCR.

  8. 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

  9. 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

  10. 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

  11. 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

  12. [Regulation of osteogenic differentiation of mesenchimal stem sells of bone marrow].

    PubMed

    Zakharov, Iu M; Makarova, E B

    2013-04-01

    The review describes the mechanisms of hormonal, autocrine, paracrine and intracellular regulation in osteogenic differentiation of mesenchymal stem cells, the role of negative feedbacks in this process.

  13. Asiaticoside induces type I collagen synthesis and osteogenic differentiation in human periodontal ligament cells.

    PubMed

    Nowwarote, Nunthawan; Osathanon, Thanaphum; Jitjaturunt, Peachaya; Manopattanasoontorn, Sukuman; Pavasant, Prasit

    2013-03-01

    Asiaticoside, an active ingredient extracted from Centella asiatica, has been widely used to promote wound healing. In this study, the effects of asiaticoside on proliferation, protein synthesis, and osteogenic differentiation in human periodontal ligament cells (HPDLs) were investigated. HPDLs were treated with asiaticoside at concentrations of 25, 50, and 100 µg/mL. Cell number was determined by MTT assay. The mRNA expression was analyzed by reverse transcription-polymerase chain reaction. Western blot analysis and immunocytochemistry were used to confirm protein synthesis. Osteogenic differentiation was determined by alkaline phosphatase activity, osteoblast marker gene expression, and in vitro mineralization. The results showed that asiaticoside treatment, ranging from 25 to 100 mg/mL, had no effect on cytotoxicity or cell proliferation. When HPDLs were treated with asiaticoside in serum-free medium, dose-dependent increases in the levels of fibronectin and collagen type I mRNA and protein were observed at 72 h. Moreover, asiaticoside attenuated matrix metalloproteinase-1 but enhanced tissue inhibitor of metalloproteinase-1 mRNA expression. The addition of asiaticoside to osteogenic medium resulted in an increase in alkaline phosphatase enzymatic activity, up-regulation of osteoblast marker gene mRNA expression, and enhancement of mineralization by HPDLs. These results suggest the potential application of asiaticoside for enhancing periodontal tissue healing.

  14. 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

  15. 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

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

    PubMed Central

    Bravenboer, Nathalie

    2016-01-01

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

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

    PubMed Central

    Bravenboer, Nathalie

    2016-01-01

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

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

    PubMed

    Bastidas-Coral, Angela P; Bakker, Astrid D; Zandieh-Doulabi, Behrouz; Kleverlaan, Cornelis J; Bravenboer, Nathalie; Forouzanfar, Tim; Klein-Nulend, Jenneke

    2016-01-01

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

  19. 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

  20. Osteogenic differentiation of preosteoblasts on a hemostatic gelatin sponge

    PubMed Central

    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

  1. Exogenous nitric oxide enhances calcification in embryonic stem cell-derived osteogenic cultures.

    PubMed

    Ehnes, D D; Geransar, R M; Rancourt, D E; Zur Nieden, N I

    2015-01-01

    While the involvement of nitric oxide in bone formation, homeostasis and healing has been extensively characterized, its role in directing pluripotent stem cells to the osteogenic lineage has not been described. Yet, the identification of chemical inducers that improve differentiation output to a particular lineage is highly valuable to the development of such cells for the cell-based treatment of osteo-degenerative diseases. This study aimed at investigating the instructive role of nitric oxide (NO) and its synthesizing enzymes on embryonic stem cell (ESC) osteogenic differentiation. Our findings showed that NO levels may support osteogenesis, but that the effect of nitric oxide on osteoblast differentiation may be specific to a particular time phase during the development of osteoblasts in vitro. Endogenously, nitric oxide was specifically secreted by osteogenic cultures during the calcification period. Simultaneously, messenger RNAs for both the endothelial and inducible nitric oxide synthase isoforms (eNOS and iNOS) were upregulated during this late phase development. However, the specific eNOS inhibitor L-N(5)-(1-Iminoethyl)ornithine dihydrochloride attenuated calcification more so than the specific iNOS inhibitor diphenyleneiodonium. Exogenous stage-specific supplementation of culture medium with the NO donor S-nitroso-N-acetyl-penicillamine increased the percentage of cells differentiating into osteoblasts and enhanced calcification. Our results point to a primary role for eNOS as a pro-osteogenic trigger in ESC differentiation and expand on the variety of supplements that may be used to direct ESC fate to the osteogenic lineage, which will be important in the development of cell-based therapies for osteo-degenerative diseases.

  2. 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.

  3. 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

  4. Osteogenic differentiation of mesenchymal stem cells from dental bud: Role of integrins and cadherins.

    PubMed

    Di Benedetto, Adriana; Brunetti, Giacomina; Posa, Francesca; Ballini, Andrea; Grassi, Felice Roberto; Colaianni, Graziana; Colucci, Silvia; Rossi, Enzo; Cavalcanti-Adam, Elisabetta A; Lo Muzio, Lorenzo; Grano, Maria; Mori, Giorgio

    2015-11-01

    Several studies have reported the beneficial effects of mesenchymal stem cells (MSCs) in tissue repair and regeneration. New sources of stem cells in adult organisms are continuously emerging; dental tissues have been identified as a source of postnatal MSCs. Dental bud is the immature precursor of the tooth, is easy to access and we show in this study that it can yield a high number of cells with ≥95% expression of mesenchymal stemness makers and osteogenic capacity. Thus, these cells can be defined as Dental Bud Stem Cells (DBSCs) representing a promising source for bone regeneration of stomatognathic as well as other systems. Cell interactions with the extracellular matrix (ECM) and neighboring cells are critical for tissue morphogenesis and architecture; such interactions are mediated by integrins and cadherins respectively. We characterized DBSCs for the expression of these adhesion receptors and examined their pattern during osteogenic differentiation. Our data indicate that N-cadherin and cadherin-11 were expressed in undifferentiated DBSCs and their expression underwent changes during the osteogenic process (decreasing and increasing respectively), while expression of E-cadherin and P-cadherin was very low in DBSCs and did not change during the differentiation steps. Such expression pattern reflected the mesenchymal origin of DBSCs and confirmed their osteoblast-like features. On the other hand, osteogenic stimulation induced the upregulation of single subunits, αV, β3, α5, and the formation of integrin receptors α5β1 and αVβ3. DBSCs differentiation toward osteoblastic lineage was enhanced when cells were grown on fibronectin (FN), vitronectin (VTN), and osteopontin (OPN), ECM glycoproteins which contain an integrin-binding sequence, the RGD motif. In addition we established that integrin αVβ3 plays a crucial role during the commitment of MSCs to osteoblast lineage, whereas integrin α5β1 seems to be dispensable. These data suggest that

  5. Effect of substrate stiffness on the osteogenic differentiation of bone marrow stem cells and bone-derived cells.

    PubMed

    Witkowska-Zimny, Malgorzata; Walenko, Katarzyna; Wrobel, Edyta; Mrowka, Piotr; Mikulska, Agnieszka; Przybylski, Jacek

    2013-06-01

    There is a profound dependence of cell behaviour on the stiffness of its microenvironment. To gain a better understanding of the regulation of cellular differentiation by mechanical cues, we investigated the influence of matrix stiffness (E = 1.46 kPa and E = 26.12 kPa) on differentiated osteogenic cell lineage of bone marrow stem cells (BM-MSCs) and bone-derived cells (BDCs) using flexible collagen-coated polyacrylamide substrates. Differentiation potential was determined by measuring alkaline phosphatase activity, expression of osteoblast-specific markers including alkaline phosphatase, osteocalcin, Runx2 and collagen type I, as well as assessment of mineralisation (Alizarin Red S staining). We found that osteogenic differentiation can be regulated by the rigidity of the substrate, which may depend on the commitment in multi- or uni-potent targeting cells. Osteogenic differentiation of BM-MSCs was enhanced on a stiff substrate compared to a soft one, whereas BDCs osteogenic differentiation did not vary depending on the substrate stiffness. The data help in understanding the role of the external mechanical determinants in stem cell differentiation, and can also be useful in translational approach in functional tissue engineering.

  6. 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

  7. 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.

  8. The Transcription Factor DLX3 Regulates the Osteogenic Differentiation of Human Dental Follicle Precursor Cells

    PubMed Central

    Viale-Bouroncle, Sandra; Felthaus, Oliver; Schmalz, Gottfried; Brockhoff, Gero; Reichert, Torsten E

    2012-01-01

    The transcription factor DLX3 plays a decisive role in bone development of vertebrates. In neural-crest derived stem cells from the dental follicle (DFCs), DLX3 is differentially expressed during osteogenic differentiation, while other osteogenic transcription factors such as DLX5 or RUNX2 are not highly induced. DLX3 has therefore a decisive role in the differentiation of DFCs, but its actual biological effects and regulation are unknown. This study investigated the DLX3-regulated processes in DFCs. After DLX3 overexpression, DFCs acquired a spindle-like cell shape with reorganized actin filaments. Here, marker genes for cell morphology, proliferation, apoptosis, and osteogenic differentiation were significantly regulated as shown in a microarray analysis. Further experiments showed that DFCs viability is directly influenced by the expression of DLX3, for example, the amount of apoptotic cells was increased after DLX3 silencing. This transcription factor stimulates the osteogenic differentiation of DFCs and regulates the BMP/SMAD1-pathway. Interestingly, BMP2 did highly induce DLX3 and reverse the inhibitory effect of DLX3 silencing in osteogenic differentiation. However, after DLX3 overexpression in DFCs, a BMP2 supplementation did not improve the expression of DLX3 and the osteogenic differentiation. In conclusion, DLX3 influences cell viability and regulates osteogenic differentiation of DFCs via a BMP2-dependent pathway and a feedback control. PMID:22107079

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

    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.

  10. 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

  11. 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

  12. 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

  13. 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

  14. Carbon nanotube array inducing osteogenic differentiation of human mesenchymal stem cells.

    PubMed

    Xu, Baiyao; Ju, Yang; Cui, Yanbin; Song, Guanbin

    2015-06-01

    Carbon nanotubes (CNTs) are a kind of nanomaterials which have been shown a promising application for biomedicine. There are a lot of studies to use CNTs to induce the differentiation of mesenchymal stem cells (MSCs). However, the cellular behavior of MSCs on the top layer of CNT array was still not well understood. In this study, we evaluated the morphology, the gene expressions of the osteogenic differentiation related markers, and the gene expressions of collagen type II (Col II, a marker of chondrogenesis), PPARγ (a marker of adipogenesis) and scleraxis (SCX, a marker of tenogenesis) in human mesenchymal stem cells (hMSCs) cultured on multi-walled carbon nanotube (MWCNT) array. The effect of MWCNT array on the mineralization of hMSCs which were cultured in osteogenic differentiation medium (ODM) was further assayed. Our results showed that the hMSCs cultured on MWCNT array spread well, formed numerous spiral shaped cell colons and showed perinuclear morphology. Compared to hMSCs cultured on dish, the gene expression of osteocalcin (OCN) was increased while the gene expressions of collagen type II (Col II), PPARγ and scleraxis (SCX) were decreased in hMSCs which were cultured on MWCNT array without any differentiation factors. Furthermore, compared with hMSCs on dish, the gene expressions of collagen type I (Col I), osteocalcin (OCN), osteopontin (OPN) and RUNX2, and the mineralization of hMSCs on MWCNT array were enhanced when they were cultured in osteogenic differentiation medium (ODM). Our results indicated that MWCNT array was able to promote the osteogenesis of hMSCs.

  15. Contrasting effect of perlecan on adipogenic and osteogenic differentiation of mesenchymal stem cells in vitro.

    PubMed

    Nakamura, Ryosuke; Nakamura, Fumio; Fukunaga, Shigeharu

    2014-03-01

    Perlecan, a basement membrane component, shows diverse functions in different organs and tissues. However, the role of perlecan in differentiation of mesenchymal stem cells (MSCs) has been barely investigated. In this study, we examined the effect of perlecan on adipogenic and osteogenic differentiation of MSCs in vitro by adding extrinsic perlecan to culture media or blocking the function of intrinsic perlecan expressed into culture media by differentiating MSCs. Extrinsic perlecan suppressed adipogenic differentiation; however, it promoted osteogenic differentiation. These functions were further confirmed by a study of blocking intrinsic perlecan. Perlecan treated with heparitinase-I also showed the suppressive effect on adipogenic differentiation. In contrast, the promotive effect on osteogenic differentiation was found to be heparan sulfate-dependent. Intrinsic perlecan was suggested to be effective at the late stage of adipogenic differentiation by a study of perlecan-blocking performed at distinct periods, but was suggested to be effective at the early stage of osteogenic differentiation. Our results showed perlecan has contrasting effect on adipogenic and osteogenic differentiation of MSCs due to its diverse actions. Based on these outcomes, we recognized that employing extrinsic perlecan or blocking intrinsic perlecan is effective for regulating adipogenic and osteogenic differentiation of MSCs by restricting its direction.

  16. 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.

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

    PubMed

    Keller, Vivian; Deiwick, Andrea; Pflaum, Michael; Schlie-Wolter, Sabrina

    2016-10-01

    The correlation between extracellular matrix (ECM) components, cell shape, and stem cell guidance can shed light in understanding and mimicking the functionality of stem cell niches for various applications. This interplay on osteogenic guidance of human adipose-derived stem cells (hASCs) was focus of this study. Proliferation and osteogenic markers like alkaline phosphatase activity and calcium mineralization were slightly increased by the ECM components laminin (LA), collagen I (COL), and fibronectin (FIB); with control medium no differentiation occurred. ECM guided differentiation was rather dependent on osterix than on Runx2 pathway. FIB significantly enhanced cell elongation even in presence of actin polymerization blockers cytochalasin D (CytoD) and ROCK inhibitor Y-27632, which generally caused more rounded cells. Except for the COL surface, both inhibitors increased the extent of osterix, while the Runx2 pathway was more sensitive to the culture condition. Both inhibitors did not affect hASC proliferation. CytoD enabled osteogenic differentiation independently from the ECM, while it was rather blocked via Y-27632 treatment; on FIB the general highest extent of differentiation occurred. Taken together, the ECM effect on hASCs occurs indirectly and selectively via a dominant role of FIB: it sustains osteogenic differentiation in case of a tension-dependent control of actin polymerization. PMID:27590529

  18. Role of MSX1 in Osteogenic Differentiation of Human Dental Pulp Stem Cells

    PubMed Central

    Fujii, Sakiko; Ida-Yonemochi, Hiroko; Noshiro, Mitsuhide; Shukunami, Chisa; Kozai, Katsuyuki

    2016-01-01

    Msh homeobox 1 (MSX1) encodes a transcription factor implicated in embryonic development of limbs and craniofacial tissues including bone and teeth. Although MSX1 regulates osteoblast differentiation in the cranial bone of young animal, little is known about the contribution of MSX1 to the osteogenic potential of human cells. In the present study, we investigate the role of MSX1 in osteogenic differentiation of human dental pulp stem cells isolated from deciduous teeth. When these cells were exposed to osteogenesis-induction medium, runt-related transcription factor-2 (RUNX2), bone morphogenetic protein-2 (BMP2), alkaline phosphatase (ALPL), and osteocalcin (OCN) mRNA levels, as well as alkaline phosphatase activity, increased on days 4–12, and thereafter the matrix was calcified on day 14. However, knockdown of MSX1 with small interfering RNA abolished the induction of the osteoblast-related gene expression, alkaline phosphatase activity, and calcification. Interestingly, DNA microarray and PCR analyses revealed that MSX1 knockdown induced the sterol regulatory element-binding protein 2 (SREBP2) transcriptional factor and its downstream target genes in the cholesterol synthesis pathway. Inhibition of cholesterol synthesis enhances osteoblast differentiation of various mesenchymal cells. Thus, MSX1 may downregulate the cholesterol synthesis-related genes to ensure osteoblast differentiation of human dental pulp stem cells.

  19. Role of MSX1 in Osteogenic Differentiation of Human Dental Pulp Stem Cells.

    PubMed

    Goto, Noriko; Fujimoto, Katsumi; Fujii, Sakiko; Ida-Yonemochi, Hiroko; Ohshima, Hayato; Kawamoto, Takeshi; Noshiro, Mitsuhide; Shukunami, Chisa; Kozai, Katsuyuki; Kato, Yukio

    2016-01-01

    Msh homeobox 1 (MSX1) encodes a transcription factor implicated in embryonic development of limbs and craniofacial tissues including bone and teeth. Although MSX1 regulates osteoblast differentiation in the cranial bone of young animal, little is known about the contribution of MSX1 to the osteogenic potential of human cells. In the present study, we investigate the role of MSX1 in osteogenic differentiation of human dental pulp stem cells isolated from deciduous teeth. When these cells were exposed to osteogenesis-induction medium, runt-related transcription factor-2 (RUNX2), bone morphogenetic protein-2 (BMP2), alkaline phosphatase (ALPL), and osteocalcin (OCN) mRNA levels, as well as alkaline phosphatase activity, increased on days 4-12, and thereafter the matrix was calcified on day 14. However, knockdown of MSX1 with small interfering RNA abolished the induction of the osteoblast-related gene expression, alkaline phosphatase activity, and calcification. Interestingly, DNA microarray and PCR analyses revealed that MSX1 knockdown induced the sterol regulatory element-binding protein 2 (SREBP2) transcriptional factor and its downstream target genes in the cholesterol synthesis pathway. Inhibition of cholesterol synthesis enhances osteoblast differentiation of various mesenchymal cells. Thus, MSX1 may downregulate the cholesterol synthesis-related genes to ensure osteoblast differentiation of human dental pulp stem cells. PMID:27648077

  20. Role of MSX1 in Osteogenic Differentiation of Human Dental Pulp Stem Cells

    PubMed Central

    Fujii, Sakiko; Ida-Yonemochi, Hiroko; Noshiro, Mitsuhide; Shukunami, Chisa; Kozai, Katsuyuki

    2016-01-01

    Msh homeobox 1 (MSX1) encodes a transcription factor implicated in embryonic development of limbs and craniofacial tissues including bone and teeth. Although MSX1 regulates osteoblast differentiation in the cranial bone of young animal, little is known about the contribution of MSX1 to the osteogenic potential of human cells. In the present study, we investigate the role of MSX1 in osteogenic differentiation of human dental pulp stem cells isolated from deciduous teeth. When these cells were exposed to osteogenesis-induction medium, runt-related transcription factor-2 (RUNX2), bone morphogenetic protein-2 (BMP2), alkaline phosphatase (ALPL), and osteocalcin (OCN) mRNA levels, as well as alkaline phosphatase activity, increased on days 4–12, and thereafter the matrix was calcified on day 14. However, knockdown of MSX1 with small interfering RNA abolished the induction of the osteoblast-related gene expression, alkaline phosphatase activity, and calcification. Interestingly, DNA microarray and PCR analyses revealed that MSX1 knockdown induced the sterol regulatory element-binding protein 2 (SREBP2) transcriptional factor and its downstream target genes in the cholesterol synthesis pathway. Inhibition of cholesterol synthesis enhances osteoblast differentiation of various mesenchymal cells. Thus, MSX1 may downregulate the cholesterol synthesis-related genes to ensure osteoblast differentiation of human dental pulp stem cells. PMID:27648077

  1. 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

  2. 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

  3. 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

  4. 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

  5. Effects of Polygonatum sibiricum polysaccharide on the osteogenic differentiation of bone mesenchymal stem cells in mice

    PubMed Central

    Zong, Shaohui; Zeng, Gaofeng; Zou, Bin; Li, Keke; Fang, Ye; Lu, Li; Xiao, Deqiang; Zhang, Zhiyong

    2015-01-01

    Polygonatum sibiricum polysaccharide (PSP) is a traditional Chinese medicine and is widely used to treat many diseases for hundreds of years conventionally. This study was to access the effects of PSP on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in the mice. Cells collected from BALB/C mice in the bone marrow were isolated and cultured with osteogenic medium (OM) with different concentrations of PSP. The proliferation and morphological changes of BMSCs were observed using an inverted microscope. Flow cytometric analysis was used to identify the BMSCs. MTT test was performed to analyze the proliferation and viability of the cells. ELISA was used to determine the expression levels of alkaline phosphatase (ALP), osteocalcin (OC), N-terminal propeptide of type I procollagen (PINP) and bone morphogenetic protein-2 (BMP-2). Immunocytochemistry and western blot were respectively used to determine the expressions of bone sialoprotein (BSP) and SPARC/osteonectin (OSN). The growth curves of the proliferation and differentiation of the Control, OM, 17β-E2 and PSP groups were increased. Compared to the Control and OM groups, the expression levels of ALP, OC, PINP and BMP-2 were significantly increased in the PSP induced group (P<0.05). Immunocytochemistry and western blot showed that BSP and SPARC were increased after induction of PSP compared to the OM group (P<0.05). The study demonstrates that PSP promotes the proliferation and enhances the viability of BMSCs during osteogenic differentiation. Therefore, PSP may be a potential treatment of osteoporosis in the clinic. PMID:26261494

  6. 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.

  7. 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

  8. 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

  9. A proteomic analysis during serial subculture and osteogenic differentiation of human mesenchymal stem cell.

    PubMed

    Sun, Hyun Jin; Bahk, Young Yil; Choi, Yon Rak; Shim, Jung Hye; Han, Seung Hwan; Lee, Jin Woo

    2006-11-01

    Although previous studies have reported the effects of extensive subculturing on proliferation rates and osteogenic potential of human mesenchymal stem cells (hMSCs), the results remain controversial. The aim of our study was to characterize the proliferation and osteogenic potential of hMSCs during serial subculture, and also to identify proteins that are differentially regulated in hMSCs during serial subculture and osteogenic differentiation using proteome analysis. Here we show that the proliferation and osteogenic capacity of hMSCs decrease during serial subculturing. Several proteins were shown to be differentially regulated during serial subculture; among these the expression of T-complex protein 1 alpha subunit (TCP-1alpha), a protein known to be associated with cell proliferation, cell cycle, morphological changes, and apoptosis, gradually decreased during serial subculture. Among proteins that were differentially regulated during osteogenic differentiation, chloride intracellular channel 1 (CLIC1) was downregulated only during the early passages eukaryotic translation elongation factor, and acidic ribosomal phosphoprotein P0 was downregulated during the middle passages, while annexin V, LIM, and SH3 domain protein 1 (LASP-1), and 14-3-3 protein gamma (YWHAG) were upregulated during the later passage. These studies suggest that differentially regulated passage-specific proteins may play a role in the decrease of osteogenic differentiation potential under serial subculturing.

  10. Matrix stiffness regulation of integrin-mediated mechanotransduction during osteogenic differentiation of human mesenchymal stem cells.

    PubMed

    Shih, Yu-Ru V; Tseng, Kuo-Fung; Lai, Hsiu-Yu; Lin, Chi-Hung; Lee, Oscar K

    2011-04-01

    Mesenchymal stem cells (MSCs) cultured on extracellular matrices with different stiffness have been shown to possess diverse lineage commitment owing to the extracellular mechanical stimuli sensed by the cells. The aim of this study was to further delineate how matrix stiffness affects intracellular signaling through the mechanotransducers Rho kinase (ROCK) and focal adhesion kinase (FAK) and subsequently regulates the osteogenic phenotype of MSCs. MSCs were cultured in osteogenic medium on tunable polyacrylamide hydrogels coated with type I collagen with elasticities corresponding to Young's modulus of 7.0 ± 1.2 and 42.1 ± 3.2 kPa. Osteogenic differentiation was increased on stiffer matrices, as evident by type I collagen, osteocalcin, and Runx2 gene expressions and alizarin red S staining for mineralization. Western blot analysis demonstrated an increase in kinase activities of ROCK, FAK, and ERK1/2 on stiffer matrices. Inhibition of FAK, an important mediator of osteogenic differentiation, and inhibition of ROCK, a known mechanotransducer of matrix stiffness during osteogenesis, resulted in decreased expression of osteogenic markers during osteogenic induction. In addition, FAK affects osteogenic differentiation through ERK1/2, whereas ROCK regulates both FAK and ERK1/2. Furthermore, α(2)-integrin was upregulated on stiffer matrices during osteogenic induction, and its knockdown by siRNA downregulated the osteogenic phenotype through ROCK, FAK, and ERK1/2. Taken together, our results provide evidence that the matrix rigidity affects the osteogenic outcome of MSCs through mechanotransduction events that are mediated by α(2)-integrin.

  11. Morphology, proliferation, and osteogenic differentiation of mesenchymal stem cells cultured on titanium, tantalum, and chromium surfaces.

    PubMed

    Stiehler, Maik; Lind, Martin; Mygind, Tina; Baatrup, Anette; Dolatshahi-Pirouz, Alireza; Li, Haisheng; Foss, Morten; Besenbacher, Flemming; Kassem, Moustapha; Bünger, Cody

    2008-08-01

    Metallic implants are widely used in orthopedic surgery and dentistry. Durable osseous fixation of an implant requires that osteoprogenitor cells attach and adhere to the implant, proliferate, differentiate into osteoblasts, and produce mineralized matrix. In the present study, we investigated the interactions between human mesenchymal stem cells (MSCs) and smooth surfaces of titanium (Ti), tantalum (Ta), and chromium (Cr). Mean cellular area was quantified using fluorescence microscopy (4 h). Cellular proliferation was assessed by (3)H-thymidine incorporation and methylene blue cell counting assays (4 days). Osteogenic differentiation response was quantified by cell-specific alkaline phosphatase activity (ALP) assay (4 days), expression analysis of bone-related genes (4 days), and mineralization assay (21 days). Undifferentiated and osteogenically stimulated MSCs cultured on the different surfaces showed the same tendencies for proliferation and differentiation. MSCs exposed to Ti surfaces demonstrated enhanced proliferation compared with Ta and Cr surfaces. Cultivation of MSCs on Ta surfaces resulted in significantly increased mean cellular area and cell-specific ALP activity compared with the other surfaces tested. Cells cultured on Cr demonstrated reduced spreading and proliferation. In conclusion, Ta metal, as an alternative for Ti, can be considered as a promising biocompatible material, whereas further studies are needed to fully understand the role of Cr and its alloys in bone implants.

  12. Different roles of GNAS and cAMP signaling during early and late stages of osteogenic differentiation.

    PubMed

    Zhang, S; Kaplan, F S; Shore, E M

    2012-09-01

    Progressive osseous heteroplasia (POH) and fibrous dysplasia (FD) are genetic diseases of bone formation at opposite ends of the osteogenic spectrum: imperfect osteogenesis of the skeleton occurs in FD, while heterotopic ossification in skin, subcutaneous fat, and skeletal muscle forms in POH. POH is caused by heterozygous inactivating germline mutations in GNAS, which encodes G-protein subunits regulating the cAMP pathway, while FD is caused by GNAS somatic activating mutations. We used pluripotent mouse ES cells to examine the effects of Gnas dysregulation on osteoblast differentiation. At the earliest stages of osteogenesis, Gnas transcripts Gsα, XLαs and 1A are expressed at low levels and cAMP levels are also low. Inhibition of cAMP signaling (as in POH) by 2',5'-dideoxyadenosine enhanced osteoblast differentiation while conversely, increased cAMP signaling (as in FD), induced by forskolin, inhibited osteoblast differentiation. Notably, increased cAMP was inhibitory for osteogenesis only at early stages after osteogenic induction. Expression of osteogenic and adipogenic markers showed that increased cAMP enhanced adipogenesis and impaired osteoblast differentiation even in the presence of osteogenic factors, supporting cAMP as a critical regulator of osteoblast and adipocyte lineage commitment. Furthermore, increased cAMP signaling decreased BMP pathway signaling, indicating that G protein-cAMP pathway activation (as in FD) inhibits osteoblast differentiation, at least in part by blocking the BMP-Smad pathway, and suggesting that GNAS inactivation as occurs in POH enhances osteoblast differentiation, at least in part by stimulating BMP signaling. These data support that differences in cAMP levels during early stages of cell differentiation regulate cell fate decisions. Supporting information available online at http:/www.thieme-connect.de/ejournals/toc/hmr. PMID:22903279

  13. 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.

  14. Regulation of osteogenic differentiation of human adipose-derived stem cells by controlling electromagnetic field conditions

    PubMed Central

    Kang, Kyung Shin; Hong, Jung Min; Kang, Jo A; Rhie, Jong-Won; Jeong, Young Hun; Cho, Dong-Woo

    2013-01-01

    Many studies have reported that an electromagnetic field can promote osteogenic differentiation of mesenchymal stem cells. However, experimental results have differed depending on the experimental and environmental conditions. Optimization of electromagnetic field conditions in a single, identified system can compensate for these differences. Here we demonstrated that specific electromagnetic field conditions (that is, frequency and magnetic flux density) significantly regulate osteogenic differentiation of adipose-derived stem cells (ASCs) in vitro. Before inducing osteogenic differentiation, we determined ASC stemness and confirmed that the electromagnetic field was uniform at the solenoid coil center. Then, we selected positive (30/45 Hz, 1 mT) and negative (7.5 Hz, 1 mT) osteogenic differentiation conditions by quantifying alkaline phosphate (ALP) mRNA expression. Osteogenic marker (for example, runt-related transcription factor 2) expression was higher in the 30/45 Hz condition and lower in the 7.5 Hz condition as compared with the nonstimulated group. Both positive and negative regulation of ALP activity and mineralized nodule formation supported these responses. Our data indicate that the effects of the electromagnetic fields on osteogenic differentiation differ depending on the electromagnetic field conditions. This study provides a framework for future work on controlling stem cell differentiation. PMID:23306704

  15. Regulation of osteogenic differentiation of human adipose-derived stem cells by controlling electromagnetic field conditions.

    PubMed

    Kang, Kyung Shin; Hong, Jung Min; Kang, Jo A; Rhie, Jong-Won; Jeong, Young Hun; Cho, Dong-Woo

    2013-01-18

    Many studies have reported that an electromagnetic field can promote osteogenic differentiation of mesenchymal stem cells. However, experimental results have differed depending on the experimental and environmental conditions. Optimization of electromagnetic field conditions in a single, identified system can compensate for these differences. Here we demonstrated that specific electromagnetic field conditions (that is, frequency and magnetic flux density) significantly regulate osteogenic differentiation of adipose-derived stem cells (ASCs) in vitro. Before inducing osteogenic differentiation, we determined ASC stemness and confirmed that the electromagnetic field was uniform at the solenoid coil center. Then, we selected positive (30/45 Hz, 1 mT) and negative (7.5 Hz, 1 mT) osteogenic differentiation conditions by quantifying alkaline phosphate (ALP) mRNA expression. Osteogenic marker (for example, runt-related transcription factor 2) expression was higher in the 30/45 Hz condition and lower in the 7.5 Hz condition as compared with the nonstimulated group. Both positive and negative regulation of ALP activity and mineralized nodule formation supported these responses. Our data indicate that the effects of the electromagnetic fields on osteogenic differentiation differ depending on the electromagnetic field conditions. This study provides a framework for future work on controlling stem cell differentiation.

  16. Induction of osteogenic differentiation of adipose derived stem cells by microstructured nitinol actuator-mediated mechanical stress.

    PubMed

    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.

  17. 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.

  18. The effects of acellular amniotic membrane matrix on osteogenic differentiation and ERK1/2 signaling in human dental apical papilla cells.

    PubMed

    Chen, Yi-Jane; Chung, Min-Chun; Jane Yao, Chung-Chen; Huang, Chien-Hsun; Chang, Hao-Hueng; Jeng, Jiiang-Huei; Young, Tai-Horng

    2012-01-01

    The amniotic membrane (AM) has been widely used in the field of tissue engineering because of the favorable biological properties for scaffolding material. However, little is known about the effects of an acellular AM matrix on the osteogenic differentiation of mesenchymal stem cells. In this study, it was found that both basement membrane side and collagenous stroma side of the acellular AM matrix were capable of providing a preferential environment for driving the osteogenic differentiation of human dental apical papilla cells (APCs) with proven stem cell characteristics. Acellular AM matrix potentiated the induction effect of osteogenic supplements (OS) such as ascorbic acid, β-glycerophosphate, and dexamethasone and enhanced the osteogenic differentiation of APCs, as seen by increased core-binding factor alpha 1 (Cbfa-1) phosphorylation, alkaline phosphatase activity, mRNA expression of osteogenic marker genes, and mineralized matrix deposition. Even in the absence of soluble OS, acellular AM matrix also could exert the substrate-induced effect on initiating APCs' differentiation. Especially, the collagenous stroma side was more effective than the basement membrane side. Moreover, the AM-induced effect was significantly inhibited by U0126, an inhibitor of extracellular signaling-regulated kinase 1/2 (ERK1/2) signaling. Taken together, the osteogenic differentiation promoting effect on APCs is AM-specific, which provides potential applications of acellular AM matrix in bone/tooth tissue engineering.

  19. Caffeine regulates osteogenic differentiation and mineralization of primary adipose-derived stem cells and a bone marrow stromal cell line.

    PubMed

    Su, Shu-Jem; Chang, Kee-Lung; Su, Shu-Hui; Yeh, Yao-Tsung; Shyu, Huey-Wen; Chen, Kuan-Ming

    2013-06-01

    Caffeine consumption reportedly influences bone mineral density and body weight. However, the effects of caffeine on bone metabolism are still controversial, and whether the dosage of caffeine influences osteogenic differentiation is yet to be clarified. In the present study, we cultured primary adipose-derived stem cells (ADSCs) and a bone marrow stromal cell line (M2-10B4) in osteogenic differentiation media containing varying concentrations of caffeine. Caffeine had biphasic effects: 0.1 mM caffeine significantly enhanced mineralization and alkaline phosphatase (ALP) activity. Consistent with these observations, a caffeine concentration of 0.1 mM upregulated the osteogenic differentiation marker genes ALP and osteocalcin (OCN), and elevated osteoprotegerin (OPG), Runt-related transcription factor 2 (RUNX2) and Sirtuin 1 (SIRT1) levels. However, a concentration of caffeine greater than 0.3 mM suppressed the differentiation of both the cell types. These findings indicate that caffeine has a beneficial effect on ADSCs and bone marrow stromal cells, enhancing differentiation to osteoblasts; this effect, which is mediated via RUNX2 activation at low doses is significantly suppressed at high doses.

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

    PubMed

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

    2014-10-01

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

  1. 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

  2. Design of experiments approach to engineer cell-secreted matrices for directing osteogenic differentiation.

    PubMed

    Decaris, Martin L; Leach, J Kent

    2011-04-01

    The presentation of extracellular matrix (ECM) proteins provides an opportunity to instruct the phenotype and behavior of responsive cells. Decellularized cell-secreted matrix coatings (DM) represent a biomimetic culture surface that retains the complexity of the natural ECM. Microenvironmental culture conditions alter the composition of these matrices and ultimately the ability of DMs to direct cell fate. We employed a design of experiments (DOE) multivariable analysis approach to determine the effects and interactions of four variables (culture duration, cell seeding density, oxygen tension, and media supplementation) on the capacity of DMs to direct the osteogenic differentiation of human mesenchymal stem cells (hMSCs). DOE analysis revealed that matrices created with extended culture duration, ascorbate-2-phosphate supplementation, and in ambient oxygen tension exhibited significant correlations with enhanced hMSC differentiation. We validated the DOE model results using DMs predicted to have superior (DM1) or lesser (DM2) osteogenic potential for naïve hMSCs. Compared to cells on DM2, hMSCs cultured on DM1 expressed 2-fold higher osterix levels and deposited 3-fold more calcium over 3 weeks. Cells on DM1 coatings also exhibited greater proliferation and viability compared to DM2-coated substrates. This study demonstrates that DOE-based analysis is a powerful tool for optimizing engineered systems by identifying significant variables that have the greatest contribution to the target output.

  3. 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

  4. 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

  5. Cartilage Oligomeric Matrix Protein Gene Multilayers Inhibit Osteogenic Differentiation and Promote Chondrogenic Differentiation of Mesenchymal Stem Cells

    PubMed Central

    Guo, Peng; Shi, Zhong-Li; Liu, An; Lin, Tiao; Bi, Fang-Gang; Shi, Ming-Min; Yan, Shi-Gui

    2014-01-01

    There are still many challenges to acquire the optimal integration of biomedical materials with the surrounding tissues. Gene coatings on the surface of biomaterials may offer an effective approach to solve the problem. In order to investigate the gene multilayers mediated differentiation of mesenchymal stem cells (MSCs), gene functionalized films of hyaluronic acid (HA) and lipid-DNA complex (LDc) encoding cartilage oligomeric matrix protein (COMP) were constructed in this study via the layer-by-layer self-assembly technique. Characterizations of the HA/DNA multilayered films indicated the successful build-up process. Cells could be directly transfected by gene films and a higher expression could be obtained with the increasing bilayer number. The multilayered films were stable for a long period and DNA could be easily released in an enzymatic condition. Real-time polymerase chain reaction (RT-PCR) assay presented significantly higher (p < 0.01) COMP expression of MSCs cultured with HA/COMP multilayered films. Compared with control groups, the osteogenic gene expression levels of MSCs with HA/COMP multilayered films were down-regulated while the chondrogenic gene expression levels were up-regulated. Similarly, the alkaline phosphatase (ALP) staining and Alizarin red S staining of MSCs with HA/COMP films were weakened while the alcian blue staining was enhanced. These results demonstrated that HA/COMP multilayered films could inhibit osteogenic differentiation and promote chondrogenic differentiation of MSCs, which might provide new insight for physiological ligament-bone healing. PMID:25380520

  6. Upregulation of miR-22 Promotes Osteogenic Differentiation and Inhibits Adipogenic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stem Cells by Repressing HDAC6 Protein Expression

    PubMed Central

    Huang, Shan; Wang, Shihua; Bian, Chunjing; Yang, Zhuo; Zhou, Hong; Zeng, Yang; Li, Hongling; Han, Qin

    2012-01-01

    Mesenchmal stem cells (MSCs) can be differentiated into either adipocytes or osteoblasts, and a reciprocal relationship exists between adipogenesis and osteogenesis. Multiple transcription factors and signaling pathways have been reported to regulate adipogenic or osteogenic differentiation, respectively, yet the molecular mechanism underlying the cell fate alteration between adipogenesis and osteogenesis still remains to be illustrated. MicroRNAs are important regulators in diverse biological processes by repressing protein expression of their targets. Here, miR-22 was found to regulate adipogenic and osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hADMSCs) in opposite directions. Our data showed that miR-22 decreased during the process of adipogenic differentiation but increased during osteogenic differentiation. On one hand, overexpression of miR-22 in hADMSCs could inhibit lipid droplets accumulation and repress the expression of adipogenic transcription factors and adipogenic-specific genes. On the other hand, enhanced alkaline phosphatase activity and matrix mineralization, as well as increased expression of osteo-specific genes, indicated a positive role of miR-22 in regulating osteogenic differentiation. Target databases prediction and validation by Dual Luciferase Reporter Assay, western blot, and real-time polymerase chain reaction identified histone deacetylase 6 (HDAC6) as a direct downstream target of miR-22 in hADMSCs. Inhibition of endogenous HDAC6 by small-interfering RNAs suppressed adipogenesis and stimulated osteogenesis, consistent with the effect of miR-22 overexpression in hADMSCs. Together, our results suggested that miR-22 acted as a critical regulator of balance between adipogenic and osteogenic differentiation of hADMSCs by repressing its target HDAC6. PMID:22375943

  7. Osteogenic Differentiation of Human Amniotic Fluid Mesenchymal Stem Cells Is Determined by Epigenetic Changes

    PubMed Central

    2016-01-01

    Osteogenic differentiation of human amniotic fluid derived mesenchymal stem cells (AF-MSCs) has been widely studied in vitro and in vivo as a potential tool for regenerative medicine and tissue engineering. While most of the studies analyze changes in transcriptional profile during differentiation to date there is not much information regarding epigenetic changes in AF-MSCs during differentiation. The aim of our study was to evaluate epigenetic changes during osteogenic differentiation of AF-MS cells. Isolated AF-MSCs were characterized morphologically and osteogenic differentiation was confirmed by cell staining and determining expression of alkaline phosphatase and osteopontin by RT-qPCR. Variation in gene expression levels of pluripotency markers and specific microRNAs were also evaluated. Analysis of epigenetic changes revealed that levels of chromatin modifying enzymes such as Polycomb repressive complex 2 (PRC2) proteins (EZH2 and SUZ12), DNMT1, HDAC1, and HDAC2 were reduced after osteogenic differentiation of AF-MSCs. We demonstrated that the level of specific histone markers keeping active state of chromatin (H3K4me3, H3K9Ac, and others) increased and markers of repressed state of chromatin (H3K27me3) decreased. Our results show that osteogenic differentiation of AF-MSCs is conducted by various epigenetic alterations resulting in global chromatin remodeling and provide insights for further epigenetic investigations in human AF-MSCs.

  8. Myocilin Stimulates Osteogenic Differentiation of Mesenchymal Stem Cells through Mitogen-activated Protein Kinase Signaling*

    PubMed Central

    Kwon, Heung Sun; Johnson, Thomas V.; Tomarev, Stanislav I.

    2013-01-01

    Myocilin is a secreted glycoprotein that is expressed in ocular and non-ocular tissues. Mutations in the MYOCILIN gene may lead to juvenile- and adult-onset primary open-angle glaucoma. Here we report that myocilin is expressed in bone marrow-derived mesenchymal stem cells (MSCs) and plays a role in their differentiation into osteoblasts in vitro and in osteogenesis in vivo. Expression of myocilin was detected in MSCs derived from mouse, rat, and human bone marrow, with human MSCs exhibiting the highest level of myocilin expression. Expression of myocilin rose during the course of human MSC differentiation into osteoblasts but not into adipocytes, and treatment with exogenous myocilin further enhanced osteogenesis. MSCs derived from Myoc-null mice had a reduced ability to differentiate into the osteoblastic lineage, which was partially rescued by exogenous extracellular myocilin treatment. Myocilin also stimulated osteogenic differentiation of wild-type MSCs, which was associated with activation of the p38, Erk1/2, and JNK MAP kinase signaling pathways as well as up-regulated expression of the osteogenic transcription factors Runx2 and Dlx5. Finally, cortical bone thickness and trabecular volume, as well as the expression level of osteopontin, a known factor of bone remodeling and osteoblast differentiation, were reduced dramatically in the femurs of Myoc-null mice compared with wild-type mice. These data suggest that myocilin should be considered as a target for improving the bone regenerative potential of MSCs and may identify a new role for myocilin in bone formation and/or maintenance in vivo. PMID:23629661

  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. Differential effect of amelogenin peptides on osteogenic differentiation in vitro: identification of possible new drugs for bone repair and regeneration.

    PubMed

    Amin, Harsh D; Olsen, Irwin; Knowles, Jonathan C; Donos, Nikolaos

    2012-06-01

    Enamel matrix proteins (EMP) have been shown to promote regeneration of periodontal ligament and root cementum, and sometimes to enhance the differentiation of bone-forming cells in vitro and new bone growth in vivo. However, the inconsistent and unpredictable effects of EMP that have been reported for bone regeneration may be due to the highly variable composition of this heterogeneous material, which is comprised mainly of amelogenin and amelogenin-derived peptides. The present study has therefore examined the effects of naturally occurring low-molecular-weight (LMW) and high-molecular-weight (HMW) fractions of Emdogain(®) (EMD; Institut Straumann, Basel, Switzerland), a commercially available form of EMP, on osteogenic differentiation of bone precursor cells in vitro. In addition, the effects of chemically synthesized specific components of LMW and HMW-namely, the tyrosine-rich amelogenin peptide (TRAP), a specific amelogenin isoform derived by proteolytic clipping, and a leucine-rich amelogenin peptide (LRAP), an isoform derived by alternative splicing-on bone-forming cell activity were also investigated. Our findings demonstrate that while TRAP suppressed the formation of bone-like mineralized nodules, LRAP upregulated osteogenic differentiation. Furthermore, synthetically produced TRAP and its unique C-terminal 12 amino acid sequence (TCT) also suppressed bone-forming cells, whereas LRAP and its unique C-terminal 23 amino acid sequence (LCT) markedly enhanced terminal differentiation of bone-forming cells. These findings suggest that the differential effects of amelogenin-derived peptide sequences present in EMP could be of potential clinical value, with the novel bioactive TCT peptide as a useful tool for limiting pathological bone cell growth and the unique LCT sequence having therapeutic benefits in the treatment of periodontal and orthopedic diseases. PMID:22320389

  11. 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.

  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. Graphene/single-walled carbon nanotube hybrids promoting osteogenic differentiation of mesenchymal stem cells by activating p38 signaling pathway

    PubMed Central

    Yan, Xinxin; Yang, Wen; Shao, Zengwu; Yang, Shuhua; Liu, Xianzhe

    2016-01-01

    Carbon nanomaterials are becoming increasingly significant in biomedical fields since they exhibit exceptional physicochemical and biocompatible properties. Today, the stem cells offer potentially new therapeutic approaches in tissue engineering and regenerative medicine. However, the induction of differentiation into specific lineages remains challenging, which provoked us to explore the biomedical applications of carbon nanomaterials in stem cells. In this study, we investigated the interactions between graphene/single-walled carbon nanotube (G/SWCNT) hybrids and rat mesenchymal stem cells (rMSCs) and focused on the proliferation and differentiation of rMSCs treated with G/SWCNT hybrids. Cell viability and morphology were evaluated using cell counting kit-8 assay and immunofluorescence staining, respectively. Osteogenic differentiation evaluated by alkaline phosphatase activity of MSCs proved to be higher after treatment with G/SWCNT hybrids, and the mineralized matrix nodule formation was also enhanced. In addition, the expression levels of osteogenic-associated genes were upregulated, while the adipocyte-specific markers were downregulated. Consistent with these results, we illustrated that the effect of G/SWCNT hybrids on the process of osteogenic differentiation of rMSCs can be modulated by activating the p38 signaling pathway and inhibiting the extracellular signal-regulated kinase 1/2 pathway. Nevertheless, our study suggests that carbon nanomaterials offer a promising platform for regenerative medicine in the near future. PMID:27799770

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. The influence of biodegradable magnesium alloys on the osteogenic differentiation of human mesenchymal stem cells.

    PubMed

    Li, Rachel W; Kirkland, Nicholas T; Truong, John; Wang, Jian; Smith, Paul N; Birbilis, Nick; Nisbet, David R

    2014-12-01

    The postdegradation effect of pure Mg, Mg-1Y, Mg-5Al, and Mg-2Ca alloys on the differentiation, proliferation and gene expression of human mesenchymal stem cells (hMSCs) was investigated. It was revealed that that Mg(2+) ions result in an increase in cell proliferation. However, we observed a maximum concentration (approximately 8.0 × 10(-4) M) that was favourable to ATP production, above which ATP production began to decrease. In contrast to proliferation, no maximum concentration for osteogenic differentiation was observed, with increasing concentration of Mg(2+) ions resulting in an increase in osteogenic differentiation across the entire tested range. Interestingly, the Mg-2Ca alloy had minimal effect on osteogenic differentiation, with Mg-1Y and pure Mg having a superior effect on the proliferation and differentiation of hMSCs. This was also observed from gene expression data, where these alloys upregulated TGFβ-1, SMAD4, FGF-2, FGF-10, and BMP-2, while SOX-2, SOX-9, and TNF-α were downregulated. Increased expression of TGFβ-1, SMAD4, BMPs, and COLIA1 protein provided further evidence to support osteogenic differentiation and that the influence of the alloying extracts on differentiation may be via the SMAD signaling pathway.

  20. 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

  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. 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

  3. Asperosaponin VI promotes bone marrow stromal cell osteogenic differentiation through the PI3K/AKT signaling pathway in an osteoporosis model

    PubMed Central

    Ke, Ke; Li, Qi; Yang, Xiaofeng; Xie, Zhijian; Wang, Yu; Shi, Jue; Chi, Linfeng; Xu, Weijian; Hu, Lingling; Shi, Huali

    2016-01-01

    Asperosaponin VI (ASA VI), a natural compound isolated from the well-known traditional Chinese herb Radix Dipsaci, has an important role in promoting osteoblast formation. However, its effects on osteoblasts in the context of osteoporosis is unknown. This study aimed to investigate the effects and mechanism of ASA VI action on the proliferation and osteogenic differentiation of bone marrow stromal cells isolated from the ovariectomized rats (OVX rBMSCs). The toxicity of ASA VI and its effects on the proliferation of OVX rBMSCs were measured using a CCK-8 assay. Various osteogenic differentiation markers were also analyzed, such as ALP activity, calcified nodule formation, and the expression of osteogenic genes, i.e., ALP, OCN, COL 1 and RUNX2. The results indicated that ASA VI promoted the proliferation of OVX rBMSCs and enhanced ALP activity and calcified nodule formation. In addition, while ASA VI enhanced the expression of ALP, OCN, Col 1 and RUNX2, treatment with LY294002 reduced all of these osteogenic effects and reduced the p-AKT levels induced by ASA VI. These results suggest that ASA VI promotes the osteogenic differentiation of OVX rBMSCs by acting on the phosphatidylinositol—3 kinase/AKT signaling pathway. PMID:27756897

  4. Intercellular adhesion molecule-1 inhibits osteogenic differentiation of mesenchymal stem cells and impairs bio-scaffold-mediated bone regeneration in vivo.

    PubMed

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

    2014-10-01

    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.

  5. 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.

  6. Effects of bioactive glass S53P4 or beta-tricalcium phosphate and bone morphogenetic protein-2 and bone morphogenetic protein-7 on osteogenic differentiation of human adipose stem cells

    PubMed Central

    Patrikoski, Mimmi; Juntunen, Miia; Kujala, Kasperi; Kääriäinen, Minna; Kuokkanen, Hannu; Sándor, George K; Vapaavuori, Outi; Suuronen, Riitta; Mannerström, Bettina; von Rechenberg, Brigitte; Miettinen, Susanna

    2012-01-01

    The effects of bioactive glass S53P4 or beta-tricalcium phosphate; and bone morphogenetic proteins bone morphogenetic protein-2, bone morphogenetic protein-7, or bone morphogenetic protein-2 + 7 on osteogenic differentiation of human adipose stem cells were compared in control medium, osteogenic medium, and bone morphogenetic protein-supplemented osteogenic medium to assess suitability for bone tissue engineering. Cell amount was evaluated with qDNA measurements; osteogenic differentiation using marker gene expression, alkaline phosphate activity, and angiogenic potential was measured by vascular endothelial growth factor expression. As compared to beta-tricalcium phosphate, cell amount was significantly greater for bioactive glass in control medium after 7 days and in osteogenic medium after 14 days, and alkaline phosphate activity was always significantly greater for bioactive glass in control medium. However, alkaline phosphate activity increased for beta-tricalcium phosphate and decreased for bioactive glass granules in osteogenic medium. For both biomaterials, bone morphogenetic protein supplementation decreased cell amount and osteogenic differentiation of human adipose stem cells, and vascular endothelial growth factor expressions correlated with cell amounts. Effects of culture medium on human adipose stem cells are biomaterial dependent; bioactive glass in control medium enhanced osteogenic differentiation most effectively. PMID:23316275

  7. 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

  8. 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.

  9. Three-dimensional graphene foams promote osteogenic differentiation of human mesenchymal stem cells

    NASA Astrophysics Data System (ADS)

    Crowder, Spencer W.; Prasai, Dhiraj; Rath, Rutwik; Balikov, Daniel A.; Bae, Hojae; Bolotin, Kirill I.; Sung, Hak-Joon

    2013-05-01

    Graphene is a novel material whose application in biomedical sciences has only begun to be realized. In the present study, we have employed three-dimensional graphene foams as culture substrates for human mesenchymal stem cells and provide evidence that these materials can maintain stem cell viability and promote osteogenic differentiation.

  10. Mechanisms of stem cell osteogenic differentiation on TiO2 nanotubes.

    PubMed

    Yu, Weiqiang; Qian, Chao; Jiang, Xinquan; Zhang, Fuqiang; Weng, Weimin

    2015-12-01

    TiO2 nanotubes could stimulate osteogenic differentiation of stem cells, but the molecular mechanisms underlying the interactions between nanotubes and stem cells remain unclear. In this study, we investigated the response of bone marrow stromal cells to nanotubes of different diameters using microarray-based bioinformatics approach. Gene ontology (GO) and GO enrichment network analysis indicated that larger TiO2 nanotubes were more potent than smaller nanotubes in inducing the expression of genes involved in cell proliferation, differentiation, and immune responses, and inhibiting that of genes responsible for cell adhesion. The analysis of the signaling network containing significantly affected genes suggested that Na(+)/K(+) transporting ATPases ATP1A2 (alpha 2 polypeptide) and ATP1A3 (alpha 3 polypeptide), and MAP3K11 (mitogen-activated protein kinase kinase kinase 11) were important for inducing osteogenic differentiation of bone marrow stromal cells without additional osteogenic stimuli. The upregulation of the ATP1A2 and MAP3K11 genes confirmed by real-time PCR indicates that the response of bone marrow stromal cells to nanotube cues may be mediated by the pathways previously implicated in transducing mechanical stress signals. Our results revealed some molecular mechanisms by which TiO2 nanotubes may direct osteogenic differentiation of stem cells.

  11. 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

  12. 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

  13. Polysaccharides immobilized in polypyrrole matrices are able to induce osteogenic differentiation in mouse mesenchymal stem cells.

    PubMed

    Moreno, Judith Serra; Sabbieti, Maria Giovanna; Agas, Dimitrios; Marchetti, Luigi; Panero, Stefania

    2014-12-01

    Bone marrow mesenchymal stem cells (MSCs) have attracted considerable interest due to their ability to differentiate and contribute to the regeneration of mesenchymal tissues. The present study illustrates that the proper immobilization of heparin (Hep) and hyaluronic acid (HA) into a polypyrrole (PPy) matrix by electropolymerization results in an optimal interface for MSC differentiation towards osteoblast lineage. The obtained thin films showed good thermal stability, hydrophilicity and slow controlled polysaccharide release. The in vitro tests showed the main role of the interface chemical composition. Indeed, PPyHep and PPyHA thin films were able to induce osteogenic differentiation as determined by levels of specific early osteogenic markers (Runx2 and osterix) even in the absence of differentiating medium. Increased levels of ALP and Alizarin red staining, both indicating mineralization processes, confirmed the presence of mature osteoblasts.

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. The collagen I mimetic peptide DGEA enhances an osteogenic phenotype in mesenchymal stem cells when presented from cell-encapsulating hydrogels.

    PubMed

    Mehta, Manav; Madl, Christopher M; Lee, Shimwoo; Duda, Georg N; Mooney, David J

    2015-11-01

    Interactions between cells and the extracellular matrix (ECM) are known to play critical roles in regulating cell phenotype. The identity of ECM ligands presented to mesenchymal stem cells (MSCs) has previously been shown to direct the cell fate commitment of these cells. To enhance osteogenic differentiation of MSCs, alginate hydrogels were prepared that present the DGEA ligand derived from collagen I. When presented from hydrogel surfaces in 2D, the DGEA ligand did not facilitate cell adhesion, while hydrogels presenting the RGD ligand derived from fibronectin did encourage cell adhesion and spreading. However, the osteogenic differentiation of MSCs encapsulated within alginate hydrogels presenting the DGEA ligand was enhanced when compared with unmodified alginate hydrogels and hydrogels presenting the RGD ligand. MSCs cultured in DGEA-presenting gels exhibited increased levels of osteocalcin production and mineral deposition. These data suggest that the presentation of the collagen I-derived DGEA ligand is a feasible approach for selectively inducing an osteogenic phenotype in encapsulated MSCs.

  1. 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

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

    PubMed

    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

    2015-08-17

    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.

  3. 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

  4. 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

  5. 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

  6. Matrix-mediated retention of osteogenic differentiation potential by human adult bone marrow stromal cells during ex vivo expansion.

    PubMed

    Mauney, Joshua R; Kaplan, David L; Volloch, Vladimir

    2004-07-01

    During prolonged cultivation ex vivo, adult bone marrow stromal stem cells (BMSCs) undergo two probably interdependent processes, replicative aging and a decline in differentiation potential. Recently, our results with primary human fibroblasts indicated that growth on denatured collagen (DC) matrix results in the reduction of the rate of cellular aging. The present study has been undertaken to test whether the growth of human BMSCs under the same conditions would translate into preservation of cellular aging-attenuated functions, such as the ability to express HSP70 in response to stress as well as of osteogenic differentiation potential. We report here that growth of BMSCs on a DC matrix versus tissue culture polystyrene significantly reduced one of the main manifestations of cellular aging, the attenuation of the ability to express a major protective stress response component, HSP70, increased the proliferation capacity of ex vivo expanded BMSCs, reduced the rate of morphological changes, and resulted in a dramatic increase in the retention of the potential to express osteogenic-specific functions and markers upon treatment with osteogenic stimulants. BMSCs are a promising and increasingly important cell source for tissue engineering as well as cell and gene therapeutic strategies. For use of BMSCs in these applications, ex vivo expansion is necessary to obtain a sufficient, therapeutically useful, number of cells; however, this results in the loss of differentiation potential. This problem is especially acute in older patients where more extensive in vitro expansion of smaller number of stem/progenitor cells is needed. The finding that growth on certain biomaterials preserves aging-attenuated functions, enhances proliferation capacity, and maintains differentiation potential of BMSCs indicates a promising approach to address this problem.

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. 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

  12. [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

  13. 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.

  14. Surface hydrophilicity of PLGA fibers governs in vitro mineralization and osteogenic differentiation.

    PubMed

    Thomas, Minnah; Arora, Aditya; Katti, Dhirendra S

    2014-12-01

    Interfacial properties of biomaterials play an important role in governing their interaction with biological microenvironments. This work investigates the role of surface hydrophilicity of electrospun poly(lactide-co-glycolide) (PLGA) fibers in determining their biological response. For this, PLGA is blended with varying amounts of Pluronic®F-108 and electrospun to fabricate microfibers with varying surface hydrophilicity. The results of mineralization study in simulated body fluid (SBF) demonstrate a significant enhancement in mineralization with an increase in surface hydrophilicity. While presence of serum proteins in SBF reduces absolute mineral content, mineralization continues to be higher on samples with higher surface hydrophilicity. The results from in vitro cell culture studies demonstrate a marked improvement in mesenchymal stem cell-adhesion, elongation, proliferation, infiltration, osteogenic differentiation and matrix mineralization on hydrophilized fibers. Therefore, hydrophilized PLGA fibers are advantageous both in terms of mineralization and elicitation of favorable cell response. Since most of the polymeric materials being used in orthopedics are hydrophobic in nature, the results from this study have strong implications in the future design of interfaces of such hydrophobic materials. In addition, the work proposes a facile method for the modification of electrospun fibers of hydrophobic polymers by blending with a poloxamer for improved bone tissue regeneration. PMID:25491835

  15. Surface hydrophilicity of PLGA fibers governs in vitro mineralization and osteogenic differentiation.

    PubMed

    Thomas, Minnah; Arora, Aditya; Katti, Dhirendra S

    2014-12-01

    Interfacial properties of biomaterials play an important role in governing their interaction with biological microenvironments. This work investigates the role of surface hydrophilicity of electrospun poly(lactide-co-glycolide) (PLGA) fibers in determining their biological response. For this, PLGA is blended with varying amounts of Pluronic®F-108 and electrospun to fabricate microfibers with varying surface hydrophilicity. The results of mineralization study in simulated body fluid (SBF) demonstrate a significant enhancement in mineralization with an increase in surface hydrophilicity. While presence of serum proteins in SBF reduces absolute mineral content, mineralization continues to be higher on samples with higher surface hydrophilicity. The results from in vitro cell culture studies demonstrate a marked improvement in mesenchymal stem cell-adhesion, elongation, proliferation, infiltration, osteogenic differentiation and matrix mineralization on hydrophilized fibers. Therefore, hydrophilized PLGA fibers are advantageous both in terms of mineralization and elicitation of favorable cell response. Since most of the polymeric materials being used in orthopedics are hydrophobic in nature, the results from this study have strong implications in the future design of interfaces of such hydrophobic materials. In addition, the work proposes a facile method for the modification of electrospun fibers of hydrophobic polymers by blending with a poloxamer for improved bone tissue regeneration.

  16. 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

  17. 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

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

    PubMed Central

    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

  19. 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

  20. Crucial role of Notch signaling in osteogenic differentiation of periodontal ligament stem cells in osteoporotic rats.

    PubMed

    Li, Ying; Li, S Q; Gao, Y M; Li, Jin; Zhang, Bin

    2014-06-01

    Estrogen deficiency-induced osteoporosis typically occurs in postmenopausal women and has been strongly associated with periodontal diseases. Periodontal ligament stem cells (PDLSCs) isolated from the periodontal ligament can differentiate into many types of specialized cells, including osteoblast-like cells that contribute to periodontal tissue repair. The Notch signaling pathway is highly conserved and associated with self-renewal potential and cell-fate determination. Recently, several studies have focused on the relationship between Notch signaling and osteogenic differentiation. However, the precise mechanisms underlying this relationship are largely unknown. We have successfully isolated PDLSCs from both ovariectomized (OVX) and sham-operated rats. Both the mRNA and protein levels of Notch1 and Jagged1 were upregulated when PDLSCs were cultured in osteogenic induction media. Mineralization assays showed decreased calcium deposits in OVX-PDLSCs treated with a γ-secretase inhibitor compared with control cells. Thus Notch signaling is important in maintaining the osteogenic differentiation of PDLSCs in osteoporotic rats, which help in the development of a potential therapeutic strategy for periodontal disease in postmenopausal women.

  1. Osteogenic differentiation of hypertrophic chondrocytes involves asymmetric cell divisions and apoptosis

    PubMed Central

    1995-01-01

    We have investigated the early cellular events that take place during the change in lineage commitment from hypertrophic chondrocytes to osteoblast-like cells. We have induced this osteogenic differentiation by cutting through the hypertrophic cartilage of embryonic chick femurs and culturing the explants. Immunocytochemical characterization, [3H]thymidine pulse-chase labeling, in situ nick translation or end labeling of DNA breaks were combined with ultrastructural studies to characterize the changing pattern of differentiation. The first responses to the cutting, seen after 2 d, were upregulation of alkaline phosphatase activity, synthesis of type I collagen and single-stranded DNA breaks, probably indicating a metastable state. Associated with the change from chondrogenic to osteogenic commitment was an asymmetric cell division with diverging fates of the two daughter cells, where one daughter cell remained viable and the other one died. The available evidence suggests that the viable daughter cell then divided and generated osteogenic cells, while the other daughter cell died by apoptosis. The results suggest a new concept of how changes in lineage commitment of differentiated cells may occur. The concepts also reconcile previously opposing views of the fate of the hypertrophic chondrocyte. PMID:7593173

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. Application of low-frequency alternating current electric fields via interdigitated electrodes: effects on cellular viability, cytoplasmic calcium, and osteogenic differentiation of human adipose-derived stem cells.

    PubMed

    McCullen, Seth D; McQuilling, John P; Grossfeld, Robert M; Lubischer, Jane L; Clarke, Laura I; Loboa, Elizabeth G

    2010-12-01

    Electric stimulation is known to initiate signaling pathways and provides a technique to enhance osteogenic differentiation of stem and/or progenitor cells. There are a variety of in vitro stimulation devices to apply electric fields to such cells. Herein, we describe and highlight the use of interdigitated electrodes to characterize signaling pathways and the effect of electric fields on the proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs). The advantage of the interdigitated electrode configuration is that cells can be easily imaged during short-term (acute) stimulation, and this identical configuration can be utilized for long-term (chronic) studies. Acute exposure of hASCs to alternating current (AC) sinusoidal electric fields of 1 Hz induced a dose-dependent increase in cytoplasmic calcium in response to electric field magnitude, as observed by fluorescence microscopy. hASCs that were chronically exposed to AC electric field treatment of 1 V/cm (4 h/day for 14 days, cultured in the osteogenic differentiation medium containing dexamethasone, ascorbic acid, and β-glycerol phosphate) displayed a significant increase in mineral deposition relative to unstimulated controls. This is the first study to evaluate the effects of sinusoidal AC electric fields on hASCs and to demonstrate that acute and chronic electric field exposure can significantly increase intracellular calcium signaling and the deposition of accreted calcium under osteogenic stimulation, respectively.

  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. 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.

  11. 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

  12. 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.

  13. Human amnion mesenchymal stem cells promote proliferation and osteogenic differentiation in human bone marrow mesenchymal stem cells.

    PubMed

    Wang, Yuli; Yin, Ying; Jiang, Fei; Chen, Ning

    2015-02-01

    Human amnion mesenchymal stem cells (HAMSCs) can be obtained from human amniotic membrane, a highly abundant and readily available tissue. HAMSC sources present fewer ethical issues, have low immunogenicity, anti-inflammatory properties, considerable advantageous characteristics, and are considered an attractive potential treatment material in the field of regenerative medicine. We used a co-culture system to determine whether HAMSCs could promote osteogenesis in human bone marrow mesenchymal stem cells (HBMSCs). We isolated HAMSCs from discarded amnion samples and collected them using pancreatin/collagenase digestion. We cultured HAMSCs and HBMSCSs in basal medium. Activity of alkaline phosphatase (ALP), an early osteogenesis marker, was increased in the co-culture system compared to the control single cultures, which we also confirmed by ALP staining. We used immunofluorescence testing to investigate the effects of co-culturing with HAMSCs on HBMSC proliferation, which revealed that the co-culturing enhanced EdU expression in HBMSCs. Western blotting and quantitative real-time PCR indicated that co-culturing promoted osteogenesis in HBMSCs. Furthermore, Alizarin red S staining revealed that extracellular matrix calcium levels in mineralized nodule formation produced by the co-cultures were higher than that in the controls. Using the same co-culture system, we further observed the effects of HAMSCs on osteogenic differentiation in primary osteoblasts by Western blotting, which better addressed the mechanism for HAMSCs in bone regeneration. The results showed HAMSCs are osteogenic and not only play a role in promoting HBMSC proliferation and osteogenic differentiation but also in osteoblasts, laying the foundation for new regenerative medicine methods.

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

    PubMed

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

    2005-09-01

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

  15. 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

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

    PubMed

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

    2013-11-01

    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.

  17. 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.

  18. Effects of pulsed electromagnetic field frequencies on the osteogenic differentiation of human mesenchymal stem cells.

    PubMed

    Luo, Fei; Hou, Tianyong; Zhang, Zehua; Xie, Zhao; Wu, Xuehui; Xu, Jianzhong

    2012-04-01

    The purpose of this study was to evaluate the effect of different frequencies of pulsed electromagnetic fields on the osteogenic differentiation of human mesenchymal stem cells. Third-generation human mesenchymal stem cells were irradiated with different frequencies of pulsed electromagnetic fields, including 5, 25, 50, 75, 100, and 150 Hz, with a field intensity of 1.1 mT, for 30 minutes per day for 21 days. Changes in human mesenchymal stem cell morphology were observed using phase contrast microscopy. Alkaline phosphatase activity and osteocalcin expression were also determined to evaluate human mesenchymal stem cell osteogenic differentiation.Different effects were observed on human mesenchymal stem cell osteoblast induction following exposure to different pulsed electromagnetic field frequencies. Levels of human mesenchymal stem cell differentiation increased when the pulsed electromagnetic field frequency was increased from 5 hz to 50 hz, but the effect was weaker when the pulsed electromagnetic field frequency was increased from 50 Hz to 150 hz. The most significant effect on human mesenchymal stem cell differentiation was observed at of 50 hz.The results of the current study show that pulsed electromagnetic field frequency is an important factor with regard to the induction of human mesenchymal stem cell differentiation. Furthermore, a pulsed electromagnetic field frequency of 50 Hz was the most effective at inducing human mesenchymal stem cell osteoblast differentiation in vitro.

  19. 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.

  20. 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

  1. The effect of gold nanoparticle size on osteogenic differentiation of adipose-derived stem cells.

    PubMed

    Ko, Wan-Kyu; Heo, Dong Nyoung; Moon, Ho-Jin; Lee, Sang Jin; Bae, Min Soo; Lee, Jung Bok; Sun, In-Cheol; Jeon, Hoon Bong; Park, Hun Kuk; Kwon, Il Keun

    2015-01-15

    There have been many medical applications based on gold nanoparticles (GNPs) over the past several centuries. Recently, researchers have focused on bone tissue engineering applications utilizing GNPs. The effect of various sizes of gold nanoparticles on the differentiation of human adipose-derived stem cells (ADSCs) into osteoblasts was investigated. The concentration of gold nanoparticles was fixed at 1 μM and varying sizes of 15, 30, 50, 75 and 100 nm (spherical GNPs) were used. The lack of cytotoxicity was confirmed by establishing viability of ADSCs using cell counting kit-8 (CCK-8) and live/dead assays. The results showed that each size of GNPs had no significant toxicity on ADSCs during 1 week of incubation. Osteogenic differentiation of ADSCs was confirmed by alkaline phosphatase (ALP) staining, ALP activity, calcium deposition, and real time PCR experiments. It was found, through dark field assays and microscope cell images, that 30 nm and 50 nm GNPs were preferentially up taken into the ADSCs. As expected, all sizes of gold nanoparticles promoted the differentiation of ADSCs toward osteoblasts more than control. Among all sizes, 30 and 50 nm GNPs appeared to have the highest differentiation rates. The data consistently demonstrated that 30 and 50 nm GNPs are the most effective in promoting osteogenic differentiation of ADSCs.

  2. Live-cell, temporal gene expression analysis of osteogenic differentiation in adipose-derived stem cells.

    PubMed

    Desai, Hetal V; Voruganti, Indu S; Jayasuriya, Chathuraka; Chen, Qian; Darling, Eric M

    2014-03-01

    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.

  3. 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.

  4. 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

  5. Glimepiride Promotes Osteogenic Differentiation in Rat Osteoblasts via the PI3K/Akt/eNOS Pathway in a High Glucose Microenvironment

    PubMed Central

    Xiong, Wei; Tan, Baosheng; Geng, Wei; Li, Jun; Liu, Hongchen

    2014-01-01

    Our previous studies demonstrated that glimepiride enhanced the proliferation and differentiation of osteoblasts and led to activation of the PI3K/Akt pathway. Recent genetic evidence shows that endothelial nitric oxide synthase (eNOS) plays an important role in bone homeostasis. In this study, we further elucidated the roles of eNOS, PI3K and Akt in bone formation by osteoblasts induced by glimepiride in a high glucose microenvironment. We demonstrated that high glucose (16.5 mM) inhibits the osteogenic differentiation potential and proliferation of rat osteoblasts. Glimepiride activated eNOS expression in rat osteoblasts cultured with two different concentrations of glucose. High glucose-induced osteogenic differentiation was significantly enhanced by glimepiride. Down-regulation of PI3K P85 levels by treatment with LY294002 (a PI3K inhibitor) led to suppression of P-eNOS and P-AKT expression levels, which in turn resulted in inhibition of RUNX2, OCN and ALP mRNA expression in osteoblasts induced by glimepiride at both glucose concentrations. ALP activity was partially inhibited by 10 µM LY294002. Taken together, our results demonstrate that glimepiride-induced osteogenic differentiation of osteoblasts occurs via eNOS activation and is dependent on the PI3K/Akt signaling pathway in a high glucose microenvironment. PMID:25391146

  6. Glimepiride promotes osteogenic differentiation in rat osteoblasts via the PI3K/Akt/eNOS pathway in a high glucose microenvironment.

    PubMed

    Ma, Pan; Gu, Bin; Xiong, Wei; Tan, Baosheng; Geng, Wei; Li, Jun; Liu, Hongchen

    2014-01-01

    Our previous studies demonstrated that glimepiride enhanced the proliferation and differentiation of osteoblasts and led to activation of the PI3K/Akt pathway. Recent genetic evidence shows that endothelial nitric oxide synthase (eNOS) plays an important role in bone homeostasis. In this study, we further elucidated the roles of eNOS, PI3K and Akt in bone formation by osteoblasts induced by glimepiride in a high glucose microenvironment. We demonstrated that high glucose (16.5 mM) inhibits the osteogenic differentiation potential and proliferation of rat osteoblasts. Glimepiride activated eNOS expression in rat osteoblasts cultured with two different concentrations of glucose. High glucose-induced osteogenic differentiation was significantly enhanced by glimepiride. Down-regulation of PI3K P85 levels by treatment with LY294002 (a PI3K inhibitor) led to suppression of P-eNOS and P-AKT expression levels, which in turn resulted in inhibition of RUNX2, OCN and ALP mRNA expression in osteoblasts induced by glimepiride at both glucose concentrations. ALP activity was partially inhibited by 10 µM LY294002. Taken together, our results demonstrate that glimepiride-induced osteogenic differentiation of osteoblasts occurs via eNOS activation and is dependent on the PI3K/Akt signaling pathway in a high glucose microenvironment. PMID:25391146

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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

  13. AFM studies of cellular mechanics during osteogenic differentiation of human amniotic fluid-derived stem cells.

    PubMed

    Chen, Qian; Xiao, Pan; Chen, Jia-Nan; Cai, Ji-Ye; Cai, Xiao-Fang; Ding, Hui; Pan, Yun-Long

    2010-01-01

    Amniotic fluid-derived stem cells (AFSCs) are becoming an important source of cells for regenerative medicine given with apparent advantages of accessibility, renewal capacity and multipotentiality. In this study, the mechanical properties of human amniotic fluid-derived stem cells (hAFSCs), such as the average Young's modulus, were determined by atomic force microscopy (3.97 ± 0.53 kPa for hAFSCs vs. 1.52 ± 0.63 kPa for fully differentiated osteoblasts). These differences in cell elasticity result primarily from differential actin cytoskeleton organization in these two cell types. Furthermore, ultrastructures, nanostructural details on the surface of cell, were visualized by atomic force microscopy (AFM). It was clearly shown that surface of osteoblasts were covered by mineralized particles, and the histogram of particles size showed that most of the particles on the surface of osteoblasts distributed from 200 to 400 nm in diameter, while the diameter of hAFSCs particles ranged from 100 to 200 nm. In contrast, there were some dips on the surface of hAFSCs, and particles were smaller than that of osteoblasts. Additionally, as osteogenic differentiation of hAFSCs progressed, more and more stress fibers were replaced by a thinner actin network which is characteristic of mature osteoblasts. These results can improve our understanding of the mechanical properties of hAFSCs during osteogenic differentiation. AFM can be used as a powerful tool for detecting ultrastructures and mechanical properties.

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

    PubMed

    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.

  15. 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

  16. 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

  17. 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

  18. Positive Selection for Bone Morphogenetic Protein Receptor Type-IB Promotes Differentiation and Specification of Human Adipose-Derived Stromal Cells Toward an Osteogenic Lineage

    PubMed Central

    McArdle, Adrian; Chung, Michael T.; Paik, Kevin J.; Duldulao, Chris; Chan, Charles; Rennert, Robert; Walmsley, Graham G.; Senarath-Yapa, Kshemendra; Hu, Michael; Seo, Elly; Lee, Min

    2014-01-01

    Background: Adipose tissue represents an abundant and easily accessible source of multipotent cells that may serve as an excellent building block for tissue engineering. However, adipose-derived stromal cells (ASCs) are a heterogeneous group and subpopulations may be identified with enhanced osteogenic potential. Methods: Human ASC subpopulations were prospectively isolated based on expression of bone morphogenetic protein receptor type-IB (BMPR-IB). Unsorted, BMPR-IB(+), and BMPR-IB(−) cells were analyzed for their osteogenic capacity through histological staining and gene expression. To evaluate their in vivo osteogenic potential, critical-sized calvarial defects were created in immunocompromised mice and treated with unsorted, BMPR-IB(+), or BMPR-IB(−) cells. Healing was assessed using microcomputed tomography and pentachrome staining of specimens at 8 weeks. Results: Increased osteogenic differentiation was noted in the BMPR-IB(+) subpopulation, as demonstrated by alkaline phosphatase staining at day 7 and extracellular matrix mineralization with Alizarin red staining at day 14. This was also associated with increased expression for osteocalcin, a late marker of osteogenesis. Radiographic analysis demonstrated significantly enhanced healing of critical-sized calvarial defects treated with BMPR-IB(+) ASCs compared with unsorted or BMPR-IB(−) cells. This was confirmed through pentachrome staining, which revealed more robust bone regeneration in the BMPR-IB(+) group. Conclusion: BMPR-IB(+) human ASCs have an enhanced ability to form bone both in vitro and in vivo. These data suggest that positive selection for BMPR-IB(+) and manipulation of the BMP pathway in these cells may yield a highly osteogenic subpopulation of cells for bone tissue engineering. PMID:24854876

  19. 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

  20. 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

  1. 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

  2. Effects of simvastatin on the osteogenic differentiation and immunomodulation of bone marrow mesenchymal stem cells.

    PubMed

    Niu, Jianyi; Ding, Gang; Zhang, Li

    2015-12-01

    The present study aimed to investigate the effects of simvastatin on the bone differentiation capacity and immunological characteristics of bone marrow mesenchymal stem cells (BMSCs). BMSCs were isolated and cultured in medium containing 1.0 µmol/ml simvastatin. The alkaline phosphatase activity, mRNA expression levels of osteocalcin and bone sialoprotein, and calcium nodule formation were assessed to determine the osteogenic differentiation capability of BMSCs. To investigate alterations in the immunological properties of simvastation‑treated BMSCs, the immunogenicity of these cells and the effect of BMSCs on phytohemagglutinin‑stimulated lymphocyte proliferation were also assessed. Following treatment with simvastatin, the alkaline phosphatase activity, and mRNA expression levels of osteocalcin and bone sialoprotein were increased significantly in the BMSCs. In addition, von Kossa staining revealed a brown calcium‑positive reaction zone in simvastatin‑treated cells. Simvastatin‑induced BMSCs revealed no affect on the proliferation of allogeneic lymphocytes, however, inhibited phytohemagglutinin‑induced lymphocyte proliferation. Collectively, simvastatin promoted the osteogenic differentiation of BMSCs significantly without affecting their immunosuppressive properties.

  3. Microcarrier Culture for Efficient Expansion and Osteogenic Differentiation of Human Fetal Mesenchymal Stem Cells

    PubMed Central

    Goh, Tony Kwang-Poh; Zhang, Zhi-Yong; Chen, Allen Kuan-Liang; Reuveny, Shaul; Choolani, Mahesh

    2013-01-01

    Abstract Stirred microcarrier (MC) culture has been suggested as the method of choice for supplying large volumes of mesenchymal stem cells (MSCs) for bone tissue engineering. In this study, we show that in addition to the improvement in cell expansion capacity, MSCs propagated and harvested from MC culture also demonstrate higher osteogenic potency when differentiated in vivo or in vitro in three-dimensional (3D) scaffold cultures as compared with traditional monolayer (MNL) cultures. Cytodex 3 microcarrier-expanded human fetal MSC (hfMSC) cultures (MC-hfMSCs) achieved 12- to 16-fold expansion efficiency (6×105–8×105 cells/mL) compared to 4- to 6-fold (1.2×105–1.8×105 cells/mL) achieved by traditional MNL-expanded hfMSC culture (MNL-hfMSCs; p<0.05). Both MC-hfMSCs and MNL-hfMSCs maintained similar colony-forming capacity, doubling times, and immunophenotype postexpansion. However, when differentiated under in vitro two-dimensional (2D) osteogenic conditions, MC-hfMSCs exhibited a 45-fold reduction in alkaline phosphatase level and a 37.5% decrease in calcium deposition compared with MNL-hfMSCs (p<0.05). Surprisingly, when MC-hfMSCs and MNL-hfMSCs were seeded on 3D macroporous scaffold culture or subcutaneously implanted into nonobese diabetic/severe combined immunodeficient mice, MC-hfMSCs deposited 63.5% (p<0.05) more calcium and formed 47.2% (p<0.05) more bone volume, respectively. These results suggest that the mode of hfMSC growth in the expansion phase affects the osteogenic potential of hfMSCs differently in various differentiation platforms. In conclusion, MC cultures are advantageous over MNL cultures in bone tissue engineering because MC-hfMSCs have improved cell expansion capacity and exhibit higher osteogenic potential than MNL-hfMSCs when seeded in vitro into 3D scaffolds or implanted in vivo. PMID:23593561

  4. 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.

  5. Mechanism of HIV protein induced modulation of mesenchymal stem cell osteogenic differentiation

    PubMed Central

    Cotter, Eoin J; Ip, Herbert Shi Ming; Powderly, William G; Doran, Peter P

    2008-01-01

    Background A high incidence of decreased bone mineral density (BMD) has been associated with HIV infection. Normal skeletal homeostasis is controlled, at least in part, by the maturation and activity of mature osteoblasts. Previous studies by our group have demonstrated the ability of HIV proteins to perturb osteoblast function, and the degree of osteogenesis in differentiating mesenchymal stem cells (MSCs). This study attempts to further dissect the dynamics of this effect. Methods MSCs were cultured under both osteogenic (cultured in commercially available differentiation media) and quiescent (cultured in basal medium) conditions. Both cell populations were exposed to HIV p55-gag and HIV rev (100 ng/ml). Time points were taken at 3, 6, 9, and 15 days for osteogenic conditions, while quiescent cells were treated for 1 week. Cell function (alkaline phosphatase [ALP] activity, calcium deposition, and lipid levels) and the activity of the key MSC transcription factors, RUNX-2 and PPARgamma were determined post-exposure. Also, in cells cultured in differentiating conditions, cellular levels of connective tissue growth factor (CTGF) were analysed using whole cell ELISA, while BMP-2 secretion was also examined. Results In differentiating MSCs, exposure to HIV proteins caused significant changes in both the timing and magnitude of key osteogenic events and signals. Treatment with REV increased the overall rate of mineralization, and induced earlier increases in CTGF levels, RUNX-2 activity and BMP-2 secretion, than those observed in the normal course of differntiation. In contrast, p55-gag reduced the overall level of osteogenesis, and reduced BMP-2 secretion, RUNX-2 activity, CTGF levels and ALP activity at many of the timepoints examined. Finally, in cells cultured in basal conditions, treatment with HIV proteins did not in and of itself induce a significant degree of differentiation over the time period examined. Conclusion These data demonstrate that the effect of

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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

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

    PubMed

    Duttenhoefer, Fabian; de Freitas, Rafael Lara; Loibl, Markus; Bittermann, Gido; Richards, R Geoff; 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

  14. The osteogenic differentiation stimulating activity of Sea cucumber methanolic crude extraction on rat bone marrow mesenchymal stem cells

    PubMed Central

    Baharara, Javad; Amini, Elaheh; Kerachian, Mohammad Amin; Soltani, Mozhgan

    2014-01-01

    Objective(s): Sea cucumber derived bioactive compound is considered efficient in treatment of bone disorders. This study was performed to evaluate the effect of this extract on differentiation of rat bone marrow mesenchymal stem cells (rBMMSc) into osteogenic lineage. Materials and Methods: Isolated rBMMSc were grown in DMEM supplemented with 10% FBS. The cells were exposed to different concentration of extract. After 21 days, Alizarin red staining, alkaline phosphatase assay and RT-PCR were performed. The results were analyzed by ANOVA software and P value <0.05 was considered significant. Results: Morphological methods revealed that appropriate concentrations of extract increased osteogenic differentiation in a dose-dependent manner. RT-PCR revealed that extract without or with osteogenic medium due to osteopontin expression had a potential role in osteogenesis. Conclusion: Based on our data it concluded that S. cucumber extract stimulated Bone marrow mesenchymal cells to differentiate into osteogenic lineage without existence of osteogenic medium. PMID:25422758

  15. 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

  16. The potential functional interaction of biglycan and WISP-1 in controlling differentiation and proliferation of osteogenic cells.

    PubMed

    Inkson, Colette A; Ono, Mitsuaki; Bi, Yanming; Kuznetsov, Sergei A; Fisher, Larry W; Young, Marian F

    2009-01-01

    Biglycan (BGN) and WISP-1 are 2 extracellular matrix proteins that bind to each other and colocalize in mineralizing tissue. Here we show that WISP-1 abrogates the repression of proliferation in bone marrow stromal cells induced by BGN. We also demonstrate that WISP-1 and its variant WISP-1va can alleviate the repressed osteogenic differentiation caused by the absence of BGN. These preliminary data suggest that WISP-1 and BGN may functionally interact and control each other's activity, thus regulating the differentiation and proliferation of osteogenic cells.

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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.

  4. 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

  5. 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

  6. Bone regeneration with osteogenically enhanced mesenchymal stem cells and their extracellular matrix proteins.

    PubMed

    Clough, Bret H; McCarley, Matthew R; Krause, Ulf; Zeitouni, Suzanne; Froese, Jeremiah J; McNeill, Eoin P; Chaput, Christopher D; Sampson, H Wayne; Gregory, Carl A

    2015-01-01

    Although bone has remarkable regenerative capacity, about 10% of long bone fractures and 25% to 40% of vertebral fusion procedures fail to heal. In such instances, a scaffold is employed to bridge the lesion and accommodate osteoprogenitors. Although synthetic bone scaffolds mimic some of the characteristics of bone matrix, their effectiveness can vary because of biological incompatibility. Herein, we demonstrate that a composite prepared with osteogenically enhanced mesenchymal stem cells (OEhMSCs) and their extracellular matrix (ECM) has an unprecedented capacity for the repair of critical-sized defects of murine femora. Furthermore, OEhMSCs do not cause lymphocyte activation, and ECM/OEhMSC composites retain their in vivo efficacy after cryopreservation. Finally, we show that attachment to the ECM by OEhMSCs stimulates the production of osteogenic and angiogenic factors. These data demonstrate that composites of OEhMSCs and their ECM could be utilized in the place of autologous bone graft for complex orthopedic reconstructions.

  7. 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

  8. Directed osteogenic differentiation of human mesenchymal stem/precursor cells on silicate substituted calcium phosphate.

    PubMed

    Cameron, Kate; Travers, Paul; Chander, Chaman; Buckland, Tom; Campion, Charlie; Noble, Brendon

    2013-01-01

    Insufficient, underactive, or inappropriate osteoblast function results in serious clinical conditions such as osteoporosis, osteogenesis imperfecta and fracture nonunion and therefore the control of osteogenesis is a medical priority. In vitro mesenchymal stem cells (MSCs) can be directed to form osteoblasts through the addition of soluble factors such as β-glycerophosphate, ascorbic acid, and dexamethasone; however this is unlikely to be practical in the clinical setting. An alternative approach would be to use a scaffold or matrix engineered to provide cues for differentiation without the need for soluble factors. Here we describe studies using Silicate-substituted calcium phosphate (Si-CaP) and unmodified hydroxyapatite (HA) to test whether these materials are capable of promoting osteogenic differentiation of MSCs in the absence of soluble factors. Si-CaP supported attachment and proliferation of MSCs and induced osteogenesis to a greater extent than HA, as evidenced through upregulation of the osteoblast-related genes: Runx2 (1.2 fold), Col1a1 (2 fold), Pth1r (1.5 fold), and Bglap (1.7 fold) Dmp1 (1.1 fold), respectively. Osteogenic-associated proteins, alkaline phosphatase (1.4 fold), RUNX2, COL1A1, and BGLAP, were also upregulated and there was an increased production of mineralized bone matrix (1.75 fold), as detected by the Von Kossa Assay. These data indicate that inorganic substrates are capable of directing the differentiation programme of stem cells in the absence of known chemical drivers and therefore may provide the basis for bone repair in the clinical setting.

  9. Biomimetic hybrid nanofibrous substrates for mesenchymal stem cells differentiation into osteogenic cells.

    PubMed

    Gandhimathi, Chinnasamy; Venugopal, Jayarama Reddy; Tham, Allister Yingwei; Ramakrishna, Seeram; Kumar, Srinivasan Dinesh

    2015-04-01

    Mimicking native extracellular matrix with electrospun porous bio-composite nanofibrous scaffolds has huge potential in bone tissue regeneration. The aim of this study is to fabricate porous poly(l-lactic acid)-co-poly-(ε-caprolactone)/silk fibroin/ascorbic acid/tetracycline hydrochloride (PLACL/SF/AA/TC) and nanohydroxyapatite (n-HA) was deposited by calcium-phosphate dipping method for bone tissue engineering (BTE). Fabricated nanofibrous scaffolds were characterized for fiber morphology, hydrophilicity, porosity, mechanical test and chemical properties by FT-IR and EDX analysis. The results showed that the fiber diameter and pore size of scaffolds observed around 228±62-320±22nm and 1.5-6.9μm respectively. Resulting nanofibrous scaffolds are highly porous (87-94%) with ultimate tensile strength observed in the range of 1.51-4.86MPa and also showed better hydrophilic properties after addition of AA, TC and n-HA. Human mesenchymal stem cells (MSCs) cultured on these bio-composite nanofibrous scaffolds and stimulated to osteogenic differentiation in the presence of AA/TC/n-HA for BTE. The cell proliferation and biomaterial interactions were studied using MTS assay, SEM and CMFDA dye exclusion methods. Osteogenic differentiation of MSCs was proven by using alkaline phosphatase activity, mineralization and double immunofluorescence staining of both CD90 and osteocalcin. The observed results suggested that the fabricated PLACL/SF/AA/TC/n-HA biocomposite hybrid nanofibrous scaffolds have good potential for the differentiation of MSCs into osteogenesis for bone tissue engineering. PMID:25687008

  10. Inkjet printed periodical micropatterns made of inert alumina ceramics induce contact guidance and stimulate osteogenic differentiation of mesenchymal stromal cells.

    PubMed

    Lauria, Ines; Kramer, Michael; Schröder, Teresa; Kant, Sebastian; Hausmann, Anne; Böke, Frederik; Leube, Rudolf; Telle, Rainer; Fischer, Horst

    2016-10-15

    Bioinert high performance ceramics exhibit detrimental features for implant components with direct bone contact because of their low osseointegrating capability. We hypothesized that periodical microstructures made of inert alumina ceramics can influence the osteogenic differentiation of human mesenchymal stromal cells (hMSC). In this study, we manufactured pillared arrays made of alumina ceramics with periodicities as low as 100μm and pillar heights of 40μm employing direct inkjet printing (DIP) technique. The response of hMSC to the microstructured surfaces was monitored by measuring cell morphology, viability and formation of focal adhesion complexes. Osteogenic differentiation of hMSCs was investigated by alkaline phosphatase activity, mineralization assays and expression analysis of respective markers. We demonstrated that MSCs react to the pillars with contact guidance. Subsequently, cells grow onto and form connections between the microstructures, and at the same time are directly attached to the pillars as shown by focal adhesion stainings. Cells build up tissue-like constructs with heights up to the micropillars resulting in increased cell viability and osteogenic differentiating properties. We conclude that periodical micropatterns on the micrometer scale made of inert alumina ceramics can mediate focal adhesion dependent cell adhesion and stimulate osteogenic differentiation of hMSCs.

  11. 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

  12. miR-21 synergizes with BMP9 in osteogenic differentiation by activating the BMP9/Smad signaling pathway in murine multilineage cells

    PubMed Central

    SONG, QILING; ZHONG, LIANG; CHEN, CHU; TANG, ZUCHUAN; LIU, HONGXIA; ZHOU, YIQIN; TANG, MIN; ZHOU, LAN; ZUO, GUOWEI; LUO, JINYONG; ZHANG, YAN; SHI, QIONG; WENG, YAGUANG

    2015-01-01

    Bone morphogenetic proteins (BMPs), particularly BMP9, have been shown to promote the osteogenic differentiation of murine multilineage cells (MMCs) and to promote bone formation in bone diseases; however, the mechanisms involved remain poorly understood. MicroRNAs (miRNAs or miRs) have been proven to regulate mesenchymal stem cell (MSC) differentiation. In this study, we identified a novel mechanism that unravels the functional axis of a key miRNA (miR-21) which contributes to BMP9-induced osteogenic differentiation. We screened differentially expressed miRNAs in MMCs during BMP9-induced osteogenic differentiation and found that miR-21 was significantly upregulated by BMP9 during the osteogenesis of MMCs. Furthermore, miR-21 was confirmed to promote the osteogenic differentiation of the MMCs by suppressing Smad7, which negatively regulates the osteogenic differentiation of MMCs. The upregulation of miR-21 may promote the osteogenic differentiation of MMCs in synergy with BMP9. The findings of our study revealed a novel function of miR-21, and suggest that the overexpression of miR-21 contributes to bone formation by promoting BMP9-induced osteogenic differentiation. Our data may provide a molecular basis for the development of novel therapeutic strategies to treat bone diseases, such as osteoporosis and other inflammatory bone diseases. PMID:26460584

  13. 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

  14. Application of Green Tea Catechin for Inducing the Osteogenic Differentiation of Human Dedifferentiated Fat Cells in Vitro.

    PubMed

    Kaida, Koji; Honda, Yoshitomo; Hashimoto, Yoshiya; Tanaka, Masahiro; Baba, Shunsuke

    2015-11-25

    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.

  15. 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

  16. 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.

  17. Expansion and osteogenic differentiation of bone marrow-derived mesenchymal stem cells on a vitamin C functionalized polymer.

    PubMed

    Wang, Yongzhong; Singh, Amarjit; Xu, Peng; Pindrus, Mariya A; Blasioli, Dominick J; Kaplan, David L

    2006-06-01

    In human body ascorbic acid plays an essential role in the synthesis and function of skeletal tissues and immune system factors. Ascorbic acid is also a major physiological antioxidant, repairing oxidatively damaged biomolecules, preventing the formation of excessive reactive oxygen species or scavenging these species. We recently reported the synthesis of ascorbic acid-functionalized polymers in which the antioxidant features of the pendant ascorbic acid groups was preserved. In the present work we demonstrate that ascorbic acid-functionalized poly(methyl methacrylate) (AA-PMMA) can modulate the proliferation and osteogenic differentiation of early and late-passage bone marrow-derived human mesenchymal stem cells (MSCs). The covalently coupled ascorbic acid impacted MSCs differently than when ascorbic acid was presented to the cells in soluble form. At optimal concentration, the covalently coupled ascorbic acid and soluble ascorbic acid synergistically promoted and retained the ability of MSCs to respond to osteogenic stimulation over extensive cell expansions in vitro. In the presence of soluble ascorbic acid, AA-PMMA films prepared at optimal concentrations (0.1 mg/ml in the present study) showed a significant promotive effect over other concentrations and tissue culture plastic (TCP) with respect to osteogenic differentiation of both EP (young) and LP (old) MSCs. These results suggest that the coupled ascorbic acid is acting mainly at the extracellular level and, at optimal concentrations, the immobilized extracellular ascorbic acid and soluble ascorbic acid synergistically promote osteogenic differentiation of MSCs. Importantly, the covalently coupled ascorbic acid on the films of optimal concentration was able to preserve the capacity of MSCs to undergo osteogenic differentiation in vitro. These results suggest an important role for functionalized biomaterials with antioxidant features in control of cell physiology and cell aging phenomena.

  18. Osteogenic and osteoclastogenic differentiation of co-cultured cells in polylactic acid-nanohydroxyapatite fiber scaffolds.

    PubMed

    Morelli, Sabrina; Salerno, Simona; Holopainen, Jani; Ritala, Mikko; De Bartolo, Loredana

    2015-06-20

    The design of bone substitutes involves the creation of a microenvironment supporting molecular cross-talk between cells and scaffolds during tissue formation and remodelling. Bone remodelling process includes the cooperation of bone-building cells and bone-resorbing cells. In this paper we developed polylactic acid (PLA) and composite PLA-nanohydroxyapatite (nHA) scaffolds with 20 and 50wt.% of nHA by electrospinning technique to be used in bone tissue engineering. The developed scaffolds have different fiber diameter, porosity with interconnected pores and mechanical properties. Taking cues from the bone environment features we investigated the differentiation of human mesenchymal stem cells (hMSCs) from bone marrow in osteoblasts and the osteoclastogenesis in the developed scaffolds in homotypic and in co-culture up to 46 days. PLA and composite PLA-nHA scaffolds induced osteogenic and osteoclastogenic differentiation. Both osteoblasts and osteoclasts displayed high expression of specific markers (osteopontin, osteocalcin, RANK, RANKL) and functions such as secretion of ALP, cathepsin K and TRAP activity on composite scaffolds especially on PLA-nHA containing 20wt.% of nHA. The heterotypic interactions between osteoblasts and osteoclasts co-cultured in the developed scaffolds triggered their functional differentiation and activation.

  19. 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

  20. Shockwaves induce osteogenic differentiation of human mesenchymal stem cells through ATP release and activation of P2X7 receptors.

    PubMed

    Sun, Dahui; Junger, Wolfgang G; Yuan, Changji; Zhang, Wenyan; Bao, Yi; Qin, Daming; Wang, Chengxue; Tan, Lei; Qi, Baochang; Zhu, Dong; Zhang, Xizheng; Yu, Tiecheng

    2013-06-01

    Shockwave treatment promotes bone healing of nonunion fractures. In this study, we investigated whether this effect could be due to adenosine 5'-triphosphate (ATP) release-induced differentiation of human mesenchymal stem cells (hMSCs) into osteoprogenitor cells. Cultured bone marrow-derived hMSCs were subjected to shockwave treatment and ATP release was assessed. Osteogenic differentiation and mineralization of hMSCs were evaluated by examining alkaline phosphatase activity, osteocalcin production, and calcium nodule formation. Expression of P2X7 receptors and c-fos and c-jun mRNA was determined with real-time reverse transcription polymerase chain reaction and Western blotting. P2X7-siRNA, apyrase, P2 receptor antagonists, and p38 MAPK inhibitors were used to evaluate the roles of ATP release, P2X7 receptors, and p38 MAPK signaling in shockwave-induced osteogenic hMSCs differentiation. Shockwave treatment released significant amounts (≈ 7 μM) of ATP from hMSCs. Shockwaves and exogenous ATP induced c-fos and c-jun mRNA transcription, p38 MAPK activation, and hMSC differentiation. Removal of ATP with apyrase, targeting of P2X7 receptors with P2X7-siRNA or selective antagonists, or blockade of p38 MAPK with SB203580 prevented osteogenic differentiation of hMSCs. Our findings indicate that shockwaves release cellular ATP that activates P2X7 receptors and downstream signaling events that caused osteogenic differentiation of hMSCs. We conclude that shockwave therapy promotes bone healing through P2X7 receptor signaling, which contributes to hMSC differentiation.

  1. Shockwaves Induce Osteogenic Differentiation of Human Mesenchymal Stem Cells Through ATP Release and Activation of P2X7 Receptors

    PubMed Central

    Sun, Dahui; Junger, Wolfgang G.; Yuan, Changji; Zhang, Wenyan; Bao, Yi; Qin, Daming; Wang, Chengxue; Tan, Lei; Qi, Baochang; Zhu, Dong; Zhang, Xizheng; Yu, Tiecheng

    2014-01-01

    Shockwave fractures treatment promotes bone healing of nonunion fractures. In this study, we investigated whether this effect could be due to adenosine 5’-triphosphate (ATP) release-induced differentiation of human mesenchymal stem cells (hMSCs) into osteoprogenitor cells. Cultured bone marrow-derived hMSCs were subjected to shockwave treatment and ATP release was assessed. Osteogenic differentiation and mineralization of hMSCs were evaluated by examining alkaline phosphatase activity, osteocalcin production, and calcium nodule formation. Expression of P2X7 receptors and c-fos and c-jun mRNA was determined with real-time reverse transcription polymerase chain reaction and Western blotting. P2X7-siRNA, apyrase, P2 receptor antagonists, and p38 MAPK inhibitors were used to evaluate the roles of ATP release, P2X7 receptors, and p38 MAPK sig naling in shockwave-induced osteogenic hMSCs differentiation. Shockwave treatment released significant amounts (~7 μM) of ATP from hMSCs. Shockwaves and exogenous ATP induced c-fos and c-jun mRNA transcription, p38 MAPK activation, and hMSC differentiation. Removal of ATP with apyrase, targeting of P2X7 receptors with P2X7-siRNA or selective antagonists, or blockade of p38 MAPK with SB203580 prevented osteogenic differentiation of hMSCs. Our findings indicate that shockwaves release cellular ATP that activates P2X7 receptors and downstream signaling events that caused osteogenic differentiation of hMSCs. We conclude that shockwave therapy promotes bone healing through P2X7 receptor signaling, which contributes to hMSC differentiation. PMID:23404811

  2. Deleterious effects of freezing on osteogenic differentiation of human adipose-derived stromal cells in vitro and in vivo.

    PubMed

    James, Aaron W; Levi, Benjamin; Nelson, Emily R; Peng, Michelle; Commons, George W; Lee, Min; Wu, Benjamin; Longaker, Michael T

    2011-03-01

    Human adipose-derived stromal cells (hASCs) represent a multipotent stromal cell type with a proven capacity to undergo osteogenic differentiation. Many hurdles exist, however, between current knowledge of hASC osteogenesis and their potential future use in skeletal tissue regeneration. The impact of frozen storage on hASC osteogenic differentiation, for example, has not been studied in detail. To examine the effects of frozen storage, hASCs were harvested from lipoaspirate and either maintained in standard culture conditions or frozen for 2 weeks under standard conditions (90% fetal bovine serum, 10% dimethyl sulfoxide). Next, in vitro parameters of cell morphology (surface electron microscopy [EM]), cell viability and growth (trypan blue; bromodeoxyuridine incorporation), osteogenic differentiation (alkaline phosphatase, alizarin red, and quantitative real-time (RT)-polymerase chain reaction), and adipogenic differentiation (Oil red O staining and quantitative RT-polymerase chain reaction) were performed. Finally, in vivo bone formation was assessed using a critical-sized cranial defect in athymic mice, utilizing a hydroxyapatite (HA)-poly(lactic-co-glycolic acid) scaffold for ASC delivery. Healing was assessed by serial microcomputed tomography scans and histology. Freshly derived ASCs differed significantly from freeze-thaw ASCs in all markers examined. Surface EM showed distinct differences in cellular morphology. Proliferation, and osteogenic and adipogenic differentiation were all significantly hampered by the freeze-thaw process in vitro (*P < 0.01). In vivo, near complete healing was observed among calvarial defects engrafted with fresh hASCs. This was in comparison to groups engrafted with freeze-thaw hASCs that showed little healing (*P < 0.01). Finally, recombinant insulin-like growth factor 1 or recombinant bone morphogenetic protein 4 was observed to increase or rescue in vitro osteogenic differentiation among frozen hASCs (*P < 0.01). The

  3. 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

  4. Collagen/Wollastonite nanowire hybrid scaffolds promoting osteogenic differentiation and angiogenic factor expression of mesenchymal stem cells.

    PubMed

    Zhang, Qin; Nakamoto, Tomoko; Chen, Shangwu; Kawazoe, Naoki; Lin, Kaili; Chang, Jiang; Chen, Guoping

    2014-04-01

    Porous materials and scaffolds have wide applications in biomedical and biological fields. They can provide biological and physical cues to promote cell adhesion, proliferation, differentiation and extracellular matrix secretion to guide new tissue formation. Hybrid scaffolds of collagen and wollastonite nanowires with well controlled pore structures were prepared by using ice particulates as a porogen material. The hybrid scaffolds had interconnected large spherical pores with wollastonite nanowires embedded in the walls of the pores. The wollastonite nanowires reinforced the hybrid scaffolds and showed some stimulatory effects on cell functions. Human bone marrow-derived mesenchymal stem cells showed higher proliferation and osteogenic differentiation and expressed higher level of genes encoding angiogenesis-related genes in the hybrid scaffolds than did in the collagen scaf-. fold. The results suggest the hybrid scaffolds could facilitate osteogenic differentiation and induce angiogenesis and will be useful for bone tissue engineering. PMID:24734758

  5. 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

  6. Osteogenic differentiation and proliferation of bone marrow-derived mesenchymal stromal cells on PDLLA + BMP-2-coated titanium alloy surfaces.

    PubMed

    Haversath, Marcel; Hülsen, Tobias; Böge, Carolin; Tassemeier, Tjark; Landgraeber, Stefan; Herten, Monika; Warwas, Sebastian; Krauspe, Rüdiger; Jäger, Marcus

    2016-01-01

    RhBMP-2 is clinically applied to enhance bone healing and used in combination with titanium fixation implants. The purpose of this in vitro study was to compare the osteogenic differentiation and proliferation of hMSC on native polished versus sandblasted titanium surfaces (TS) and to test their behavior on pure poly-D,L-lactide (PDLLA) coated as well as PDLLA + rhBMP-2 coated TS. Furthermore, the release kinetics of PDLLA + rhBMP-2-coated TS was investigated. Human bone marrow cells were obtained from three different donors (A: male, 16 yrs; B: male, 27 yrs, C: male, 49 yrs) followed by density gradient centrifugation and flow cytometry with defined antigens. The cells were seeded on native polished and sandblasted TS, PDLLA-coated TS and PDLLA + rhBMP-2-coated TS. Osteogenic differentiation (ALP specific activity via ALP and BCA assay) and proliferation (LDH cytotoxicity assay) was examined on day 7 and 14 and release kinetics of rhBMP-2 was investigated on day 3, 7, 10, and 14. We found significant higher ALP specific activity and LDH activity on native polished compared to native sandblasted surfaces. PDLLA led to decreased ALP specific and LDH activity on both surface finishes. Additional rhBMP-2 slightly diminished this effect. RhBMP-2-release from coated TS decreased nearly exponentially with highest concentrations at the beginning of the cultivation period. The results of this in vitro study suggest that native TS stimulate hMSC significantly stronger toward osteogenic differentiation and proliferation than rhBMP-2 + PDLLA-layered TS in the first 14 days of cultivation. The PDLLA-layer seems to inhibit local hMSC differentiation and proliferation.

  7. 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

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

    PubMed

    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.

  9. In vitro osteogenic differentiation of HOS cells on two types of collagen gels.

    PubMed

    Takitoh, Takako; Kato, Yoichi; Nakasu, Asako; Tadokoro, Mika; Bessho, Masahiko; Hirose, Motohiro; Ohgushi, Hajime; Mori, Hideki; Hara, Masayuki

    2010-10-01

    HOS cell is a model strain of human osteoblasts derived from human osteosarcoma. We cultured the HOS cells on both the conventional collagen gel (neutral gel), and the gamma-crosslinked collagen gel without collagen fibrils (acidic gel). The shape of HOS cells on the neutral gel was similar to that on the culture dish. However, HOS cells on acidic gel had an elongated shape and attached each other to form a mesh-like pattern. The cells attached to the surface of both gels but scarcely penetrated their depths. We measured the biochemical markers for osteogenic differentiation in the HOS cells cultured on both the neutral gel and the acidic gel. The expressions of alkaline phosphatase and osteocalcin were detected in the HOS cells on both types of collagen gel. Deposition of the calcium also occurred on both gels although it was higher in the neutral gel than the acidic one. These results indicate the importance of collagen for the differentiation of HOS cells, but it is not dependent on the molecular structure (fibril formation) of collagen.

  10. 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

  11. 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

  12. Proliferation and osteogenic differentiation of human bone marrow stromal cells on alginate-gelatine-hydroxyapatite scaffolds with anisotropic pore structure.

    PubMed

    Bernhardt, A; Despang, F; Lode, A; Demmler, A; Hanke, T; Gelinsky, M

    2009-01-01

    Porous mineralized scaffolds are required for various applications in bone engineering. In particular, tube-like pores with controlled orientation inside the scaffold may support homogeneous cell seeding as well as sufficient nutrient supply and may facilitate blood vessel ingrowth. Scaffolds with parallely orientated tube-like pores were generated by diffusion-controlled ionotropic gelation of alginate. Incorporation of hydroxyapatite (HA) during the gelation process yielded stable scaffolds with an average pore diameter of approximately 90 microm. To evaluate the potential use of alginate-gelatine-HA scaffolds for bone tissue engineering, in vitro tests with human bone marrow stromal cells (hBMSCs) were carried out. We analysed biocompatibility and cell penetration into the capillary pores by microscopic methods. hBMSCs were also cultivated on alginate-gelatine-HA scaffolds for 3 weeks in the presence and absence of osteogenic supplements. We studied proliferation and osteogenic differentiation in terms of total lactate dehydrogenase (LDH) activity, DNA content and alkaline phosphatase (ALP) activity and found a 10-14-fold increase of cell number after 2 weeks of cultivation, as well as an increase of specific ALP activity for osteogenic-induced hBMSCs. Furthermore, the expression of bone-related genes [ALP, bone sialoprotein II (BSPII)] was analysed. We found an increase of ALP as well as BSPII expression for osteogenic-induced hBMSCs on alginate-gelatin-HA scaffolds.

  13. 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

  14. Methods for analyzing microRNA expression and function during osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells.

    PubMed

    Kim, Yeon Jeong; Jung, Jin Sup

    2011-01-01

    MicroRNAs (miRNA) are single-stranded RNA molecules of 21-23 nucleotides in length that regulate gene expression at the posttranscriptional level. They may play important roles during osteogenic differentiation of adipose tissue-derived mesenchymal stem cells (hASC). In this chapter, we focus on the methods and strategies for elucidating miRNA function during osteogenic differentiation. We describe a miRNA expression analysis protocol, and a lentiviral vector strategy for the ectopic expression of miRNA in hASC to determine the role of miRNA during osteogenic differentiation. We also describe miRNA inhibition to further determine the role of miRNA during osteogenic differentiation, and a luciferase assay to demonstrate direct binding between a specific miRNA and its putative target.

  15. 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.

  16. 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

  17. Influence of platelet-rich plasma on osteogenic differentiation of mesenchymal stem cells and ectopic bone formation in calcium phosphate ceramics.

    PubMed

    Kasten, Philip; Vogel, Julia; Luginbühl, Reto; Niemeyer, Philipp; Weiss, Stefan; Schneider, Sven; Kramer, Martin; Leo, Albrecht; Richter, Wiltrud

    2006-01-01

    Platelet-rich plasma (PRP) contains a mixture of growth factors that play an important role in wound and fracture healing. While PRP enhanced bone formation by autogenous cancellous bone grafts, its influence in combination with different bone substitutes remained unknown. This study evaluated the effect of PRP on osteogenic differentiation and ectopic bone formation of human mesenchymal stem cells (MSC) in distinct resorbable calcium phosphate ceramics. Calcium-deficient hydroxyapatite (CDHA) blocks with a large specific surface area (48 m2/g) and beta-tricalcium phosphate (beta-TCP) with a low specific surface area (<0.5 m2/g) were loaded with 2 x 10(5) bone marrow-derived MSC. Half of the specimens were treated with 5-fold concentrated PRP. Biocomposites were implanted subcutaneously into SCID mice or kept under osteogenic culture conditions for 2 weeks before implantation. The addition of PRP increased the specific alkaline phosphatase (ALP) activity (p = 0.012) in undifferentiated MSC/CDHA composites but not in MSC/beta-TCP composites. Osteogenic preinduction was ineffective for CDHA and reduced ALP activity of beta-TCP composites significantly at explantation. Ectopic bone formation was stronger in MSC/CDHA (7/32) compared to MSC/beta-TCP (2/30) composites, but no influence of PRP was evident. In conclusion, the effect of PRP depended on the type of ceramic and the differentiation status of the MSC, and enhanced ALP activity of MSC on the high surface scaffold CDHA only, but PRP did not improve osteogenesis in our setting.

  18. 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.

  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. 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

  1. 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

  2. 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

  3. 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.

  4. Famotidine suppresses osteogenic differentiation of tendon cells in vitro and pathological calcification of tendon in vivo.

    PubMed

    Yamamoto, Kenichi; Hojo, Hironori; Koshima, Isao; Chung, Ung-il; Ohba, Shinsuke

    2012-12-01

    Heterotopic ossification or calcification follows any type of musculoskeletal trauma and is known to occur after arthroplasties of hip, knee, shoulder, or elbow; fractures; joint dislocations; or tendon ruptures. Histamine receptor H2 (Hrh2) has been shown to be effective for reducing pain and decreasing calcification in patients with calcifying tendinitis, which suggested that H2 blockers were effective for the treatment of tendon ossification or calcification. However, the detailed mechanisms of its action on tendon remain to be clarified. We investigated the mechanisms underlying H2 blocker-mediated suppression of tendon calcification, with a focus on the direct action of the drug on tendon cells. Famotidine treatment suppressed the mRNA expressions of Col10a1 and osteocalcin, ossification markers, in a tendon-derived cell line TT-D6, as well as a preosteoblastic one MC3T3-E1. Both of the cell lines expressed Hrh2; histamine treatment induced osteocalcin expression in these cells. Famotidine administration suppressed calcification in the Achilles tendon of ttw mice, a mouse model of ectopic ossification. These data suggest that famotidine inhibits osteogenic differentiation of tendon cells in vitro, and this inhibition may underlie the anti-calcification effects of the drug in vivo. This study points to the use of H2 blockers as a promising strategy for treating heterotopic ossification or calcification in tendon, and provides evidence in support of the clinical use of famotidine.

  5. Azelnidipine inhibits Msx2-dependent osteogenic differentiation and matrix mineralization of vascular smooth muscle cells.

    PubMed

    Shimizu, Takehisa; Tanaka, Toru; Iso, Tatsuya; Kawai-Kowase, Keiko; Kurabayashi, Masahiko

    2012-01-01

    Vascular calcification is an active and regulated process that is similar to bone formation. While calcium channel blockers (CCBs) have been shown to improve outcomes in atherosclerotic vascular disease, it remains unknown whether CCBs have an effect on the process of vascular calcification. Here we investigated whether CCBs inhibit osteogenic differentiation and matrix mineralization of vascular smooth muscle cells induced by Msx2, a key factor of vascular calcification. Human aortic smooth muscle cells (HASMCs) were transduced with adenovirus expressing MSX2 and were treated with 3 distinct CCBs. Azelnidipine, a dihydropyridine subclass of CCBs, significantly decreased alkaline phosphatase (ALP) activity of Msx2-overexpressed HASMCs, whereas verapamil and diltiazem had no effect. Furthermore, azelnidipine, but not verapamil and diltiazem, significantly decreased matrix mineralization of Msx2-overexpressing HASMCs. Azelnidipine significantly attenuated the induction of ALP gene expression by Msx2, a key transcription factor in osteogenesis, while it did not reduce enzymatic activity of ALP. Furthermore, azelnidipine inhibited the ability of Msx2 to activate the ALP gene, but had no effect on Notch-induced Msx2 expression. Given that L-type calcium channels are equally blocked by these CCBs, our results suggest that azelnidipine inhibits the Msx2-dependent process of vascular calcification by mechanisms other than inhibition of calcium channel activity.

  6. 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)

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

    PubMed

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

    2015-01-01

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

  8. 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

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

    PubMed

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

    2015-01-01

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

  10. 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

  11. 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

  12. Extracellular Purines Promote the Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells to the Osteogenic and Adipogenic Lineages

    PubMed Central

    Zini, Roberta; Rossi, Lara; Salvestrini, Valentina; Ferrari, Davide; Manfredini, Rossella; Lemoli, Roberto M.

    2013-01-01

    Extracellular nucleotides are potent signaling molecules mediating cell-specific biological functions, mostly within the processes of tissue damage and repair and flogosis. We previously demonstrated that adenosine 5′-triphosphate (ATP) inhibits the proliferation of human bone marrow-derived mesenchymal stem cells (BM-hMSCs), while stimulating, in vitro and in vivo, their migration. Here, we investigated the effects of ATP on BM-hMSC differentiation capacity. Molecular analysis showed that ATP treatment modulated the expression of several genes governing adipogenic and osteoblastic (ie, WNT-pathway-related genes) differentiation of MSCs. Functional studies demonstrated that ATP, under specific culture conditions, stimulated adipogenesis by significantly increasing the lipid accumulation and the expression levels of the adipogenic master gene PPARγ (peroxisome proliferator-activated receptor-gamma). In addition, ATP stimulated osteogenic differentiation by promoting mineralization and expression of the osteoblast-related gene RUNX2 (runt-related transcription factor 2). Furthermore, we demonstrated that ATP stimulated adipogenesis via its triphosphate form, while osteogenic differentiation was induced by the nucleoside adenosine, resulting from ATP degradation induced by CD39 and CD73 ectonucleotidases expressed on the MSC membrane. The pharmacological profile of P2 purinergic receptors (P2Rs) suggests that adipogenic differentiation is mainly mediated by the engagement of P2Y1 and P2Y4 receptors, while stimulation of the P1R adenosine-specific subtype A2B is involved in adenosine-induced osteogenic differentiation. Thus, we provide new insights into molecular regulation of MSC differentiation. PMID:23259837

  13. 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.

  14. 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

  15. Histone Acetyltransferase GCN5 Regulates Osteogenic Differentiation of Mesenchymal Stem Cells by Inhibiting NF-κB.

    PubMed

    Zhang, Ping; Liu, Yunsong; Jin, Chanyuan; Zhang, Min; Tang, Fuchou; Zhou, Yongsheng

    2016-02-01

    As the most well-studied histone acetyltransferase (HAT) in yeast and mammals, general control nonderepressible 5 (GCN5) was documented to play essential roles in various developmental processes. However, little is known about its role in osteogenic differentiation of mesenchymal stem cells (MSCs). Here, we detected the critical function of GCN5 in osteogenic commitment of MSCs. In this role, the HAT activity of GCN5 was not required. Mechanistically, GCN5 repressed nuclear factor kappa B (NF-κB)-dependent transcription and inhibited the NF-κB signaling pathway. The impaired osteogenic differentiation by GCN5 knockdown was blocked by inhibition of NF-κB. Most importantly, the expression of GCN5 was decreased significantly in the bone tissue sections of ovariectomized mice or aged mice. Collectively, these results may point to the GCN5-NF-κB pathway as a novel potential molecular target for stem cell mediated regenerative medicine and the treatment of metabolic bone diseases such as osteoporosis.

  16. 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

  17. Effects of hydroxyapatite nanostructure on channel surface of porcine acellular dermal matrix scaffold on cell viability and osteogenic differentiation of human periodontal ligament stem cells.

    PubMed

    Ge, Shaohua; Zhao, Ning; Wang, Lu; Liu, Hong; Yang, Pishan

    2013-01-01

    A new nanostructured hydroxyapatite-coated porcine acellular dermal matrix (HAp-PADM) was fabricated by a biomimetic mineralization method. Human periodontal ligament stem cells were seeded on HAp-PADM and the effects of this scaffold on cell shape, cytoskeleton organization, cell viability, and osteogenic differentiation were examined. Periodontal ligament stem cells cultured on HAp-PADM exhibited different cell shape when compared with those on pure PADM. Moreover, HAp-PADM promoted cell viability and alkaline phosphatase activity significantly. Based on quantitative real-time polymerase chain reaction, the expression of bone-related markers runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteocalcin (OCN) upregulated in the HAp-PADM scaffold. The enhancement of osteogenic differentiation of periodontal ligament stem cells on the HAp-PADM scaffold was proposed based on the research results. The results of this study highlight the micro-nano, two-level, three-dimensional HAp-PADM composite as a promising scaffold for periodontal tissue engineering.

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

    2016-04-01

    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

  3. 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

  4. 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

  5. Altered Membrane Dynamics of Quantum Dot-Conjugated Integrins during Osteogenic Differentiation of Human Bone Marrow Derived Progenitor Cells

    PubMed Central

    Chen, Hongfeng; Titushkin, Igor; Stroscio, Michael; Cho, Michael

    2007-01-01

    Functionalized quantum dots offer several advantages for tracking the motion of individual molecules on the cell surface, including selective binding, precise optical identification of cell surface molecules, and detailed examination of the molecular motion without photobleaching. We have used quantum dots conjugated with integrin antibodies and performed studies to quantitatively demonstrate changes in the integrin dynamics during osteogenic differentiation of human bone marrow derived progenitor cells (BMPCs). Consistent with the unusually strong BMPC adhesion previously observed, integrins on the surface of undifferentiated BMPC were found in clusters and the lateral diffusion was slow (e.g., ∼10−11 cm2/s). At times as early as those after a 3-day incubation in the osteogenic differentiation media, the integrin diffusion coefficients increased by an order of magnitude, and the integrin dynamics became indistinguishable from that measured on the surface of terminally differentiated human osteoblasts. Furthermore, microfilaments in BMPCs consisted of atypically thick bundles of stress fibers that were responsible for restricting the integrin lateral mobility. Studies using laser optical tweezers showed that, unlike fully differentiated osteoblasts, the BMPC cytoskeleton is weakly associated with its cell membrane. Based on these findings, it appears likely that the altered integrin dynamics is correlated with BMPC differentiation and that the integrin lateral mobility is restricted by direct links to microfilaments. PMID:17114225

  6. Altered membrane dynamics of quantum dot-conjugated integrins during osteogenic differentiation of human bone marrow derived progenitor cells.

    PubMed

    Chen, Hongfeng; Titushkin, Igor; Stroscio, Michael; Cho, Michael

    2007-02-15

    Functionalized quantum dots offer several advantages for tracking the motion of individual molecules on the cell surface, including selective binding, precise optical identification of cell surface molecules, and detailed examination of the molecular motion without photobleaching. We have used quantum dots conjugated with integrin antibodies and performed studies to quantitatively demonstrate changes in the integrin dynamics during osteogenic differentiation of human bone marrow derived progenitor cells (BMPCs). Consistent with the unusually strong BMPC adhesion previously observed, integrins on the surface of undifferentiated BMPC were found in clusters and the lateral diffusion was slow (e.g., approximately 10(-11) cm2/s). At times as early as those after a 3-day incubation in the osteogenic differentiation media, the integrin diffusion coefficients increased by an order of magnitude, and the integrin dynamics became indistinguishable from that measured on the surface of terminally differentiated human osteoblasts. Furthermore, microfilaments in BMPCs consisted of atypically thick bundles of stress fibers that were responsible for restricting the integrin lateral mobility. Studies using laser optical tweezers showed that, unlike fully differentiated osteoblasts, the BMPC cytoskeleton is weakly associated with its cell membrane. Based on these findings, it appears likely that the altered integrin dynamics is correlated with BMPC differentiation and that the integrin lateral mobility is restricted by direct links to microfilaments.

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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

  13. Effect of advanced oxidation protein products on the proliferation and osteogenic differentiation of rat mesenchymal stem cells.

    PubMed

    Sun, Nan; Yang, Li; Li, Yingbin; Zhang, Hua; Chen, Hong; Liu, Duan; Li, Qingnan; Cai, Dehong

    2013-08-01

    Advanced oxidation protein products (AOPPs) as a novel marker of oxidative stress, are involved in a variety of diseases, including osteoporosis. Although a number of studies have shown the possible functions of AOPPs in biological processes, little is known about the role of AOPPs in the pathogenesis of osteoporosis. In this study, we aimed to investigate the effect of AOPPs on the proliferation and osteogenic differentiation of rat mesenchymal stem cells (MSCs). MSCs, isolated from bone marrow, were cultured in the absence or presence of AOPPs (50, 100, 200 and 400 mg/ml). MTT assay was used to determine the proliferative ability of the cells. Alkaline phosphatase (ALP) activity, the mRNA expression of ALP and collagen I and bone nodule formation were detected to assess osteogenic differentiation. Reactive oxygen species (ROS) generation was analyzed with the probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The expression of receptor of advanced glycation end-products (RAGE) at the mRNA and protein level was detected by real-time PCR and western blot analysis, respectively. Compared with the control group, AOPPs inhibited MSC proliferation in a dose- and time-dependent manner. Moreover, AOPPs induced a significant reduction in ALP activity, as well as a decrease in ALP and collagen I mRNA levels in the MSCs; bone nodule formation was also inhibited. Furthermore, AOPPs increased ROS generation in the MSCs, and upregulated the expression of RAGE at the mRNA and protein level. These results suggest that AOPPs inhibit the proliferation and osteogenic differentiation of MSCs, possibly through the AOPPs-RAGE-ROS pathway; this may be an important mechanism in the development of osteoporosis.

  14. 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

  15. 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.

  16. Osteogenic differentiation of human mesenchymal stem cells on α5 integrin binding peptide hydrogels is dependent on substrate elasticity

    PubMed Central

    Gandavarapu, Navakanth R.; Alge, Daniel L.; Anseth, Kristi S.

    2014-01-01

    The extracellular matrix plays a crucial role in controlling human mesenchymal stem cell (hMSC) biology including differentiation, and α5β1 integrin signaling plays an important role during osteogenic differentiation of hMSCs. Here, peptide-functionalized hydrogels were used to examine the role of α5β1 integrin signaling in inducing osteogenic differentiation in hMSCs. Further, the role of substrate elasticity was also studied. A thiolene chemistry was used to functionalize poly(ethylene glycol) hydrogels with a pendant peptide moieity, c(RRETAWA), as previous studies have shown that RRETAWA containing peptides bind to the α5β1 integrin with very high specificity. Notably, hMSC attachment to c(RRETAWA)-functionalized hydrogels was found to occur primarily through α5 integrins, as the number of attached cells was significantly reduced to ~20% upon blocking the α5 integrin during culture. To investigate the interplay between stiffness and c(RRETAWA) concentration, hydrogels were formulated with Young’s moduli of ~2 kPa (soft) and ~25 kPa (stiff) and c(RRETAWA) concentrations of 0.1 mM and 1 mM. Stiff substrates led to ~3.5 fold higher hMSC attachment and ~3 fold higher cell area in comparison to soft substrates. hMSCs formed robust and larger focal adhesions on stiff substrates at 1 mM c(RRETAWA) compared to soft substrates. Alkaline phosphatase (ALP) activity in hMSCs cultured on stiff gels at 0.1 mM and 1 mM c(RRETAWA) was increased 2.5 and 3.5 fold, respectively after 14 days in growth media. hMSCs did not show an increase in ALP activity when cultured on soft gels. Further, gene expression of osteogenic related genes, core binding factor-1, osteopontin and Collagen-1a at day 14 in hMSCs cultured on stiff gels at 1 mM c(RRETAWA) were increased 10, 7 and 4 fold, respectively, while on soft gels, gene expression was at basal levels. Collectively, these results demonstrate that the combination of high substrate stiffness and α5β1 integrin signaling

  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.

  18. Osteogenic differentiation of stem cells from human exfoliated deciduous teeth on poly(ε-caprolactone) nanofibers containing strontium phosphate.

    PubMed

    Su, Wen-Ta; Wu, Pai-Shuen; Huang, Te-Yang

    2015-01-01

    Mimicking the architecture of the extracellular matrix is an effective strategy for tissue engineering. Composite nanofibers similar to natural bone structure can be prepared via an electrospinning technique and used in biomedical applications. Stem cells from human exfoliated deciduous teeth (SHEDs) can differentiate into multiple cell lineages, such as cells that are alternative sources of stem cells for tissue engineering. Strontium has important functions in bone remodeling; for example, this element can simulate bone formation and decrease bone resorption. Incorporating strontium phosphate into nanofibers provides a potential material for bone tissue engineering. This study investigated the potential of poly(ε-caprolactone) (PCL) nanofibers coated or blended with strontium phosphate for the osteogenic differentiation of SHEDs. Cellular morphology and MTT assay revealed that nanofibers effectively support cellular attachment, spreading, and proliferation. Strontium-loaded PCL nanofibers exhibited higher expressions of collagen type I, alkaline phosphatase, biomineralization, and bone-related genes than pure PCL nanofibers during the osteogenic differentiation of SHEDs. This study demonstrated that strontium can be an effective inducer of osteogenesis for SHEDs. Understanding the function of bioceramics (such as strontium) is useful in designing and developing strategies for bone tissue engineering.

  19. 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

  20. 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

  1. 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

  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. 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).

  4. 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

  5. Runx1 and Runx3 Are Downstream Effectors of Nanog in Promoting Osteogenic Differentiation of the Mouse Mesenchymal Cell Line C3H10T1/2.

    PubMed

    Saito, Tadahito; Ohba, Shinsuke; Yano, Fumiko; Seto, Ichiro; Yonehara, Yoshiyuki; Takato, Tsuyoshi; Ogasawara, Toru

    2015-06-01

    Previously, we reported that the transcription factor Nanog, which maintains the self-renewal of embryonic stem cells (ESCs), promotes the osteogenic differentiation of the mouse mesenchymal cell line C3H10T1/2 through a genome reprogramming process. In the present study, to clarify the mechanism underlying the multipotency of mesenchymal stem cells (MSCs) and to develop a novel approach to bone regenerative medicine, we attempted to identify the downstream effectors of Nanog in promoting osteogenic differentiation of mouse mesenchymal cells. We demonstrated that Runx1 and Runx3 are the downstream effectors of Nanog, especially in the early and intermediate osteogenic differentiation of the mouse mesenchymal cell line C3H10T1/2. PMID:26053522

  6. 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

  7. 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

  8. Loss of the Osteogenic Differentiation Potential during Senescence Is Limited to Bone Progenitor Cells and Is Dependent on p53

    PubMed Central

    Despars, Geneviève; Carbonneau, Cynthia L.; Bardeau, Pascal; Coutu, Daniel L.; Beauséjour, Christian M.

    2013-01-01

    DNA damage can lead to the induction of cellular senescence. In particular, we showed that exposure to ionizing radiation (IR) leads to the senescence of bone marrow-derived multipotent stromal cells (MSC) and osteoblast-like stromal cells (OB–SC), a phenotype associated with bone loss. The mechanism by which IR leads to bone dysfunction is not fully understood. One possibility involves that DNA damage-induced senescence limits the regeneration of bone progenitor cells. Another possibility entails that bone dysfunction arises from the inability of accumulating senescent cells to fulfill their physiological function. Indeed, we show here that exposure to IR prevented the differentiation and mineralization functions of MSC, an effect we found was limited to this population as more differentiated OB–SC could still form mineralize nodules. This is in contrast to adipogenesis, which was inhibited in both IR-induced senescent MSC and 3T3-L1 pre-adipocytes. Furthermore, we demonstrate that IR-induced loss of osteogenic potential in MSC was p53-dependent, a phenotype that correlates with the inability to upregulate key osteogenic transcription factors. These results are the first to demonstrate that senescence impacts osteogenesis in a cell type dependent manner and suggest that the accumulation of senescent osteoblasts is unlikely to significantly contribute to bone dysfunction in a cell autonomous manner. PMID:24009740

  9. Osteogenic differentiation as a result of BMP-2 plasmid DNA based gene therapy in vitro and in vivo.

    PubMed

    Wegman, F; Bijenhof, A; Schuijff, L; Oner, F C; Dhert, W J A; Alblas, J

    2011-03-15

    Bone regeneration is one of the major focus points in the field of regenerative medicine. A well-known stimulus of bone formation is bone morphogenetic protein-2 (BMP-2), which has already been extensively used in clinical applications. We investigated the possibility of achieving osteogenic differentiation both in vitro and in vivo as a result of prolonged presence of BMP-2 using plasmid DNA-based gene therapy. By delivering BMP-2 cDNA in an alginate hydrogel, a versatile formulation is developed. High transfection efficiencies of up to 95% were obtained in both human multipotent stromal cells (MSCs) and MG-63 cells using naked DNA in vitro. Over a period of 5 weeks, an increasing amount of biologically active BMP-2 was released from the cells and remained present in the gel. In vivo, transfected cells were found after both two and six weeks implantation in naked mice, even in groups without seeded cells, thus indicating in vivo transfection of endogenous cells. The protein levels were effective in inducing osteogenic differentiation in vitro, as seen by elevated alkaline phosphatase (ALP) production and in vivo, as demonstrated by the production of collagen I and osteocalcin in a mineralised alginate matrix. We conclude that BMP-2 cDNA incorporated in alginate hydrogel appears to be a promising new strategy for minimal-invasive delivery of growth factors in bone regeneration.

  10. 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

  11. 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.

  12. 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

  13. 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

    2016-04-12

    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.

  14. 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.

  15. 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

  16. 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

  17. 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

  18. Gene expression patterns related to osteogenic differentiation of bone marrow-derived mesenchymal stem cells during ex vivo expansion.

    PubMed

    Granchi, Donatella; Ochoa, Gorka; Leonardi, Elisa; Devescovi, Valentina; Baglìo, Serena Rubina; Osaba, Lourdes; Baldini, Nicola; Ciapetti, Gabriela

    2010-06-01

    Bone marrow is commonly used as a source of adult multipotent mesenchymal stem cells (MSCs), defined for their ability to differentiate in vitro into multiple lineages. The ex vivo-expanded MSCs are currently being evaluated as a strategy for the restoration of function in damaged skeletal tissue, both in cell therapy and tissue engineering applications. The aim of this study was to define gene expression patterns underlying the differentiation of MSCs into mature osteoblasts during the expansion in vitro, and to explore a variety of cell functions that cannot be easily evaluated using morphological, cytochemical, and biochemical assays. Cell cultures were obtained from bone marrow samples of six individuals undergoing total hip replacement, and a large-scale transcriptome analysis, using Affymetrix HG-U133A Plus 2.0 array (Affymetrix((R)), Santa Clara, CA), was performed at the occurrence of specific events, including the appearance of MSC surface markers, formation of colonies, and deposition of mineral nodules. We focused our attention on 213 differentially upregulated genes, some belonging to well-known pathways and some having one or more Gene Ontology annotations related to bone cell biology, including angiogenesis, bone-related genes, cell communication, development and morphogenesis, transforming growth factor-beta signaling, and Wnt signaling. Twenty-nine genes, whose role in bone cell pathophysiology has not been described yet, were found. In conclusion, gene expression patterns that characterize the early, intermediate, and late phases of the osteogenic differentiation process of ex vivo-expanded MSCs were defined. These signatures represent a useful tool to monitor the osteogenic process, and to analyze a broad spectrum of functions of MSCs cultured on scaffolds, especially when the constructs are conceived for releasing growth factors or other signals to promote bone regeneration.

  19. 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

  20. 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

    Lim, Kitaek; Kim, Jangho; Seonwoo, Hoon; Park, Soo Hyun; 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/cm(2) 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.

  1. Effects of apatite particle size in two apatite/collagen composites on the osteogenic differentiation profile of osteoblastic cells

    PubMed Central

    HATAKEYAMA, WATARU; TAIRA, MASAYUKI; CHOSA, NAOYUKI; KIHARA, HIDEMICHI; ISHISAKI, AKIRA; KONDO, HISATOMO

    2013-01-01

    The development of new osteoconductive bone substitute materials is expected in medicine. In this study, we attempted to produce new hydroxylapatite (HAP)/collagen (Col) composites using two HAP particles of different sizes and porcine type I collagen. The two HAP particles were either nano-sized (40 nm in average diameter; n-HAP) or had macro-pore sizes of 0.5–1.0 mm in length with fully interconnected pores (m-HAP). The aim of this study was to investigate the effects of apatite particle size in two HAP/Col composites on the osteogenic differentiation profile in osteoblast-like cells (SaOS-2). We created a collagen control sponge (Col) and two HAP/Col composite sponges (n-HAP/Col and m-HAP/Col) using freeze-drying and dehydrothermal cross-linking techniques, and then punched out samples of 6 mm in diameter and 1 mm in height. The SaOS-2 cells were cultured on three test materials for 1, 2, 3 and 4 weeks. Total RNA was extracted from the cultured cells and the expression of osteogenic differentiation-related genes was evaluated by reverse transcription PCR (RT-PCR) using primer sets of alkaline phosphatase (ALP), type 1 collagen (COL1), bone sialoprotein (BSP) and osteocalcin precursor [bone gamma-carboxyglutamate (gla) protein (BGLAP)] genes, as well as the β-actin gene. The cells were also cultured on Col, n-HAP/Col and m-HAP/Col specimens for 1 and 4 weeks, and were then observed under a scanning electron microscope (SEM). The experimental results were as follows: RT-PCR indicated that osteogenic differentiation, particularly the gene expression of BSP, was most accelerated when the cells were cultured on n-HAP/Col specimens, followed by m-HAP/Col, whilst the weakest accelaeration was observed when the cells were cultured on Col specimens. As shown by the SEM images, the SaOS-2 cells were fibroblastic when cultured on Col specimens for up to 4 weeks; they were fibroblastic when cultured on n-HAP/Col specimens for 1 week, but appeared as spheroids, while

  2. 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

  3. 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

  4. The scaffold protein Tks4 is required for the differentiation of mesenchymal stromal cells (MSCs) into adipogenic and osteogenic lineages

    PubMed Central

    Dülk, Metta; Kudlik, Gyöngyi; Fekete, Anna; Ernszt, Dávid; Kvell, Krisztián; Pongrácz, Judit E.; Merő, Balázs L.; Szeder, Bálint; Radnai, László; Geiszt, Miklós; Csécsy, Dalma E.; Kovács, Tamás; Uher, Ferenc; Lányi, Árpád; Vas, Virag; Buday, László

    2016-01-01

    The commitment steps of mesenchymal stromal cells (MSCs) to adipogenic and other lineages have been widely studied but not fully understood. Therefore, it is critical to understand which molecules contribute to the conversion of stem cells into differentiated cells. The scaffold protein Tks4 plays a role in podosome formation, EGFR signaling and ROS production. Dysfunction of Tks4 causes a hereditary disease called Frank-ter Haar syndrome with a variety of defects concerning certain mesenchymal tissues (bone, fat and cartilage) throughout embryogenic and postnatal development. In this study, we aimed to analyze how the mutation of Tks4 affects the differentiation potential of multipotent bone marrow MSCs (BM-MSCs). We generated a Tks4 knock-out mouse strain on C57Bl/6 background, and characterized BM-MSCs isolated from wild type and Tks4−/− mice to evaluate their differentiation. Tks4−/− BM-MSCs had reduced ability to differentiate into osteogenic and adipogenic lineages compared to wild type. Studying the expression profile of a panel of lipid-regulated genes during adipogenic induction revealed that the expression of adipogenic transcription factors, genes responsible for lipid droplet formation, sterol and fatty acid metabolism was delayed or reduced in Tks4−/− BM-MSCs. Taken together, these results establish a novel function for Tks4 in the regulation of MSC differentiation. PMID:27711054

  5. 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

  6. 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

  7. 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.

  8. Cytocompatibility with osteogenic cells and enhanced in vivo anti-infection potential of quaternized chitosan-loaded titania nanotubes

    PubMed Central

    Yang, Ying; Ao, Haiyong; Wang, Yugang; Lin, Wentao; Yang, Shengbing; Zhang, Shuhong; Yu, Zhifeng; Tang, Tingting

    2016-01-01

    Infection is one of the major causes of failure of orthopedic implants. Our previous study demonstrated that nanotube modification of the implant surface, together with nanotubes loaded with quaternized chitosan (hydroxypropyltrimethyl ammonium chloride chitosan, HACC), could effectively inhibit bacterial adherence and biofilm formation in vitro. Therefore, the aim of this study was to further investigate the in vitro cytocompatibility with osteogenic cells and the in vivo anti-infection activity of titanium implants with HACC-loaded nanotubes (NT-H). The titanium implant (Ti), nanotubes without polymer loading (NT), and nanotubes loaded with chitosan (NT-C) were fabricated and served as controls. Firstly, we evaluated the cytocompatibility of these specimens with human bone marrow-derived mesenchymal stem cells in vitro. The observation of cell attachment, proliferation, spreading, and viability in vitro showed that NT-H has improved osteogenic activity compared with Ti and NT-C. A prophylaxis rat model with implantation in the femoral medullary cavity and inoculation with methicillin-resistant Staphylococcus aureus was established and evaluated by radiographical, microbiological, and histopathological assessments. Our in vivo study demonstrated that NT-H coatings exhibited significant anti-infection capability compared with the Ti and NT-C groups. In conclusion, HACC-loaded nanotubes fabricated on a titanium substrate show good compatibility with osteogenic cells and enhanced anti-infection ability in vivo, providing a good foundation for clinical application to combat orthopedic implant-associated infections.

  9. Cytocompatibility with osteogenic cells and enhanced in vivo anti-infection potential of quaternized chitosan-loaded titania nanotubes

    PubMed Central

    Yang, Ying; Ao, Haiyong; Wang, Yugang; Lin, Wentao; Yang, Shengbing; Zhang, Shuhong; Yu, Zhifeng; Tang, Tingting

    2016-01-01

    Infection is one of the major causes of failure of orthopedic implants. Our previous study demonstrated that nanotube modification of the implant surface, together with nanotubes loaded with quaternized chitosan (hydroxypropyltrimethyl ammonium chloride chitosan, HACC), could effectively inhibit bacterial adherence and biofilm formation in vitro. Therefore, the aim of this study was to further investigate the in vitro cytocompatibility with osteogenic cells and the in vivo anti-infection activity of titanium implants with HACC-loaded nanotubes (NT-H). The titanium implant (Ti), nanotubes without polymer loading (NT), and nanotubes loaded with chitosan (NT-C) were fabricated and served as controls. Firstly, we evaluated the cytocompatibility of these specimens with human bone marrow-derived mesenchymal stem cells in vitro. The observation of cell attachment, proliferation, spreading, and viability in vitro showed that NT-H has improved osteogenic activity compared with Ti and NT-C. A prophylaxis rat model with implantation in the femoral medullary cavity and inoculation with methicillin-resistant Staphylococcus aureus was established and evaluated by radiographical, microbiological, and histopathological assessments. Our in vivo study demonstrated that NT-H coatings exhibited significant anti-infection capability compared with the Ti and NT-C groups. In conclusion, HACC-loaded nanotubes fabricated on a titanium substrate show good compatibility with osteogenic cells and enhanced anti-infection ability in vivo, providing a good foundation for clinical application to combat orthopedic implant-associated infections. PMID:27672479

  10. Cytocompatibility with osteogenic cells and enhanced in vivo anti-infection potential of quaternized chitosan-loaded titania nanotubes.

    PubMed

    Yang, Ying; Ao, Haiyong; Wang, Yugang; Lin, Wentao; Yang, Shengbing; Zhang, Shuhong; Yu, Zhifeng; Tang, Tingting

    2016-01-01

    Infection is one of the major causes of failure of orthopedic implants. Our previous study demonstrated that nanotube modification of the implant surface, together with nanotubes loaded with quaternized chitosan (hydroxypropyltrimethyl ammonium chloride chitosan, HACC), could effectively inhibit bacterial adherence and biofilm formation in vitro. Therefore, the aim of this study was to further investigate the in vitro cytocompatibility with osteogenic cells and the in vivo anti-infection activity of titanium implants with HACC-loaded nanotubes (NT-H). The titanium implant (Ti), nanotubes without polymer loading (NT), and nanotubes loaded with chitosan (NT-C) were fabricated and served as controls. Firstly, we evaluated the cytocompatibility of these specimens with human bone marrow-derived mesenchymal stem cells in vitro. The observation of cell attachment, proliferation, spreading, and viability in vitro showed that NT-H has improved osteogenic activity compared with Ti and NT-C. A prophylaxis rat model with implantation in the femoral medullary cavity and inoculation with methicillin-resistant Staphylococcus aureus was established and evaluated by radiographical, microbiological, and histopathological assessments. Our in vivo study demonstrated that NT-H coatings exhibited significant anti-infection capability compared with the Ti and NT-C groups. In conclusion, HACC-loaded nanotubes fabricated on a titanium substrate show good compatibility with osteogenic cells and enhanced anti-infection ability in vivo, providing a good foundation for clinical application to combat orthopedic implant-associated infections.

  11. Cytocompatibility with osteogenic cells and enhanced in vivo anti-infection potential of quaternized chitosan-loaded titania nanotubes.

    PubMed

    Yang, Ying; Ao, Haiyong; Wang, Yugang; Lin, Wentao; Yang, Shengbing; Zhang, Shuhong; Yu, Zhifeng; Tang, Tingting

    2016-01-01

    Infection is one of the major causes of failure of orthopedic implants. Our previous study demonstrated that nanotube modification of the implant surface, together with nanotubes loaded with quaternized chitosan (hydroxypropyltrimethyl ammonium chloride chitosan, HACC), could effectively inhibit bacterial adherence and biofilm formation in vitro. Therefore, the aim of this study was to further investigate the in vitro cytocompatibility with osteogenic cells and the in vivo anti-infection activity of titanium implants with HACC-loaded nanotubes (NT-H). The titanium implant (Ti), nanotubes without polymer loading (NT), and nanotubes loaded with chitosan (NT-C) were fabricated and served as controls. Firstly, we evaluated the cytocompatibility of these specimens with human bone marrow-derived mesenchymal stem cells in vitro. The observation of cell attachment, proliferation, spreading, and viability in vitro showed that NT-H has improved osteogenic activity compared with Ti and NT-C. A prophylaxis rat model with implantation in the femoral medullary cavity and inoculation with methicillin-resistant Staphylococcus aureus was established and evaluated by radiographical, microbiological, and histopathological assessments. Our in vivo study demonstrated that NT-H coatings exhibited significant anti-infection capability compared with the Ti and NT-C groups. In conclusion, HACC-loaded nanotubes fabricated on a titanium substrate show good compatibility with osteogenic cells and enhanced anti-infection ability in vivo, providing a good foundation for clinical application to combat orthopedic implant-associated infections. PMID:27672479

  12. Osterix enhances proliferation and osteogenic potential of bone marrow stromal cells

    SciTech Connect

    Tu Qisheng; Valverde, Paloma . E-mail: paloma.valverde@tufts.edu; Chen, Jake

    2006-03-24

    Osterix (Osx) is a zinc-finger-containing transcription factor that is expressed in osteoblasts of all endochondral and membranous bones. In Osx null mice osteoblast differentiation is impaired and bone formation is absent. In this study, we hypothesized that overexpression of Osx in murine bone marrow stromal cells (BMSC) would be able to enhance their osteoblastic differentiation and mineralization in vitro. Retroviral transduction of Osx in BMSC cultured in non-differentiating medium did not affect expression of Runx2/Cbfa1, another key transcription factor of osteoblast differentiation, but induced an increase in the expression of other markers associated with the osteoblastic lineage including alkaline phosphatase, bone sialoprotein, osteocalcin, and osteopontin. Retroviral transduction of Osx in BMSC also increased their proliferation, alkaline phosphatase activity, and ability to form bone nodules. These events occurred without significant changes in the expression of {alpha}1(II) procollagen or lipoprotein lipase, which are markers of chondrogenic and adipogenic differentiation, respectively.

  13. Flexible Yttrium-Stabilized Zirconia Nanofibers Offer Bioactive Cues for Osteogenic Differentiation of Human Mesenchymal Stromal Cells.

    PubMed

    Cadafalch Gazquez, Gerard; Chen, Honglin; Veldhuis, Sjoerd A; Solmaz, Alim; Mota, Carlos; Boukamp, Bernard A; van Blitterswijk, Clemens A; Ten Elshof, Johan E; Moroni, Lorenzo

    2016-06-28

    Currently, the main drawback of ceramic scaffolds used in hard tissue regeneration is their low mechanical strength. Stabilized zirconia, especially the tetragonal 3% yttrium-stabilized zirconia (YSZ) phase, has been considered as a bioinert ceramic material with high mechanical strength. In the present work, flexible nanofibrous YSZ scaffolds were prepared by electrospinning. The obtained scaffolds showed remarkable flexibility at the macroscopic scale, while retaining their stiffness at the microscopic scale. The surface nanoroughness of the scaffolds could be tailored by varying the heat treatment method. Our results demonstrate that the osteogenic differentiation and mineralization of seeded human mesenchymal stromal cells were supported by the nanofibrous YSZ scaffolds, in contrast to the well-known bioinert behavior of bulk YSZ. These findings highlight that flexible ceramic scaffolds are an appealing alternative to the current brittle ceramics for bone tissue regeneration applications. PMID:27294434

  14. Labeling and imaging of human mesenchymal stem cells with quantum dot bioconjugates during proliferation and osteogenic differentiation in long term.

    PubMed

    Shah, B; Clark, P; Stroscio, M; Mao, J

    2006-01-01

    Quantum dots (QDs) are semiconductor nanocrystals that serve as promising alternatives to organic dyes for cell labeling. Because of their unique spectral, physical and chemical properties, QDs are useful for concurrently monitoring several intercellular and intracellular interactions in live normal cells and cancer cells over periods ranging from less than a second to over several days (several divisions of cells). Here, peptide CGGGRGD is immobilized on CdSe-ZnS QDs coated with carboxyl groups by cross linking with amine groups. These conjugates are directed by the peptide to bind with selected integrins on the membrane of human Mesenchymal stem cells. Upon overnight incubation with optimal concentration, QDs effectively labeled all the cells. Here, we report long-term labeling of human bone-marrow-derived mesenchymal stem cells (hMSCs) with RGD-conjugated QDs during self replication and differentiation into osteogenic cell lineages.

  15. 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.

  16. 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.

  17. Modular poly(ethylene glycol) matrices for the controlled 3D-localized osteogenic differentiation of mesenchymal stem cells.

    PubMed

    Metzger, Stéphanie; Lienemann, Philipp S; Ghayor, Chafik; Weber, Wilfried; Martin, Ivan; Weber, Franz E; Ehrbar, Martin

    2015-03-11

    The in vitro formation of physiologically relevant engineered tissues is still limited by the availability of adequate growth-factor-presenting cell-instructive biomaterials, allowing simultaneous and three-dimensionally localized differentiation of multiple tissue progenitor cells. Together with ever improving technologies such as microfluidics, printing, or lithography, these biomaterials could provide the basis for generating provisional cellular constructs, which can differentiate to form tissue mimetics. Although state-of-the-art biomaterials are endowed with sophisticated modules for time- and space-controlled positioning and release of bioactive molecules, reports on 3D arrangements of differentiation-inducing growth factors are scarce. This paper describes the stable and localized immobilization of biotinylated bioactive molecules to a modular, Factor XIII-cross-linked poly(ethylene glycol) hydrogel platform using a genetically engineered streptavidin linker. Linker incorporation is demonstrated by Western blot, and streptavidin functionality is confirmed by capturing biotinylated alkaline phosphatase (ALP). After optimizing bone morphogenetic protein 2 (BMP-2) biotinylation, streptavidin-modified hydrogels are able to bind and present bioactive BMP-2-biotin. Finally, with this immobilization scheme for BMP-2, the specific osteogenic differentiation of mesenchymal stem cells is demonstrated by inducing ALP expression in confined 3D areas. In future, this platform together with other affinity-based strategies will be useful for the local incorporation of various growth factors for engineering cell-responsive constructs.

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. Identification of long non-coding RNA involved in osteogenic differentiation from mesenchymal stem cells using RNA-Seq data.

    PubMed

    Song, W Q; Gu, W Q; Qian, Y B; Ma, X; Mao, Y J; Liu, W J

    2015-01-01

    The aim of this study was to identify long non-coding RNA (lncRNA) associated with osteogenic differentiation from mesenchymal stem cells (MSCs) using high-throughput RNA sequencing (RNA-Seq) data. RNA-Seq dataset was obtained from the European Bioinformatics Institute (accession No. PRJEB4496), including two replicates each for immortalized mesenchymal stem cells iMSC#3 cultured in growth medium (GM) and differentiation medium (DM) for 28 days. The clean reads were aligned to a hg19 reference genome by Tophat and assembled by Cufflinks to identify the known and novel transcripts. RPKM values were calculated to screen for differentially expressed RNA. Novel lncRNA were screened based on various filter criteria. Subsequently, the underlying function of novel lncRNAs were predicted by functional annotation by ERPIN, a co-expression network was constructed by WGCNA and the KEGG pathway enriched by KOBAS. A total of 3171 RNA differentially expressed between the DM and GM groups (2597 mRNA and 574 lncRNA) were identified. Among the 574 differentially expressed lncRNA, 357 were known and 217 were novel lncRNA. Furthermore, 32 novel lncRNA were found to be miRNA precursors (including miR-689, miR-640, miR-601, and miR-544). A total of 14,275 co-expression relationships and 217 co-expression networks were obtained between novel lncRNA and mRNA. The differentially expressed lncRNA and mRNA were enriched into 6 significant pathways, including those for cancer, ECM-receptor interaction, and focal adhesion. Therefore, novel lncRNAwere identified and their underlying function predicted, which may provide the basis for future analyses of the role of lncRNA in osteoblastic differentiation.

  3. 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

  4. 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.

  5. Effect of an Experimental Direct Pulp-capping Material on the Properties and Osteogenic Differentiation of Human Dental Pulp Stem Cells

    PubMed Central

    Yu, Fan; Dong, Yan; Yang, Yan-wei; Lin, Ping-ting; Yu, Hao-han; Sun, Xiang; Sun, Xue-fei; Zhou, Huan; Huang, Li; Chen, Ji-hua

    2016-01-01

    Effective pulp-capping materials must have antibacterial properties and induce dentin bridge formation; however, many current materials do not satisfy clinical requirements. Accordingly, the effects of an experiment pulp-capping material (Exp) composed of an antibacterial resin monomer (MAE-DB) and Portland cement (PC) on the viability, adhesion, migration, and differentiation of human dental pulp stem cells (hDPSCs) were examined. Based on a Cell Counting Kit-8 assay, hDPSCs exposed to Exp extracts showed limited viability at 24 and 48 h, but displayed comparable viability to the control at 72 h. hDPSC treatment with Exp extracts enhanced cellular adhesion and migration according to in vitro scratch wound healing and Transwell migration assays. Exp significantly upregulated the expression of osteogenesis-related genes. The hDPSCs cultured with Exp exhibited higher ALP activity and calcium deposition in vitro compared with the control group. The novel material showed comparable cytocompatibility to control cells and promoted the adhesion, migration, and osteogenic differentiation of hDPSCs, indicating excellent biocompatibility. This new direct pulp-capping material containing MAE-DB and PC shows promise as a potential alternative to conventional materials for direct pulp capping. PMID:27698421

  6. 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

  7. Effect of an Experimental Direct Pulp-capping Material on the Properties and Osteogenic Differentiation of Human Dental Pulp Stem Cells

    NASA Astrophysics Data System (ADS)

    Yu, Fan; Dong, Yan; Yang, Yan-Wei; Lin, Ping-Ting; Yu, Hao-Han; Sun, Xiang; Sun, Xue-Fei; Zhou, Huan; Huang, Li; Chen, Ji-Hua

    2016-10-01

    Effective pulp-capping materials must have antibacterial properties and induce dentin bridge formation; however, many current materials do not satisfy clinical requirements. Accordingly, the effects of an experiment pulp-capping material (Exp) composed of an antibacterial resin monomer (MAE-DB) and Portland cement (PC) on the viability, adhesion, migration, and differentiation of human dental pulp stem cells (hDPSCs) were examined. Based on a Cell Counting Kit-8 assay, hDPSCs exposed to Exp extracts showed limited viability at 24 and 48 h, but displayed comparable viability to the control at 72 h. hDPSC treatment with Exp extracts enhanced cellular adhesion and migration according to in vitro scratch wound healing and Transwell migration assays. Exp significantly upregulated the expression of osteogenesis-related genes. The hDPSCs cultured with Exp exhibited higher ALP activity and calcium deposition in vitro compared with the control group. The novel material showed comparable cytocompatibility to control cells and promoted the adhesion, migration, and osteogenic differentiation of hDPSCs, indicating excellent biocompatibility. This new direct pulp-capping material containing MAE-DB and PC shows promise as a potential alternative to conventional materials for direct pulp capping.

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. Effect of micro-nano-hybrid structured hydroxyapatite bioceramics on osteogenic and cementogenic differentiation of human periodontal ligament stem cell via Wnt signaling pathway.

    PubMed

    Mao, Lixia; Liu, Jiaqiang; Zhao, Jinglei; Chang, Jiang; Xia, Lunguo; Jiang, Lingyong; Wang, Xiuhui; Lin, Kaili; Fang, Bing

    2015-01-01

    The surface structure of bioceramic scaffolds is crucial for its bioactivity and osteoinductive ability, and in recent years, human periodontal ligament stem cells have been certified to possess high osteogenic and cementogenic differential ability. In the present study, hydroxyapatite (HA) bioceramics with micro-nano-hybrid surface (mnHA [the hybrid of nanorods and microrods]) were fabricated via hydrothermal reaction of the α-tricalcium phosphate granules as precursors in aqueous solution, and the effects of mnHA on the attachment, proliferation, osteogenic and cementogenic differentiations of human periodontal ligament stem cells as well as the related mechanisms were systematically investigated. The results showed that mnHA bioceramics could promote cell adhesion, proliferation, alkaline phosphatase (ALP) activity, and expression of osteogenic/cementogenic-related markers including runt-related transcription factor 2 (Runx2), ALP, osteocalcin (OCN), cementum attachment protein (CAP), and cementum protein (CEMP) as compared to the HA bioceramics with flat and dense surface. Moreover, mnHA bioceramics stimulated gene expression of low-density lipoprotein receptor-related protein 5 (LRP5) and β-catenin, which are the key genes of canonical Wnt signaling. Moreover, the stimulatory effect on ALP activity and osteogenic and cementogenic gene expression, including that of ALP, OCN, CAP, CEMP, and Runx2 of mnHA bioceramics could be repressed by canonical Wnt signaling inhibitor dickkopf1 (Dkk1). The results suggested that the HA bioceramics with mnHA could act as promising grafts for periodontal tissue regeneration. PMID:26648716

  13. Effect of micro-nano-hybrid structured hydroxyapatite bioceramics on osteogenic and cementogenic differentiation of human periodontal ligament stem cell via Wnt signaling pathway

    PubMed Central

    Mao, Lixia; Liu, Jiaqiang; Zhao, Jinglei; Chang, Jiang; Xia, Lunguo; Jiang, Lingyong; Wang, Xiuhui; Lin, Kaili; Fang, Bing

    2015-01-01

    The surface structure of bioceramic scaffolds is crucial for its bioactivity and osteoinductive ability, and in recent years, human periodontal ligament stem cells have been certified to possess high osteogenic and cementogenic differential ability. In the present study, hydroxyapatite (HA) bioceramics with micro-nano-hybrid surface (mnHA [the hybrid of nanorods and microrods]) were fabricated via hydrothermal reaction of the α-tricalcium phosphate granules as precursors in aqueous solution, and the effects of mnHA on the attachment, proliferation, osteogenic and cementogenic differentiations of human periodontal ligament stem cells as well as the related mechanisms were systematically investigated. The results showed that mnHA bioceramics could promote cell adhesion, proliferation, alkaline phosphatase (ALP) activity, and expression of osteogenic/cementogenic-related markers including runt-related transcription factor 2 (Runx2), ALP, osteocalcin (OCN), cementum attachment protein (CAP), and cementum protein (CEMP) as compared to the HA bioceramics with flat and dense surface. Moreover, mnHA bioceramics stimulated gene expression of low-density lipoprotein receptor-related protein 5 (LRP5) and β-catenin, which are the key genes of canonical Wnt signaling. Moreover, the stimulatory effect on ALP activity and osteogenic and cementogenic gene expression, including that of ALP, OCN, CAP, CEMP, and Runx2 of mnHA bioceramics could be repressed by canonical Wnt signaling inhibitor dickkopf1 (Dkk1). The results suggested that the HA bioceramics with mnHA could act as promising grafts for periodontal tissue regeneration. PMID:26648716

  14. Differences in the Osteogenic Differentiation Capacity of Omental Adipose-Derived Stem Cells in Obese Patients With and Without Metabolic Syndrome

    PubMed Central

    Gea, Antonio Leiva; Lhamyani, Said; Coín-Aragüez, Leticia; Torres, Juan Alcaide; Bernal-López, Maria Rosa; García-Luna, Pedro Pablo; Conde, Salvador Morales; Fernández-Veledo, Sonia

    2015-01-01

    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 CD34negative-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 CD34negative-enriched fraction of omental-derived ASCs. Both the omental ASCs and the CD34negative-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/m2 or greater, only the CD34negative-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

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. Adipose-derived stem cells from the brown bear (Ursus arctos) spontaneously undergo chondrogenic and osteogenic differentiation in vitro.

    PubMed

    Fink, Trine; Rasmussen, Jeppe G; Emmersen, Jeppe; Pilgaard, Linda; Fahlman, Åsa; Brunberg, Sven; Josefsson, Johan; Arnemo, Jon M; Zachar, Vladimir; Swenson, Jon E; Fröbert, Ole

    2011-07-01

    In the den, hibernating brown bears do not develop tissue atrophy or organ damage, despite almost no physical activity. Mesenchymal stem cells could play an important role in tissue repair and regeneration in brown bears. Our objective was to determine if adipose tissue-derived stem cells (ASCs) can be recovered from wild Scandinavian brown bears and characterize their differentiation potential. Following immobilization of wild brown bears 7-10 days after leaving the den in mid-April, adipose tissue biopsies were obtained. ASCs were recovered from 6 bears, and shown to be able to undergo adipogenesis and osteogenesis in monolayer cultures and chondrogenesis in pellet cultures. Remarkably, when grown in standard cell culture medium in monolayer cultures, ASCs from yearlings spontaneously formed bone-like nodules surrounded by cartilaginous deposits, suggesting differentiation into osteogenic and chondrogenic lineages. This ability appears to be lost gradually with age. This is the first study to demonstrate stem cell recovery and growth from brown bears, and it is the first report of ASCs spontaneously forming extracellular matrix characteristic of bone and cartilage in the absence of specific inducers. These findings could have implications for the use of hibernating brown bears as a model to study disuse osteoporosis.

  20. 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

  1. Gold nanoparticles promote osteogenic differentiation in human adipose-derived mesenchymal stem cells through the Wnt/β-catenin signaling pathway

    PubMed Central

    Choi, Seon Young; Song, Min Seok; Ryu, Pan Dong; Lam, Anh Thu Ngoc; Joo, Sang-Woo; Lee, So Yeong

    2015-01-01

    Gold nanoparticles (AuNPs) are attractive materials for use in biomedicine due to their physical properties. Increasing evidence suggests that several nanoparticles induce the differentiation of human mesenchymal stem cells into osteoblasts and adipocytes. In this study, we hypothesized that chitosan-conjugated AuNPs promote the osteogenic differentiation of human adipose-derived mesenchymal stem cells. For the evaluation of osteogenic differentiation, alizarin red staining, an alamarBlue® assay, and a quantitative real-time polymerase chain reaction analysis were performed. In order to examine specific signaling pathways, immunofluorescence and a western blotting assay were performed. Our results demonstrate that chitosan-conjugated AuNPs increase the deposition of calcium content and the expression of marker genes related to osteogenic differentiation in human adipose-derived mesenchymal stem cells at nontoxic concentrations. These results indicate that chitosan-conjugated AuNPs promote osteogenesis through the Wnt/β-catenin signaling pathway. Therefore, chitosan-conjugated AuNPs can be used as a reagent for promoting bone formation. PMID:26185441

  2. 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.

  3. A High Throughput Phenotypic Screening reveals compounds that counteract premature osteogenic differentiation of HGPS iPS-derived mesenchymal stem cells

    PubMed Central

    Lo Cicero, Alessandra; Jaskowiak, Anne-Laure; Egesipe, Anne-Laure; Tournois, Johana; Brinon, Benjamin; Pitrez, Patricia R.; Ferreira, Lino; de Sandre-Giovannoli, Annachiara; Levy, Nicolas; Nissan, Xavier

    2016-01-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare fatal genetic disorder that causes systemic accelerated aging in children. Thanks to the pluripotency and self-renewal properties of induced pluripotent stem cells (iPSC), HGPS iPSC-based modeling opens up the possibility of access to different relevant cell types for pharmacological approaches. In this study, 2800 small molecules were explored using high-throughput screening, looking for compounds that could potentially reduce the alkaline phosphatase activity of HGPS mesenchymal stem cells (MSCs) committed into osteogenic differentiation. Results revealed seven compounds that normalized the osteogenic differentiation process and, among these, all-trans retinoic acid and 13-cis-retinoic acid, that also decreased progerin expression. This study highlights the potential of high-throughput drug screening using HGPS iPS-derived cells, in order to find therapeutic compounds for HGPS and, potentially, for other aging-related disorders. PMID:27739443

  4. 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.

  5. Effects of rifampicin on osteogenic differentiation and proliferation of human mesenchymal stem cells in the bone marrow.

    PubMed

    Zhang, Z; Wang, X; Luo, F; Yang, H; Hou, T; Zhou, Q; Dai, F; He, Q; Xu, J

    2014-08-25

    This study was designed to investigate the effect of different concentrations of rifampicin on osteogenic differentiation and proliferation of mesenchymal stem cells (MSCs) in human bone marrow. Rifampicin treatment at 0, 4, 8, 16, 32, 64, and 128 mg/mL was applied throughout the whole process, from stromal cells purified from human bone marrow to differentiated bone cells. The effect of rifampicin on MSC proliferation was determined using the MTT assay. The effect of rifampicin on the expressions of type I collagen (COL1A1), osteopontin/bone Gla protein (OPN/BGP), and alkaline phosphatase (ALP) in human osteoblast cells were determined by real-time polymerase chain reaction, and the expressions of COL1A1, OPN/BGP, and the runt-related transcription factor (RUNX2) were determined by Western blot. Results showed that the proliferation of MSCs was significantly inhibited when the rifampicin concentration exceeded 32 mg/mL. In addition, increased rifampicin concentrations inhibited the formation of calcium nodules, OPN/BGP, and COL1A1 in osteoblasts after 28 days of induction. The RNA expressions of OPN/BGP, COL1A1, and ALP were significantly downregulated compared to those of the control group in osteoblasts after induction. The protein expressions of RUNX2, COL1A1, and OPN/BGP were also significantly downregulated compared to those of the control group after induction. In conclusion, rifampicin at exorbitant concentration exerts adverse effects on the proliferation of MSCs in human bone marrow and the differentiation of osteoblasts.

  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. 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

  8. 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

  9. 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

  10. 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

  11. Bisphosphonate-linked hyaluronic acid hydrogel sequesters and enzymatically releases active bone morphogenetic protein-2 for induction of osteogenic differentiation.

    PubMed

    Hulsart-Billström, Gry; Yuen, Pik Kwan; Marsell, Richard; Hilborn, Jöns; Larsson, Sune; Ossipov, Dmitri

    2013-09-01

    Regeneration of bone by delivery of bone morphogenetic proteins (BMPs) from implantable scaffolds is a promising alternative to the existing autologous bone grafting procedures. Hydrogels are used extensively in biomaterials as delivery systems for different growth factors. However, a controlled release of the growth factors is necessary to induce bone formation, which can be accomplished by various chemical functionalities. Herein we demonstrate that functionalization of a hyaluronan (HA) hydrogel with covalently linked bisphosphonate (BP) ligands provides efficient sequestering of BMP-2 in the resulting HA-BP hydrogel. The HA-BP hydrogel was investigated in comparison with its analogue lacking BP groups (HA hydrogel). While HA hydrogel released 100% of BMP-2 over two weeks, less than 10% of BMP-2 was released from the HA-BP hydrogel for the same time. We demonstrate that the sequestered growth factor can still be released by enzymatic degradation of the HA-BP hydrogel. Most importantly, entrapment of BMP-2 in HA-BP hydrogel preserves the growth factor bioactivity, which was confirmed by induction of osteogenic differentiation of mesenchymal stem cells (MSCs) after the cells incubation with the enzymatic digest of the hydrogel. At the same time, the hydrogels degradation products were not toxic to MSCs and osteoblasts. Furthermore, BP-functionalization of HA hydrogels promotes adhesion of the cells to the surface of HA hydrogel. Altogether, the present findings indicate that covalent grafting of HA hydrogel with BP groups can alter the clinical effects of BMPs in bone tissue regeneration.

  12. Supplementation of fibrin gels with sodium chloride enhances physical properties and ensuing osteogenic response.

    PubMed

    Davis, H E; Miller, S L; Case, E M; Leach, J K

    2011-02-01

    Modifying the relative concentrations of fibrinogen and thrombin can control the physical properties of fibrin gels, while the viability of associated cells has been linked to the gel's final network structure. It was hypothesized that increasing the gel ionic strength during fabrication through supplementation with sodium chloride (NaCl) would provide an improved approach for tailoring the physical properties of fibrin gels and maintaining the viability and osteogenic potential of entrapped cells. Fibrin gels were formed by mixing fibrinogen, thrombin and calcium chloride with varying masses of NaCl (0-4.40% w/v), and the osteogenic potential of entrapped human mesenchymal stem cells (MSC) was examined over 14 days. Physical properties including gelation time, compressive modulus and fiber diameter were dependent upon NaCl content, with gels containing 2.60% NaCl possessing compressive moduli threefold higher than gels without NaCl. Alkaline phosphatase activity was highest for MSC entrapped in gels containing 2.15-2.60% NaCl after 14 days, and all gels exhibited increased calcium incorporation over the culture period. These data confirm that varying the salt concentration of the pre-gel solution can modulate the material properties of fibrin constructs without additional fibrinogen or thrombin, thereby offering a new approach for generating improved cell transplantation vehicles for use in bone tissue regeneration.

  13. Human serum is a suitable supplement for the osteogenic differentiation of human adipose-derived stem cells seeded on poly-3-hydroxibutyrate-co-3-hydroxyvalerate scaffolds.

    PubMed

    de Paula, Ana Cláudia Chagas; Zonari, Alessandra Arcoverde Cavalcanti; Martins, Thaís Maria da Mata; Novikoff, Silviene; da Silva, Alexandra Rodrigues Pereira; Correlo, Vitor Manuel; Reis, Rui L; Gomes, Dawidson Assis; Goes, Alfredo Miranda

    2013-01-01

    Human adipose-derived stem cells (hASCs) are currently a point of focus for bone tissue engineering applications. However, the ex vivo expansion of stem cells before clinical application remains a challenge. Fetal bovine serum (FBS) is largely used as a medium supplement and exposes the recipient to infections and immunological reactions. In this study, we evaluated the osteogenic differentiation process of hASCs in poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-HV) scaffolds with the osteogenic medium supplemented with pooled allogeneic human serum (aHS). The hASCs grown in the presence of FBS or aHS did not show remarkable differences in morphology or immunophenotype. The PHB-HV scaffolds, which were developed by the freeze-drying technique, showed an adequate porous structure and mechanical performance as observed by micro-computed tomography, scanning electron microscopy (SEM), and compression test. The three-dimensional structure was suitable for allowing cell colonization, which was revealed by SEM micrographs. Moreover, these scaffolds were not toxic to cells as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The differentiation capacity of hASCs seeded on scaffolds was confirmed by the reduction of the proliferation, the alkaline phosphatase (AP) activity, expression of osteogenic gene markers (AP, collagen type I, Runx2, and osteocalcin), and the expression of bone markers, such as osteopontin, osteocalcin, and collagen type I. The osteogenic capacity of hASCs seeded on PHB-HV scaffolds indicates that this scaffold is adequate for cell growth and differentiation and that aHS is a promising supplement for the in vitro expansion of hASCs. In conclusion, this strategy seems to be useful and safe for application in bone tissue engineering.

  14. 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

  15. Optimization of release pattern of FGF-2 and BMP-2 for osteogenic differentiation of low-population density hMSCs.

    PubMed

    Lei, Lei; Wang, Shuo; Wu, Honghui; Ju, Wei; Peng, Jian; Qahtan, Anwar Saeed Ahmed; Chen, Chen; Lu, Yanqin; Peng, Jieying; Zhang, Xing; Nie, Hemin

    2015-01-01

    In the modern design, most delivery systems for bone regeneration focus on a single growth factor (GF) or a simple mixture of multiple GFs, overlooking the coordination of proliferation and osteogenesis induced by various factors. In this study, core-shell microspheres with poly-l-lactide core-poly(lactic-co-glycolic acid) shell were fabricated, and two GFs, basic fibroblast growth factor 2 (FGF-2) and bone morphogenetic protein 2 (BMP-2) were encapsulated into the core or/and shell. The effects of different release patterns (parallel or sequential manners) of FGF-2 and BMP-2 from these core-shell microspheres on the osteogenic differentiation of low-population density human mesenchymal stem cells (hMSCs) were investigated and the temporal organization of GF release was optimized. In vitro experiments suggested that induction of osteogenic differentiation of low-population density hMSCs by the sequential delivery of FGF-2 followed by BMP-2 from the core-shell microspheres (group S2) was much more efficient than that by the parallel release of the two factors from uniform microspheres (group U). The osteogenic induction by the sequential delivery of BMP-2 followed by FGF-2 from core-shell microspheres (group S1) was even worse than that from microspheres loaded with BMP-2 in both core and shell (group B), although comparable to the cases of parallel delivery of dual GFs (group P). This study showed the advantages of group S2 microspheres in inducing osteogenic differentiation of low-population density hMSCs and the necessity of time sequence studies in tissue engineering while multiple GFs are involved.

  16. Matrix production and collagen structure are enhanced in two types of osteogenic progenitor cells by a simple fluid shear stress stimulus.

    PubMed

    Delaine-Smith, R M; MacNeil, S; Reilly, G C

    2012-08-03

    Mesenchymal progenitor cells play a vital role in bone regenerative medicine and tissue engineering strategies. To be clinically useful osteoprogenitors should be readily available with the potential to form bone matrix. While mesenchymal stromal cells from bone marrow have shown promise for tissue engineering, they are obtained in small numbers and there is risk of donor site morbidity. Osteogenic progenitor cells derived from dermal tissue may provide a more abundant and easily expandable source of cells. Bone turnover in vivo is regulated by mechanical forces, particularly oscillatory fluid shear stresses (FSS), and in vitro osteogenic progenitors have been shown to be regulated by mechanical stimuli. The aim of this study was to assess what effect osteogenic media and FSS, generated by a simple rocking platform, had on cell behaviour and matrix production in human progenitor dermal fibroblasts (HDFs) and the embryonic stem cell-derived mesenchymal progenitor cell line (hES-MP). Osteogenic media stimulated alkaline phosphatase activity (ALP) and calcium deposition in HDFs. The addition of FSS further enhanced ALP activity and mineralised matrix deposition in both progenitor cells cultured in osteogenic media. Both types of progenitor cell subjected to FSS showed increases in collagen secretion and apparent collagen organisation as imaged by second harmonic generation.

  17. Time-lapse microscopy and classification of 2D human mesenchymal stem cells based on cell shape picks up myogenic from osteogenic and adipogenic differentiation.

    PubMed

    Seiler, Christof; Gazdhar, Amiq; Reyes, Mauricio; Benneker, Lorin M; Geiser, Thomas; Siebenrock, Klaus A; Gantenbein-Ritter, Benjamin

    2014-09-01

    Current methods to characterize mesenchymal stem cells (MSCs) are limited to CD marker expression, plastic adherence and their ability to differentiate into adipogenic, osteogenic and chondrogenic precursors. It seems evident that stem cells undergoing differentiation should differ in many aspects, such as morphology and possibly also behaviour; however, such a correlation has not yet been exploited for fate prediction of MSCs. Primary human MSCs from bone marrow were expanded and pelleted to form high-density cultures and were then randomly divided into four groups to differentiate into adipogenic, osteogenic chondrogenic and myogenic progenitor cells. The cells were expanded as heterogeneous and tracked with time-lapse microscopy to record cell shape, using phase-contrast microscopy. The cells were segmented using a custom-made image-processing pipeline. Seven morphological features were extracted for each of the segmented cells. Statistical analysis was performed on the seven-dimensional feature vectors, using a tree-like classification method. Differentiation of cells was monitored with key marker genes and histology. Cells in differentiation media were expressing the key genes for each of the three pathways after 21 days, i.e. adipogenic, osteogenic and chondrogenic, which was also confirmed by histological staining. Time-lapse microscopy data were obtained and contained new evidence that two cell shape features, eccentricity and filopodia (= 'fingers') are highly informative to classify myogenic differentiation from all others. However, no robust classifiers could be identified for the other cell differentiation paths. The results suggest that non-invasive automated time-lapse microscopy could potentially be used to predict the stem cell fate of hMSCs for clinical application, based on morphology for earlier time-points. The classification is challenged by cell density, proliferation and possible unknown donor-specific factors, which affect the performance of