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Sample records for vitro osteogenic differentiation

  1. In vitro proliferation and osteogenic differentiation of endometrial stem cells and dental pulp stem cells.

    Science.gov (United States)

    Tabatabaei, Fahimeh Sadat; Torshabi, Maryam

    2017-06-01

    Stem-cell-based therapies were introduced aiming to overcome the limitations of the existing procedures for regeneration of mineralized tissues. Stem cells isolated from the endometrial tissue and dental pulp have the capacity to differentiate into various functional cells including osteoblasts. However, studies comparing their ability to regenerate mineralized tissue are lacking. The purpose of this study was to compare the proliferation and osteogenic differentiation potential of endometrial stem cells (EnSCs) and dental pulp stem cells (DPSCs) using in vitro cell culture technique. The DPSCs and EnSCs were isolated from human dental pulp and endometrium, respectively. Their proliferation and osteogenic potential were compared in the same osteogenic medium (OM) after 3, 5, 7 and 10 days using the methyl thiazol tetrazolium assay, alizarin red staining, and real-time quantitative reverse transcription polymerase chain reaction (Real-Time qRT-PCR). The EnSCs showed higher proliferation rate compared to DPSCs. Regarding osteogenesis, alizarin red-positive colonies appeared earlier and in greater amounts in DPSCs group. The real-time qRT-PCR demonstrated significantly greater osteogenic potential of DPSCs compared to EnSCs. Our findings revealed significant differences in stem cell properties based on the tissue source. The EnSCs had greater proliferation rate than DPSCs, while DPSCs showed greater osteogenic potential compared to EnSCs in the same OM.

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

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    Giovanna Calabrese

    Full Text Available 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.

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

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    Jansen Justus HW

    2010-08-01

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

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

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    Salamon, Achim, E-mail: achim.salamon@med.uni-rostock.de [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Jonitz-Heincke, Anika, E-mail: anika.jonitz@med.uni-rostock.de [Biomechanics and Implant Technology Research Laboratory, Department of Orthopedics, Rostock University Medical Center, Doberaner Straße 142, D-18057 Rostock (Germany); Adam, Stefanie, E-mail: stefanie.adam@med.uni-rostock.de [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Rychly, Joachim, E-mail: joachim.rychly@med.uni-rostock.de [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Müller-Hilke, Brigitte, E-mail: brigitte.mueller-hilke@med.uni-rostock.de [Institute of Immunology, Rostock University Medical Center, Schillingallee 68, D-18057 Rostock (Germany); Bader, Rainer, E-mail: rainer.bader@med.uni-rostock.de [Biomechanics and Implant Technology Research Laboratory, Department of Orthopedics, Rostock University Medical Center, Doberaner Straße 142, D-18057 Rostock (Germany); Lochner, Katrin, E-mail: katrin.lochner@med.uni-rostock.de [Biomechanics and Implant Technology Research Laboratory, Department of Orthopedics, Rostock University Medical Center, Doberaner Straße 142, D-18057 Rostock (Germany); Peters, Kirsten, E-mail: kirsten.peters@med.uni-rostock.de [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany)

    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

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

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    Herzmann, Nicole; Salamon, Achim [Department of Cell Biology, University Medicine Rostock, Schillingallee 69, D-18057 Rostock (Germany); Fiedler, Tomas [Institute for Medical Microbiology, Virology and Hygiene, University Medicine Rostock, Schillingallee 70, D-18057 Rostock (Germany); Peters, Kirsten, E-mail: kirsten.peters@med.uni-rostock.de [Department of Cell Biology, University Medicine Rostock, Schillingallee 69, D-18057 Rostock (Germany)

    2017-01-01

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

  6. Human serum promotes osteogenic differentiation of human dental pulp stem cells in vitro and in vivo.

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    Alessandra Pisciotta

    Full Text Available Human dental pulp is a promising alternative source of stem cells for cell-based tissue engineering in regenerative medicine, for the easily recruitment with low invasivity for the patient and for the self-renewal and differentiation potential of cells. So far, in vitro culture of mesenchymal stem cells is usually based on supplementing culture and differentiation media with foetal calf serum (FCS. FCS is known to contain a great quantity of growth factors, and thus to promote cell attachment on plastic surface as well as expansion and differentiation. Nevertheless, FCS as an animal origin supplement may represent a potential means for disease transmission besides leading to a xenogenic immune response. Therefore, a significant interest is focused on investigating alternative supplements, in order to obtain a sufficient cell number for clinical application, avoiding the inconvenients of FCS use. In our study we have demonstrated that human serum (HS is a suitable alternative to FCS, indeed its addition to culture medium induces a high hDPSCs proliferation rate and improves the in vitro osteogenic differentiation. Furthermore, hDPSCs-collagen constructs, pre-differentiated with HS-medium in vitro for 10 days, when implanted in immunocompromised rats, are able to restore critical size parietal bone defects. Therefore these data indicate that HS is a valid substitute for FCS to culture and differentiate in vitro hDPSCs in order to obtain a successful bone regeneration in vivo.

  7. In vitro osteogenic differentiation of rat bone marrow cells subcultured with and without dexamethasone.

    NARCIS (Netherlands)

    Brugge, P.J. ter; Jansen, J.A.

    2002-01-01

    The aim of our study was to investigate the osteogenic potential of subcultured rat bone marrow cells. Rat bone marrow (RBM) cells were cultured with or without dexamethasone. Subsequently, osteogenic differentiation and expression was studied. When cells were cultured continuously in the presence

  8. BMP7 transfection induces in-vitro osteogenic differentiation of dental pulp mesenchymal stem cells

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    Ka Po John Yau

    2013-01-01

    Full Text Available Objective: To assess whether in-vitro osteogenic differentiation of human dental pulp mesenchymal stem cells can be induced by transient transfection with the gene encoding human bone morphogenic protein 7 (BMP7. Materials and Methods: A mesenchymal stem cell population was isolated from the dental pulp of two extracted permanent premolars, expanded and characterized. The human BMP7 gene, as a recombinant pcDNA3.1/V5-His-TOPO-BMP7 plasmid, was transfected into the cells. Three negative controls were used: No plasmid, empty vector, and an unrelated vector encoding green fluorescent protein. After the interval of 24 and 48 h, mRNA levels of alkaline phosphatase and osteocalcin as markers of in-vitro osteogenic differentiation were measured by real-time polymerase chain reaction and standardized against β-actin mRNA levels. Results: The level of alkaline phosphatase mRNA was significantly higher for the BMP7 group than for all three negative controls 48 h after transfection (706.9 vs. 11.24 for untransfected cells, 78.05 for empty vector, and 73.10 for green fluorescent protein vector. The level of osteocalcin mRNA was significantly higher for the BMP7 group than for all three negative controls 24 h after transfection (1.0, however, decreased after another 24 h. Conclusions: In-vitro osteoblastic differentiation of human dental pulp mesenchymal stem cells, as indicated by expression of alkaline phosphatase and osteocalcin, can be induced by transient transfection with the BMP7 gene.

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

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    Thomas, Minnah; Arora, Aditya; Katti, Dhirendra S., E-mail: dsk@iitk.ac.in

    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. - Highlights: • Surface hydrophilicity of PLGA modulated by blending with Pluronic F-108. • Hydrophilized fibers support better in vitro mineralization. • Mineralization trends retained in the presence of adsorbed serum proteins. • Hydrophilized fibers promote better cell adhesion and proliferation. • Hydrophilized fibers also enable better osteogenic differentiation.

  10. Staphylococcus aureus Biofilms Decrease Osteoblast Viability, Inhibits Osteogenic Differentiation, and Increases Bone Resorption in vitro

    Science.gov (United States)

    2013-06-01

    osteogenic differentiation Human osteoblasts (PromoCell, Heidelberg, Germany) were maintained in DMEM supplemented with 10% fetal bovine serum (FBS...with calf intestinal ALP. Osteocalcin staining Osteoblasts were grown and differentiated for 14 and 21 days in 24-well plates in the presence or

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

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    Koji Kaida

    2015-11-01

    Full Text Available 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.

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

    Science.gov (United States)

    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

  13. Osteogenic Differentiation Capacity of In Vitro Cultured Human Skeletal Muscle for Expedited Bone Tissue Engineering

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    Chunlei Miao

    2017-01-01

    Full Text Available Expedited bone tissue engineering employs the biological stimuli to harness the intrinsic regenerative potential of skeletal muscle to trigger the reparative process in situ to improve or replace biological functions. When genetically modified with adenovirus mediated BMP2 gene transfer, muscle biopsies from animals have demonstrated success in regenerating bone within rat bony defects. However, it is uncertain whether the human adult skeletal muscle displays an osteogenic potential in vitro when a suitable biological trigger is applied. In present study, human skeletal muscle cultured in a standard osteogenic medium supplemented with dexamethasone demonstrated significant increase in alkaline phosphatase activity approximately 24-fold over control at 2-week time point. More interestingly, measurement of mRNA levels revealed the dramatic results for osteoblast transcripts of alkaline phosphatase, bone sialoproteins, transcription factor CBFA1, collagen type I, and osteocalcin. Calcified mineral deposits were demonstrated on superficial layers of muscle discs after an extended 8-week osteogenic induction. Taken together, these are the first data supporting human skeletal muscle tissue as a promising potential target for expedited bone regeneration, which of the technologies is a valuable method for tissue repair, being not only effective but also inexpensive and clinically expeditious.

  14. Osteogenic Differentiation Capacity of In Vitro Cultured Human Skeletal Muscle for Expedited Bone Tissue Engineering.

    Science.gov (United States)

    Miao, Chunlei; Zhou, Lulu; Tian, Lufeng; Zhang, Yingjie; Zhang, Wei; Yang, Fanghong; Liu, Tianyi; Tang, Shengjian; Liu, Fangjun

    2017-01-01

    Expedited bone tissue engineering employs the biological stimuli to harness the intrinsic regenerative potential of skeletal muscle to trigger the reparative process in situ to improve or replace biological functions. When genetically modified with adenovirus mediated BMP2 gene transfer, muscle biopsies from animals have demonstrated success in regenerating bone within rat bony defects. However, it is uncertain whether the human adult skeletal muscle displays an osteogenic potential in vitro when a suitable biological trigger is applied. In present study, human skeletal muscle cultured in a standard osteogenic medium supplemented with dexamethasone demonstrated significant increase in alkaline phosphatase activity approximately 24-fold over control at 2-week time point. More interestingly, measurement of mRNA levels revealed the dramatic results for osteoblast transcripts of alkaline phosphatase, bone sialoproteins, transcription factor CBFA1, collagen type I, and osteocalcin. Calcified mineral deposits were demonstrated on superficial layers of muscle discs after an extended 8-week osteogenic induction. Taken together, these are the first data supporting human skeletal muscle tissue as a promising potential target for expedited bone regeneration, which of the technologies is a valuable method for tissue repair, being not only effective but also inexpensive and clinically expeditious.

  15. In Vitro Osteogenic and Odontogenic Differentiation of Human Dental Pulp Stem Cells Seeded on Carboxymethyl Cellulose-Hydroxyapatite Hybrid Hydrogel.

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    Gabriella eTeti

    2015-10-01

    Full Text Available 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.

  16. Comparative epigenetic influence of autologous versus fetal bovine serum on mesenchymal stem cells through in vitro osteogenic and adipogenic differentiation

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    Fani, Nesa [Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran (Iran, Islamic Republic of); Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran (Iran, Islamic Republic of); Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Ziadlou, Reihane [Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran (Iran, Islamic Republic of); Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran (Iran, Islamic Republic of); Shahhoseini, Maryam, E-mail: m.shahhoseini@royaninstitute.org [Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran (Iran, Islamic Republic of); Baghaban Eslaminejad, Mohamadreza, E-mail: eslami@royaninstitute.org [Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran (Iran, Islamic Republic of)

    2016-06-10

    Mesenchymal stem cells (MSCs) derived from bone marrow (BM) represents a useful source of adult stem cells for cell therapy and tissue engineering. MSCs are present at a low frequency in the BM; therefore expansion is necessary before performing clinical studies. Fetal bovine serum (FBS) as a nutritional supplement for in vitro culture of MSCs is a suitable additive for human cell culture, but not regarding subsequent use of these cells for clinical treatment of human patients due to the risk of viral and prion transmission as well as xenogeneic immune responses after transplantation. Recently, autologous serum (AS) has been as a supplement to replace FBS in culture medium. We compared the effect of FBS versus AS on the histone modification pattern of MSCs through in vitro osteogenesis and adipogenesis. Differentiation of stem cells under various serum conditions to a committed state involves global changes in epigenetic patterns that are critically determined by chromatin modifications. Chromatin immunoprecipitation (ChIP) coupled with real-time PCR showed significant changes in the acetylation and methylation patterns in lysine 9 (Lys9) of histone H3 on the regulatory regions of stemness (Nanog, Sox2, Rex1), osteogenic (Runx2, Oc, Sp7) and adipogenic (Ppar-γ, Lpl, adiponectin) marker genes in undifferentiated MSCs, FBS and AS. All epigenetic changes occurred in a serum dependent manner which resulted in higher expression level of stemness genes in undifferentiated MSCs compared to differentiated MSCs and increased expression levels of osteogenic genes in AS compared to FBS. Adipogenic genes showed greater expression in FBS compared to AS. These findings have demonstrated the epigenetic influence of serum culture conditions on differentiation potential of MSCs, which suggest that AS is possibly more efficient serum for osteogenic differentiation of MSCs in cell therapy purposes. - Highlights: • Bone marrow derived MSC could proliferate in AS as well as in FBS

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

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    Zhao, Ningbo, E-mail: curl-zhao@163.com; Wang, Xin, E-mail: 394041230@qq.com; Qin, Lei, E-mail: qinlei30@126.com; Guo, Zhengze, E-mail: zhzeguo@163.com; Li, Dehua, E-mail: lidehuafmmu@163.com

    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. Preconditioning Human Mesenchymal Stem Cells with a Low Concentration of BMP2 Stimulates Proliferation and Osteogenic Differentiation In Vitro

    DEFF Research Database (Denmark)

    Lysdahl, Helle; Baatrup, Anette; Foldager, Casper Bindzus

    2014-01-01

    treatment strategy in which human bone marrow-derived mesenchymal stem cells (hMSCs) are preconditioned with low concentrations of BMP2 for a short time in vitro. hMSCs in suspension were stimulated for 15 min with 10 and 20 ng/mL of BMP2. After the BMP2 was removed, the cells were seeded and cultured......MSCs. This implies that preconditioning with BMP2 might be more effective at inducing proliferation and osteogenic differentiation of hMSCs than continuous stimulation. Preconditioning with BMP2 could benefit the clinical application of BMP2 since side effects from high-dose treatments could be avoided....

  19. Dicalcium Phosphate Coated with Graphene Synergistically Increases Osteogenic Differentiation In Vitro

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    Jun Jae Lee

    2017-12-01

    Full Text Available In recent years, graphene and its derivatives have attracted much interest in various fields, including biomedical applications. In particular, increasing attention has been paid to the effects of reduced graphene oxide (rGO on cellular behaviors. On the other hand, dicalcium phosphate (DCP has been widely used in dental and pharmaceutical fields. In this study, DCP composites coated with rGO (DCP-rGO composites were prepared at various concentration ratios (DCP to rGO concentration ratios of 5:2.5, 5:5, and 5:10 μg/mL, respectively, and their physicochemical properties were characterized. In addition, the effects of DCP-rGO hybrid composites on MC3T3-E1 preosteoblasts were investigated. It was found that the DCP-rGO composites had an irregular granule-like structure with a diameter in the range order of the micrometer, and were found to be partially covered and interconnected with a network of rGO. The zeta potential analysis showed that although both DCP microparticles and rGO sheets had negative surface charge, the DCP-rGO composites could be successfully formed by the unique structural properties of rGO. In addition, it was demonstrated that the DCP-rGO composites significantly increased alkaline phosphatase activity and extracellular calcium deposition, indicating that the DCP-rGO hybrid composites can accelerate the osteogenic differentiation by the synergistic effects of rGO and DCP. Therefore, in conclusion, it is suggested that the DCP-rGO hybrid composites can be potent factors in accelerating the bone tissue regeneration.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Ze Tang

    Full Text Available 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.

  2. In vitro study of a novel oxysterol for osteogenic differentiation on rabbit bone marrow stromal cells.

    Science.gov (United States)

    Hokugo, Akishige; Sorice, Sarah; Yalom, Anisa; Lee, James C; Li, Andrew; Zuk, Patricia; Jarrahy, Reza

    2013-07-01

    Bone morphogenetic proteins (BMPs) are powerful osteoinductive growth factors but are associated with exorbitant costs and undesirable side effects. Oxysterols are biocompatible cholesterol oxidation products with osteoinductive properties that may represent an alternative to BMP. In this study, the authors examine the osteogenic potential and mechanisms of actions of oxysterol 49, a novel oxysterol analogue, in primary rabbit bone marrow stromal cells. Bone marrow stromal cells were isolated from the iliac crests of New Zealand White rabbits and then treated with various concentrations of oxysterol 49 or BMP-2, either alone or in combination. Alkaline phosphatase activity and expression of osteocalcin and osteopontin were evaluated. The effect of treatment of cells with cyclopamine, a known hedgehog signaling pathway inhibitor, was also assessed. Alkaline phosphatase activity was increased in cells treated with 1 µM oxysterol 49 relative to cells treated with BMP-2. Expression of osteocalcin and osteopontin in cells treated with oxysterol 49 and BMP-2 was equivalent. Alkaline phosphatase activity was decreased with the addition of cyclopamine. Combined treatment with oxysterol 49 and BMP-2 resulted in additive increases in alkaline phosphatase activity and osteocalcin and osteopontin expression. Oxysterol 49 has osteoinductive properties that are similar to those of BMP-2 in rabbit bone marrow stromal cells. The mechanism of this activity is at least in part related to the hedgehog signaling pathway. The two growth factors demonstrate additive effects when used in combination. Further study is required to examine the potential role of oxysterol 49 as a complement or alternative to BMP-2 in bone tissue engineering.

  3. Comparison of immunological properties of bone marrow stromal cells and adipose tissue-derived stem cells before and after osteogenic differentiation in vitro

    DEFF Research Database (Denmark)

    Niemeyer, Philipp; Kornacker, Martin; Mehlhorn, Alexander

    2007-01-01

    Mesenchymal stem cells (MSCs) can be isolated from various tissues and represent an attractive cell population for tissue-engineering purposes. MSCs from bone marrow (bone marrow stromal cells [BMSCs]) are negative for immunologically relevant surface markers and inhibit proliferation of allogenic...... T cells in vitro. Therefore, BMSCs are said to be available for allogenic cell therapy. Although the immunological characteristics of BMSCs have been the subject of various investigations, those of stem cells isolated from adipose tissue (ASCs) have not been adequately described. In addition......, the influence of osteogenic differentiation in vitro on the immunological characteristics of BMSCs and ASCs is the subject of this article. Before and after osteogenic induction, the influence of BMSCs and ASCs on the proliferative behavior of resting and activated allogenic peripheral blood mononuclear cells...

  4. Effect of hyaluronan on osteogenic differentiation of porcine bone marrow stromal cells in vitro

    DEFF Research Database (Denmark)

    Zou, Lijin; Zou, Xuenong; Chen, Li

    2007-01-01

    Hyaluronan (HA) plays a predominant role in tissue morphogenesis, cell migration, proliferation, and cell differentiation. The aims of the present study were to investigate whether (i) prolonged presence of high concentration (4.0 mg/mL) 800 KDa HA and (ii) pretreatment with HA can modify osteoge...

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

    Science.gov (United States)

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

    2013-01-01

    Healthy mammalian cells in normal tissues are organized in complex three-dimensional (3D) networks that display nutrient and signaling gradients. Conventional techniques that grow cells in a 2D monolayer fail to reproduce the environment that is observed in vivo. In recent years, 3D culture systems have been used to mimic tumor microenvironments in cancer research and to emulate embryogenesis in stem cell cultures. However, there have been no studies exploring the ability for adipose-derived stromal (ADS) cells in a 3D culture system to undergo osteogenic differentiation. To characterize and investigate the in vitro and in vivo potential for human ADS cells in a novel 3D culture system to undergo osteogenic differentiation. Basic science and laboratory study. Human ADS cells were isolated and prepared as either a 2D monolayer or 3D multicellular aggregates (MAs). Multicellular aggregates were formed using the hanging droplet technique. Cells were treated in osteogenic medium in vitro, and cellular differentiation was investigated using gene expression, histology, and microCT at 1-, 2-, and 4-week time points. In vivo investigation involved creating a muscle pouch by developing the avascular muscular interval in the vastus lateralis of male athymic rats. Specimens were then pretreated with osteogenic medium and surgically implanted as (1) carrier (Matrigel) alone (control), (2) carrier with human ADS cells in monolayer, or (3) human ADS cells as MAs. In vivo evidence of osteogenic differentiation was evaluated with micro computed tomography and histologic sectioning at a 2-week time point. Human ADS cells cultured by the hanging droplet technique successfully formed MAs at the air-fluid interface. Adipose-derived stromal cells cultured in monolayer or as 3D MAs retain their ability to self-replicate and undergo multilineage differentiation as confirmed by increased runx2/Cbfa2, ALP, and OCN and increased matrix mineralization on histologic sectioning

  6. Hyperbaric oxygen promotes osteogenic differentiation of bone marrow stromal cells by regulating Wnt3a/β-catenin signaling—An in vitro and in vivo study

    Directory of Open Access Journals (Sweden)

    Song-Shu Lin

    2014-01-01

    Full Text Available We hypothesized that the effect of hyperbaric oxygen (HBO on bone formation is increased via osteogenic differentiation of bone marrow stromal cells (BMSCs, which is regulated by Wnt3a/β-catenin signaling. Our in vitro data showed that HBO increased cell proliferation, Wnt3a production, LRP6 phosphorylation, and cyclin D1 expression in osteogenically differentiated BMSCs. The mRNA and protein levels of Wnt3a, β-catenin, and Runx2 were upregulated while those of GSK-3β were downregulated after HBO treatment. The relative density ratio (phospho-protein/protein of Akt and GSK-3β was both up-regulated while that of β-catenin was down-regulated after HBO treatment. We next investigated whether HBO affects the accumulation of β-catenin. Our Western blot analysis showed increased levels of translocated β-catenin that stimulated the expression of target genes after HBO treatment. HBO increased TCF-dependent transcription, Runx2 promoter/Luc gene activity, and the expression of osteogenic markers of BMSCs, such as alkaline phosphatase activity, type I collagen, osteocalcin, calcium, and the intensity of Alizarin Red staining. HBO dose dependently increased the bone morphogenetic protein (BMP2 and osterix production. We further demonstrated that HBO increased the expression of vacuolar-ATPases, which stimulated Wnt3a secretion from BMSCs. Finally, we showed that the beneficial effects of HBO on bone formation were related to Wnt3a/β-catenin signaling in a rabbit model by histology, mechanical testing, and immunohistochemical assays. Accordingly, we concluded that HBO increased the osteogenic differentiation of BMSCs by regulating Wnt3a secretion and signaling.

  7. In vitro proliferation and osteogenic differentiation of human dental pulp stem cells in injectable thermo-sensitive chitosan/β-glycerophosphate/hydroxyapatite hydrogel.

    Science.gov (United States)

    Chen, Yantian; Zhang, Fengli; Fu, Qiang; Liu, Yong; Wang, Zejian; Qi, Nianmin

    2016-09-01

    Injectable thermo-sensitive hydrogels have a potential application in bone tissue engineering for their sensitivities and minimal invasive properties. Human dental pulp stem cells have been considered a promising tool for tissue reconstruction. The objective of this study was to investigate the proliferation and osteogenic differentiation of dental pulp stem cells in injectable thermo-sensitive chitosan/β-glycerophosphate/hydroxyapatite hydrogel in vitro. The chitosan /β-glycerophosphate hydrogel and chitosan/β-glycerophosphate/hydroxyapatite hydrogel were prepared using the sol-gel method. The injectability of chitosan /β-glycerophosphate hydrogel and chitosan/β-glycerophosphate/hydroxyapatite hydrogel was measured using a commercial disposable syringe. Scanning electron microscopy was used to observe the inner structure of hydrogels. Then dental pulp stem cells were seeded in chitosan /β-glycerophosphate hydrogel and chitosan/β-glycerophosphate/hydroxyapatite hydrogel, respectively. The growth of dental pulp stem cells was periodically observed under an inverted microscope. The proliferation of dental pulp stem cells was detected by using an Alamar Blue kit, while cell apoptosis was determined by using a Live/Dead Viability/Cytotoxicity kit. The osteogenic differentiations of dental pulp stem cells in chitosan /β-glycerophosphate hydrogel and chitosan/β-glycerophosphate/hydroxyapatite hydrogel were evaluated by alkaline phosphatase activity assay and mRNA expression of osteogenesis gene for 21 days in osteogenic medium. The results indicated that there was no significant difference between chitosan /β-glycerophosphate hydrogel and chitosan/β-glycerophosphate/hydroxyapatite hydrogel in injectability. Cells within the chitosan/β-glycerophosphate/hydroxyapatite hydrogel displayed a typical adherent cell morphology and rapid proliferation with high cellular viability after 14 days of culture. Dental pulp stem cells seeded in chitosan

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

    Science.gov (United States)

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

    2016-04-01

    A major bottleneck to the therapeutic applications of dental pulp stem cells (DPSC) are their limited proliferative capacity ex vivo and tendency to undergo senescence. This may be partly due to the sub-optimal in vitro culture milieu, which could be improved by an appropriate extracellular matrix substratum. This study therefore examined decellularized matrix (DECM) from stem cells derived from human exfoliated deciduous teeth (SHED) and periodontal ligament stem cells (PDLSC), as potential substrata for DPSC culture. Both SHED-DECM and PDLSC-DECM promoted rapid adhesion and spreading of newly-seeded DPSC compared to bare polystyrene (TCPS), with vinculin immunocytochemistry showing expression of more focal adhesions by newly-adherent DPSC cultured on DECM versus TCPS. Culture of DPSC on SHED-DECM and PDLSC-DECM yielded higher proliferation of cell numbers compared to TCPS. The qRT-PCR data showed significantly higher expression of nestin by DPSC cultured on DECM versus the TCPS control. Osteogenic differentiation of DPSC was enhanced by culturing on PDLSC-DECM and SHED-DECM versus TCPS, as demonstrated by alizarin red S staining for mineralized calcium deposition, alkaline phosphatase assay and qRT-PCR analysis of key osteogenic marker expression. Hence, both SHED-DECM and PDLSC-DECM could enhance the ex vivo culture of DPSC under both non-inducing and osteogenic-inducing conditions. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Reconstitution of bone-like matrix in osteogenically differentiated mesenchymal stem cell–collagen constructs: A three-dimensional in vitro model to study hematopoietic stem cell niche

    Directory of Open Access Journals (Sweden)

    WY Lai

    2013-10-01

    Full Text Available Mesenchymal stem/stromal cells (MSCs and osteoblasts are important niche cells for hematopoietic stem cells (HSCs in bone marrow osteoblastic niche. Here, we aim to partially reconstitute the bone marrow HSC niche in vitro using collagen microencapsulation for investigation of the interactions between HSCs and MSCs. Mouse MSCs (mMSCs microencapsulated in collagen were osteogenically differentiated to derive a bone-like matrix consisting of osteocalcin, osteopontin, and calcium deposits and secreted bone morphogenic protein 2 (BMP2. Decellularized bone-like matrix was seeded with fluorescence-labeled human MSCs and HSCs. Comparing with pure collagen scaffold, significantly more HSCs and HSC–MSC pairs per unit area were found in the decellularized bone-like matrix. Moreover, incubation with excess neutralizing antibody of BMP2 resulted in a significantly higher number of HSC per unit area than that without in the decellularized matrix. This work suggests that the osteogenic differentiated MSC–collagen microsphere is a valuable three-dimensional in vitro model to elucidate cell–cell and cell–matrix interactions in HSC niche.

  10. Osteogenic differentiation of amniotic epithelial cells: synergism of pulsed electromagnetic field and biochemical stimuli

    OpenAIRE

    Wang, Qian; Wu, Wenchao; Han, Xiaoyu; Zheng, Ai; Lei, Song; Wu, Jiang; Chen, Huaiqing; He, Chengqi; Luo, Fengming; Liu, Xiaojing

    2014-01-01

    Background Pulsed electromagnetic field (PEMF) is a non-invasive physical therapy used in the treatment of fracture nonunion or delayed healing. PEMF can facilitate the osteogenic differentiation of bone marrow mesenchymal stem cells in vitro. Amniotic epithelial cells (AECs) have been proposed as a potential source of stem cells for cell therapy. However, whether PEMF could modulate the osteogenic differentiation of AECs is unknown. In the present study, the effects of PEMF on the osteogenic...

  11. Novel markers of osteogenic and adipogenic differentiation of human bone marrow stromal cells identified using a quantitative proteomics approach

    Directory of Open Access Journals (Sweden)

    Cecilia Granéli

    2014-01-01

    It has been concluded from the present results that CD10 and CD92 are potential markers of osteogenic and adipogenic differentiation and that CRYaB is a potential novel osteogenic marker specifically expressed during the osteogenic differentiation of hBMSCs in vitro.

  12. Effects of Immobilized BMP-2 and Nanofiber Morphology on In Vitro Osteogenic Differentiation of hMSCs and In Vivo Collagen Assembly of Regenerated Bone.

    Science.gov (United States)

    Perikamana, Sajeesh Kumar Madhurakkat; Lee, Jinkyu; Ahmad, Taufiq; Jeong, Yonghoon; Kim, Do-Gyoon; Kim, Kyobum; Shin, Heungsoo

    2015-04-29

    Engineering bone tissue is particularly challenging because of the distinctive structural features of bone within a complex biochemical environment. In the present study, we fabricated poly(L-lactic acid) (PLLA) electrospun nanofibers with random and aligned morphology immobilized with bone morphogenic protein-2 (BMP-2) and investigated how these signals modulate (1) in vitro osteogenic differentiation of human mesenchymal stem cells (hMSCs) and (2) in vivo bone growth rate, mechanical properties, and collagen assembly of newly formed bone. The orientation of adherent cells followed the underlying nanofiber morphology; however, nanofiber alignment did not show any difference in alkaline phosphate activity or in calcium mineralization of hMSCs after 14 days of in vitro culture in osteogenic differentiation media. In vivo bone regeneration was significantly higher in the nanofiber implanted groups (approximately 65-79%) as compared to the defect-only group (11.8 ± 0.2%), while no significant difference in bone regeneration was observed between random and aligned groups. However, nanoindentation studies of regenerated bone revealed Young's modulus and contact hardness with anisotropic feature for aligned group as compared to random group. More importantly, structural analysis of collagen at de novo bone showed the ability of nanofiber morphology to guide collagen deposition. SEM and TEM images revealed regular, highly ordered collagen assemblies on aligned nanofibers as compared to random fibers, which showed irregular, randomly organized collagen deposition. Taken together, we conclude that nanofibers in the presence of osteoinductive signals are a potent tool for bone regeneration, and nanofiber alignment can be used for engineering bone tissues with structurally assembled collagen fibers with defined direction.

  13. Osteogenic Potential Differentiation of Human Amnion Mesenchymal Stem Cell with Chitosan-Carbonate Apatite Scaffold (In Vitro Study

    Directory of Open Access Journals (Sweden)

    Michael J.K. Kamadjaja

    2016-09-01

    Full Text Available Background: Tissue engineering based approaches have received much attention. Incorporation of chitosan and carbonate apatite (CA improve its capability. Human mesenchymal stem cells (hMSCs is viable for xenogenic transplantation. The purpose of this study was to fabricate and evaluate the osteogenic potential diferentiation of human amnion mesenchymal stem cell with carbonate apatite–chitosan scaffolds (CA-ChSs for tissue engineering. Method: Human amniotic membrane was procured from using cesarean section. Soncini’s protocol was employed for the isolation procedure. The cells cultured on collagen-coated dishes using Dulbecco's minimal essential medium (DMEM/F12 (1:1. A chitosan powder of medium molecular weight deacetylated chitin, poly(D(glucosamine was used and mixed with CA. Immunocytochemistry and flowcytometry used for phenotypic characterization of hAMSC. Result: Amniotic membrane obtained using cesarean section under aseptic condition did not exhibit any growth of cell cultures which were not contaminated. Immunocytochemistry testing revealed that the target cells expressed strong mesenchymal stem cell marker CD 105. Characterization at passage 10 showed that CD44 was the most significant and abundant surface receptors. The number of viable cells in chitosan-carbonate apatite was 66.59%. Scanning electron microscope (SEM observation revealed that CA-ChSs had three-dimensional structure with many pores and hAMSc could attached and proliferation among the porosity of the scaffold. The formation of calcium in the cell as an indicator of osteoblast cells was detected using Alizarin Red solution. Conclusion: hAMSc harvested from human amniotic membrane seeding in CA-ChSs had the capability for in vitro osteogenesis makes them be the one of the potential options for bone tissue engineering.

  14. Osteogenic Differentiation of Human Dental Pulp Stromal Cells on 45S5 Bioglass® Based Scaffolds In Vitro and In Vivo

    Science.gov (United States)

    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

  15. Effects of Naringin on Proliferation and Osteogenic Differentiation of Human Periodontal Ligament Stem Cells In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Lihua Yin

    2015-01-01

    Full Text Available This study is to explore the osteogenesis potential of the human periodontal ligament stem cells (hPDLSCs induced by naringin in vitro and in vitro. The results confirmed that 1 μM naringin performs the best effect and a collection of bone-related genes (RUNX2, COL1A2, OPN, and OCN had significantly higher expression levels compared to the control group. Furthermore, a typical trabecular structure was observed in vivo, surrounded by a large amount of osteoblasts. These results demonstrated that naringin, at a concentration of 1 μM, can efficiently promote the proliferation and differentiation of hPDLSCs both in vitro and in vivo.

  16. A novel strontium(II)-modified calcium phosphate bone cement stimulates human-bone-marrow-derived mesenchymal stem cell proliferation and osteogenic differentiation in vitro.

    Science.gov (United States)

    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. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    Brauer, A; Pohlemann, T; Metzger, W

    2016-01-01

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

  18. Osteogenic Differentiation in Healthy and Pathological Conditions

    OpenAIRE

    Maria Teresa Valenti; Luca Dalle Carbonare; Monica Mottes

    2016-01-01

    This review focuses on the osteogenic differentiation of mesenchymal stem cells (MSC), bone formation and turn-over in good and ill skeletal fates. The interacting molecular pathways which control bone remodeling in physiological conditions during a lifelong process are described. Then, alterations of the molecular pathways regulating osteogenesis are addressed. In the aging process, as well as in glucocorticoid-induced osteoporosis, bone loss is caused not only by an unbalanced bone resorpti...

  19. Osteogenic Differentiation in Healthy and Pathological Conditions

    Directory of Open Access Journals (Sweden)

    Maria Teresa Valenti

    2016-12-01

    Full Text Available This review focuses on the osteogenic differentiation of mesenchymal stem cells (MSC, bone formation and turn-over in good and ill skeletal fates. The interacting molecular pathways which control bone remodeling in physiological conditions during a lifelong process are described. Then, alterations of the molecular pathways regulating osteogenesis are addressed. In the aging process, as well as in glucocorticoid-induced osteoporosis, bone loss is caused not only by an unbalanced bone resorption activity, but also by an impairment of MSCs’ commitment towards the osteogenic lineage, in favour of adipogenesis. Mutations affecting the expression of key genes involved in the control of bone development occur in several heritable bone disorders. A few examples are described in order to illustrate the pathological consequences of perturbation in different steps of osteogenic commitment, osteoblast maturation, and matrix mineralization, respectively. The involvement of abnormal MSC differentiation in cancer is then discussed. Finally, a brief overview of clinical applications of MSCs in bone regeneration and repair is presented.

  20. [RECOMBINANT ADENOVIRUS-MEDIATED BONE MORPHOGENETIC PROTEIN 9 AND ERYTHROPOIETIN GENES CO-TRANSFECTION IN PROMOTING OSTEOGENIC DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS IN VITRO].

    Science.gov (United States)

    Zhang, Guangde; Su, Chengshuai; Jin, Xia; Yang, Shimao; Fang, Dianji; Guo, Yanwei

    2016-03-01

    To investigate the effect of recombinant adenovirus-mediated bone morphogenetic protein 9 (BMP-9) and erythropoietin (EPO) genes co-transfection on osteogenic differentiation of adipose-derived stem cells (ADSCs) in vitro. The inguinal adipose tissue was harvested from 4-month-old New Zealand rabbits, ADSCs were isolated with enzyme digestion and adherence method, and multipotent differentiation capacity was identified. The 3rd generation ADSCs were divided into 5 groups: normal cells (group A), empty plasmid control group (group B), BMP-9 or EPO recombinant adenovirus transfected cells (groups C and D), BMP-9 and EPO recombinant adenovirus co-transfected cells (group E). The inverted phase contrast microscope was used to observe the cell growth at 7 days; the expression of cell fluorescence was observed under a fluorescence microscope at 14 days, and viral transfection efficiency was calculated at 48 hours; Western blot was used to detect the expressions of BMP-9 and EPO proteins at 14 days. The expression of alkaline phosphatase (ALP) activity was detected at 3, 7, and 14 days after osteogenic induction, and alizarin red staining was used to detect calcium nodules formation and real-time fluorescence quantitative PCR to detect the expressions of osteopontin (OPN) and osteocalcin (OCN) at 3 weeks. At 7 days after transfected, some cells showed oval, round, and irregular shape under the inverted phase contrast microscope in groups A and B; a few fusiform cells were observed in groups C and D; oval cells increased obviously, and there were only few round cells in group E. The fluorescence microscope observation showed that BMP-9 and EPO, BMP-9/EPO recombinant adenovirus could stably transfected ADSCs, with transfection efficiency of 80%-93%. The expressions of BMP-9 and EPO proteins significantly higher in group E than the other groups by Western blot (P transfect ADSCs, which can stably express in ADSCs, BMP-9/EPO genes co-transfection can more promote the

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

    Science.gov (United States)

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

    2016-12-01

    Predictable regeneration of alveolar bone defects has always been a challenge in implant dentistry. Bone allografts are widely used bone substitutes with controversial osteoinductive activity. This in vitro study aimed to assess the osteogenic potential of some commercially available freeze-dried bone allografts supplemented with human recombinant platelet-derived growth factor-BB and transforming growth factor beta-1. Cell viability, mineralization, and osteogenic gene expression of MG-63 osteoblast-like cells were compared among the allograft alone, allograft/platelet-derived growth factor-BB, allograft/transforming growth factor beta-1, and allograft/platelet-derived growth factor-BB/transforming growth factor beta-1 groups. The methyl thiazol tetrazolium assay, real-time quantitative reverse transcription polymerase chain reaction and alizarin red staining were performed, respectively, for assessment of cell viability, differentiation, and mineralization at 24-72 h post treatment. The allograft with greater cytotoxic effect on MG-63 cells caused the lowest differentiation among the groups. In comparison with allograft alone, allograft/transforming growth factor beta-1, and allograft/transforming growth factor beta-1/platelet-derived growth factor-BB caused significant upregulation of bone sialoprotein and osteocalcin osteogenic mid-late marker genes, and resulted in significantly higher amounts of calcified nodules especially in mineralized non-cytotoxic allograft group. Supplementation of platelet-derived growth factor-BB alone in 5 ng/mL concentration had no significant effect on differentiation or mineralization markers. According to the results, transforming growth factor beta-1 acts synergistically with bone allografts to enhance the osteogenic differentiation potential. Therefore, this combination may be useful for rapid transformation of undifferentiated cells into bone-forming cells for bone regeneration. However, platelet-derived growth factor

  2. BMP2 induces chondrogenic differentiation, osteogenic differentiation and endochondral ossification in stem cells.

    Science.gov (United States)

    Zhou, Nian; Li, Qi; Lin, Xin; Hu, Ning; Liao, Jun-Yi; Lin, Liang-Bo; Zhao, Chen; Hu, Zhen-Ming; Liang, Xi; Xu, Wei; Chen, Hong; Huang, Wei

    2016-10-01

    Bone morphogenetic protein 2 (BMP2), a member of the transforming growth factor-β (TGF-β) super-family, is one of the main chondrogenic growth factors involved in cartilage regeneration. BMP2 is known to induce chondrogenic differentiation in various types of stem cells in vitro. However, BMP2 also induces osteogenic differentiation and endochondral ossification in mesenchymal stem cells (MSCs). Although information regarding BMP2-induced chondrogenic and osteogenic differentiation within the same system might be essential for cartilage tissue engineering, few studies concerning these issues have been conducted. In this study, BMP2 was identified as a regulator of chondrogenic differentiation, osteogenic differentiation and endochondral bone formation within the same system. BMP2 was used to regulate chondrogenic and osteogenic differentiation in stem cells within the same culture system in vitro and in vivo. Any changes in the differentiation markers were assessed. BMP2 was found to induce chondrogenesis and osteogenesis in vitro via the expression of Sox9, Runx2 and its downstream markers. According to the results of the subcutaneous stem cell implantation studies, BMP2 not only induced cartilage formation but also promoted endochondral ossification during ectopic bone/cartilage formation. In fetal limb cultures, BMP2 promoted chondrocyte hypertrophy and endochondral ossification. Our data reveal that BMP2 can spontaneously induce chondrogenic differentiation, osteogenic differentiation and endochondral bone formation within the same system. Thus, BMP2 can be used in cartilage tissue engineering to regulate cartilage formation but has to be properly regulated for cartilage tissue engineering in order to retain the cartilage phenotype.

  3. In vitro Evaluation of the Effect of Melatonin on Osteogenic Differen-tiation of PDL Mesenchymal Stem Cells Isolated from Chronic Perio-dontitis Affected Teeth

    Directory of Open Access Journals (Sweden)

    M. Bidgoli

    2016-04-01

    Full Text Available Introduction & Objective: In recent years stem cells have been evaluated for regenerating lost tissues. Therefore, many studies aimed to assess isolation of these cells from different origins, and culture them. The purpose of this study is to evaluate the osteogenic differentiation of stem cells derived from periodontal ligament of periodontitis affected teeth in melatonin contained media. Materials & Methods: In this experimental-laboratory study after tooth extraction and isolating the apical segment of the roots, the periodontal ligament tissue was removed. Cells were expanded and after the third passage; flow cytometery analysis was performed to evaluate the surface markers (CD 105, CD146, CD90, CD 45, CD31, CD34, CD106 and CD 73. Adherent cell layer was used for osteogenic differentiation in melatonin contained osteogenic media. Then, osteogenic differentiation was evaluated using alizarin red staining, alkaline phosphatase and calcium content tests were performed. Results: Quantitative analysis of alizarin red staining on the 28th day demonstrated that mineralized nodule formation in the group supplemented with melatonin was higher than the control group. Results from alkaline phosphatase activity test on the 7th, 14th and 21st days demonstrated that, this activity was higher in the group supplemented with melatonin. Also, the amount of calcium on the 7th, 14th, 21st and 28th days, in melatonin group, was higher than the control group. Conclusion: Melatonin (50 µM may be beneficial for differentiation of PDL stem cells into osteoblast. (Sci J Hamadan Univ Med Sci 2016; 23 (1:49-56

  4. Sox9 potentiates BMP2-induced chondrogenic differentiation and inhibits BMP2-induced osteogenic differentiation.

    Directory of Open Access Journals (Sweden)

    Junyi Liao

    Full Text Available Bone morphogenetic protein 2 (BMP2 is one of the key chondrogenic growth factors involved in the cartilage regeneration. However, it also exhibits osteogenic abilities and triggers endochondral ossification. Effective chondrogenesis and inhibition of BMP2-induced osteogenesis and endochondral ossification can be achieved by directing the mesenchymal stem cells (MSCs towards chondrocyte lineage with chodrogenic factors, such as Sox9. Here we investigated the effects of Sox9 on BMP2-induced chondrogenic and osteogenic differentiation of MSCs. We found exogenous overexpression of Sox9 enhanced the BMP2-induced chondrogenic differentiation of MSCs in vitro. Also, it inhibited early and late osteogenic differentiation of MSCs in vitro. Subcutaneous stem cell implantation demonstrated Sox9 potentiated BMP2-induced cartilage formation and inhibited endochondral ossification. Mouse limb cultures indicated that BMP2 and Sox9 acted synergistically to stimulate chondrocytes proliferation, and Sox9 inhibited BMP2-induced chondrocytes hypertrophy and ossification. This study strongly suggests that Sox9 potentiates BMP2-induced MSCs chondrogenic differentiation and cartilage formation, and inhibits BMP2-induced MSCs osteogenic differentiation and endochondral ossification. Thus, exogenous overexpression of Sox9 in BMP2-induced mesenchymal stem cells differentiation may be a new strategy for cartilage tissue engineering.

  5. Improvement of In Vitro Osteogenic Potential through Differentiation of Induced Pluripotent Stem Cells from Human Exfoliated Dental Tissue towards Mesenchymal-Like Stem Cells

    Directory of Open Access Journals (Sweden)

    Felipe Augusto Andre Ishiy

    2015-01-01

    Full Text Available Constraints for the application of MSCs for bone reconstruction include restricted self-renewal and limited cell amounts. iPSC technology presents advantages over MSCs, providing homogeneous cellular populations with prolonged self-renewal and higher plasticity. However, it is unknown if the osteogenic potential of iPSCs differs from that of MSCs and if it depends on the iPSCs originating cellular source. Here, we compared the in vitro osteogenesis between stem cells from human deciduous teeth (SHED and MSC-like cells from iPSCs from SHED (iPS-SHED and from human dermal fibroblasts (iPS-FIB. MSC-like cells from iPS-SHED and iPS-FIB displayed fibroblast-like morphology, downregulation of pluripotency markers and upregulation of mesenchymal markers. Comparative in vitro osteogenesis analysis showed higher osteogenic potential in MSC-like cells from iPS-SHED followed by MSC-like cells from iPS-FIB and SHED. CD105 expression, reported to be inversely correlated with osteogenic potential in MSCs, did not display this pattern, considering that SHED presented lower CD105 expression. Higher osteogenic potential of MSC-like cells from iPS-SHED may be due to cellular homogeneity and/or to donor tissue epigenetic memory. Our findings strengthen the rationale for the use of iPSCs in bone bioengineering. Unveiling the molecular basis behind these differences is important for a thorough use of iPSCs in clinical scenarios.

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

    Energy Technology Data Exchange (ETDEWEB)

    Viale-Bouroncle, Sandra; Voellner, Florian [Department of Operative Dentistry and Periodontology, University Hospital Regensburg, Regensburg (Germany); Moehl, Christoph; Kuepper, Kevin [Institute of Complex Systems, ICS7: Biomechanics, Forschungszentrum Juelich, Juelich (Germany); Brockhoff, Gero [Department of Gynecology and Obstetrics, University of Regensburg (Germany); Reichert, Torsten E. [Department of Oral and Maxillofacial Surgery, University Hospital Regensburg, Regensburg (Germany); Schmalz, Gottfried [Department of Operative Dentistry and Periodontology, University Hospital Regensburg, Regensburg (Germany); Morsczeck, Christian, E-mail: christian.morsczeck@klinik.uni-regensburg.de [Department of Operative Dentistry and Periodontology, University Hospital Regensburg, Regensburg (Germany)

    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.

  7. In Vitro Study of the Effect of Vitamin E on Viability, Morphological Changes and Induction of Osteogenic Differentiation in Adult Rat Bone Marrow Mesenchymal Stem Cells

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    M Soleimani Mehranjani

    2014-10-01

    Full Text Available Introduction: Vitamin E as a strong antioxidant plays an important role in inhibiting free radicals. Therefore, this study aimed to investigate the effect of vitamin E on the viability, morphology and osteogenic differentiation in bone marrow mesenchymal stem cells of an adult rat. Methods: The bone marrow mesenchymal stem cells were extracted using the flashing-out method. At the end of the third passage, cells were divided into groups of control and experimental. Experimental cells were treated withVitamin E (5,10,15,25,50,100,150μM for a period of 21 days in the osteogenic media containing 10% of fetal bovine serum. The cell viability, bone matrix mineralization, intercellular and extracellular calcium deposition, alkaline phosphatase activity, expression of genes and synthesis of proteins of osteopontin and osteocalcin as well as morphological changes of the cells were investigated. The study data was analyzed using one-way ANOVA and T-Test setting the significant P value at P<0.05. Results: Within vitamin- E treated cells, the mean viability, mean bone matrix mineralization, calcium deposition, alkaline phosphatase activity, expression and synthesis of osteopontin and osteocalcin of the mesenchymal stem cells treated with vitamin E significantly increased in a dose dependent manner. Also cytoplasm extensions were observed in the cells treated with vitamin E. Conclusion: Since vitamin E caused a significant increase in cell viability and osteogenic differentiation in the mesenchymal stem cells, therefore it can be utilized in order to increase cell differentiation and cell survival.

  8. The regulatory landscape of osteogenic differentiation.

    Science.gov (United States)

    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. © 2014 AlphaMed Press.

  9. Retinoic acids potentiate BMP9-induced osteogenic differentiation of mesenchymal progenitor cells.

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    Wenli Zhang

    2010-07-01

    Full Text Available As one of the least studied bone morphogenetic proteins (BMPs, BMP9 is one of the most osteogenic BMPs. Retinoic acid (RA signaling is known to play an important role in development, differentiation and bone metabolism. In this study, we investigate the effect of RA signaling on BMP9-induced osteogenic differentiation of mesenchymal progenitor cells (MPCs.Both primary MPCs and MPC line are used for BMP9 and RA stimulation. Recombinant adenoviruses are used to deliver BMP9, RARalpha and RXRalpha into MPCs. The in vitro osteogenic differentiation is monitored by determining the early and late osteogenic markers and matrix mineralization. Mouse perinatal limb explants and in vivo MPC implantation experiments are carried out to assess bone formation. We find that both 9CRA and ATRA effectively induce early osteogenic marker, such as alkaline phosphatase (ALP, and late osteogenic markers, such as osteopontin (OPN and osteocalcin (OC. BMP9-induced osteogenic differentiation and mineralization is synergistically enhanced by 9CRA and ATRA in vitro. 9CRA and ATRA are shown to induce BMP9 expression and activate BMPR Smad-mediated transcription activity. Using mouse perinatal limb explants, we find that BMP9 and RAs act together to promote the expansion of hypertrophic chondrocyte zone at growth plate. Progenitor cell implantation studies reveal that co-expression of BMP9 and RXRalpha or RARalpha significantly increases trabecular bone and osteoid matrix formation.Our results strongly suggest that retinoid signaling may synergize with BMP9 activity in promoting osteogenic differentiation of MPCs. This knowledge should expand our understanding about how BMP9 cross-talks with other signaling pathways. Furthermore, a combination of BMP9 and retinoic acid (or its agonists may be explored as effective bone regeneration therapeutics to treat large segmental bony defects, non-union fracture, and/or osteoporotic fracture.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Sung Eun Kim

    2015-01-01

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

  12. Review of Signaling Pathways Governing MSC Osteogenic and Adipogenic Differentiation

    Directory of Open Access Journals (Sweden)

    Aaron W. James

    2013-01-01

    Full Text Available Mesenchymal stem cells (MSC are multipotent cells, functioning as precursors to a variety of cell types including adipocytes, osteoblasts, and chondrocytes. Between osteogenic and adipogenic lineage commitment and differentiation, a theoretical inverse relationship exists, such that differentiation towards an osteoblast phenotype occurs at the expense of an adipocytic phenotype. This balance is regulated by numerous, intersecting signaling pathways that converge on the regulation of two main transcription factors: peroxisome proliferator-activated receptor-γ (PPARγ and Runt-related transcription factor 2 (Runx2. These two transcription factors, PPARγ and Runx2, are generally regarded as the master regulators of adipogenesis and osteogenesis. This review will summarize signaling pathways that govern MSC fate towards osteogenic or adipocytic differentiation. A number of signaling pathways follow the inverse balance between osteogenic and adipogenic differentiation and are generally proosteogenic/antiadipogenic stimuli. These include β-catenin dependent Wnt signaling, Hedgehog signaling, and NELL-1 signaling. However, other signaling pathways exhibit more context-dependent effects on adipogenic and osteogenic differentiation. These include bone morphogenic protein (BMP signaling and insulin growth factor (IGF signaling, which display both proosteogenic and proadipogenic effects. In summary, understanding those factors that govern osteogenic versus adipogenic MSC differentiation has significant implications in diverse areas of human health, from obesity to osteoporosis to regenerative medicine.

  13. EGR1 supports the osteogenic differentiation of dental stem cells.

    Science.gov (United States)

    Press, T; Viale-Bouroncle, S; Felthaus, O; Gosau, M; Morsczeck, C

    2015-02-01

    To evaluate whether and how the transcription factor early growth response gene 1 (EGR1) affects the osteogenic differentiation of dental stem cells. Dental stem cells from apical papilla (SCAPs) and from the dental follicle (DFCs) were transfected with EGR1-specific siRNA or EGR-1 expression plasmid. Gene regulation was verified at protein level by Western blotting. The expression of the transcription factors distal-less homeobox 3 (DLX3), alkaline phosphatase (ALP) and bone morphogenetic protein 2 (BMP2), which are all regulators and markers of the osteogenic differentiation in dental stem cells, was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). To investigate mineralization, SCAP long-term cultures were stained with alizarin red after EGR1 over-expression. EGR1 was induced in SCAPs during osteogenic differentiation. DLX3 and bone morphogenetic protein 2 (BMP2) were up-regulated after EGR1 over-expression and down-regulated after EGR1 depletion. The expression of ALP was also down-regulated after EGR1 depletion. The over-expression of EGR1 in SCAPs promoted mineralization after osteogenic differentiation. EGR1 supported the osteogenic differentiation of dental stem cells by potentially regulating the expression of DLX3 and BMP2. © 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.

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

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    Chen-Shuang Li

    2016-01-01

    Full Text Available 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.

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

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    Federica Viti

    Full Text Available 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

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

    Science.gov (United States)

    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

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

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    Jun-Ha Hwang

    Full Text Available 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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2015-12-01

    Periostitis, which is characterized by bony pain and diffuse periosteal ossification, has been increasingly reported with prolonged clinical use of voriconazole. While resolution of clinical symptoms following discontinuation of therapy suggests a causative role for voriconazole, the biological mechanisms contributing to voriconazole-induced periostitis are unknown. To elucidate potential mechanisms, we exposed human osteoblasts in vitro to voriconazole or fluconazole at 15 or 200 μg/ml (reflecting systemic or local administration, respectively), under nonosteogenic or osteogenic conditions, for 1, 3, or 7 days and evaluated the effects on cell proliferation (reflected by total cellular DNA) and osteogenic differentiation (reflected by alkaline phosphatase activity, calcium accumulation, and expression of genes involved in osteogenic differentiation). Release of free fluoride, vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) was also measured in cell supernatants of osteoblasts exposed to triazoles, with an ion-selective electrode (for free fluoride) and enzyme-linked immunosorbent assays (ELISAs) (for VEGF and PDGF). Voriconazole but not fluconazole significantly enhanced the proliferation and differentiation of osteoblasts. In contrast to clinical observations, no increases in free fluoride levels were detected following exposure to either voriconazole or fluconazole; however, significant increases in the expression of VEGF and PDGF by osteoblasts were observed following exposure to voriconazole. Our results demonstrate that voriconazole can induce osteoblast proliferation and enhance osteogenic activity in vitro. Importantly, and in contrast to the previously proposed mechanism of fluoride-stimulated osteogenesis, our findings suggest that voriconazole-induced periostitis may also occur through fluoride-independent mechanisms that enhance the expression of cytokines that can augment osteoblastic activity. Copyright © 2015

  20. Proteomic analysis of osteogenic differentiation of dental follicle precursor cells

    DEFF Research Database (Denmark)

    Morsczeck, Christian; Petersen, Jørgen; Völlner, Florian

    2009-01-01

    Recently, there has been an increased interest in unravelling the molecular mechanisms and cellular pathways controlling the differentiation and proliferation of human stem cell lines. Proteome analysis has proven to be an effective approach to comprehensive analysis of the regulatory network...... of differentiation. In the present study we applied 2-DE combined with capillary-LC-MS/MS analysis to profile differentially regulated proteins upon differentiation of dental follicle precursor cells (DFPCs). Out of 115 differentially regulated proteins, glutamine synthetase, lysosomal proteinase cathepsin B....... The bioinformatic analyses suggest that proteins associated with cell cycle progression and protein metabolism were down-regulated and proteins involved in catabolism, cell motility and biological quality were up-regulated. These results display the general physiological state of DFPCs before and after osteogenic...

  1. Induction of Osteogenic Differentiation in Human Mesenchymal Stem Cells by Crosstalk with Osteoblasts.

    Science.gov (United States)

    Glueck, Martina; Gardner, Oliver; Czekanska, Ewa; Alini, Mauro; Stoddart, Martin J; Salzmann, Gian M; Schmal, Hagen

    2015-01-01

    Natural bone healing following fractures is initiated by osteoblasts (OBs) and mesenchymal stem cells (MSCs), a cell combination with possible potential in tissue engineering techniques for bony defects. The aim of the study was to investigate MSC/OB-crosstalk, in order to determine optimal cell culture conditions for osteogenic differentiation. Human OBs and MSCs interactions were investigated in an in vitro trans-well co-culture study over a time period of 28 days. Calcification was determined by optical density (OD) at 450 nm and Alizarin red staining. Messenger RNA expression was assessed by quantitative PCR. Osteogenic medium containing 1% fetal bovine serum resulted in superior levels of calcification in MSCs in co-culture with OBs compared to 2% or 5% fetal bovine serum (p<0.05). Comparing MSCs and OBs alone with the MSC/OB co-culture, calcification, as measured by OD 450 nm, increased over time in all groups. The highest values were recorded in the co-culture (p<0.05). Osteogenic differentiation potential showed significant interindividual differences. In order to predict differentiation potential, OD 450 nm measurements and mRNA expression of alkaline phosphatase were correlated with the population doubling rate during the expansion period. For OBs and MSCs, statistically significant associations of proliferation and differentiation potential were found (p<0.001). The addition of transforming growth factor beta resulted in up-regulation of collagen type I and Sp7 mRNA, and down-regulation of alkaline phosphatase mRNA. The results suggest the idea of soluble paracrine factors being secreted by OBs to induce osteogenic differentiation of MSCs.

  2. Promotion Effects of miR-375 on the Osteogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells

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    Si Chen

    2017-03-01

    Full Text Available MicroRNA plays an important role in bone tissue engineering; however, its role and function in osteogenic differentiation warrant further investigation. In this study, we demonstrated that miR-375 was upregulated during the osteogenic differentiation of human adipose-derived mesenchymal stem cells (hASCs. Overexpression of miR-375 significantly enhanced hASCs osteogenesis both in vitro and in vivo, while knockdown of miR-375 inhibited the osteogenic differentiation of hASCs. Mechanistically, microarray analysis revealed DEPTOR as a target of miR-375 in hASCs. Knockdown of DEPTOR accelerated the osteogenic differentiation of hASCs by inhibiting AKT signaling, which mimics miR-375 overexpression. Furthermore, we confirmed that miR-375 regulated osteogenesis by targeting YAP1, and that YAP1 reversely bound to miR-375 promoter to inhibit miR-375 expression. Taken together, our results suggested that miR-375 promoted the osteogenic differentiation of hASCs via the YAP1/DEPTOR/AKT regulatory network, indicating that miR-375-targeted therapy might be a valuable approach to promote bone regeneration.

  3. In Vitro Osteogenic Potential of Green Fluorescent Protein Labelled Human Embryonic Stem Cell-Derived Osteoprogenitors

    Directory of Open Access Journals (Sweden)

    Intekhab Islam

    2016-01-01

    Full Text Available Cellular therapy using stem cells in bone regeneration has gained increasing interest. Various studies suggest the clinical utility of osteoprogenitors-like mesenchymal stem cells in bone regeneration. However, limited availability of mesenchymal stem cells and conflicting evidence on their therapeutic efficacy limit their clinical application. Human embryonic stem cells (hESCs are potentially an unlimited source of healthy and functional osteoprogenitors (OPs that could be utilized for bone regenerative applications. However, limited ability to track hESC-derived progenies in vivo greatly hinders translational studies. Hence, in this study, we aimed to establish hESC-derived OPs (hESC-OPs expressing green fluorescent protein (GFP and to investigate their osteogenic differentiation potential in vitro. We fluorescently labelled H9-hESCs using a plasmid vector encoding GFP. The GFP-expressing hESCs were differentiated into hESC-OPs. The hESC-OPsGFP+ stably expressed high levels of GFP, CD73, CD90, and CD105. They possessed osteogenic differentiation potential in vitro as demonstrated by increased expression of COL1A1, RUNX2, OSTERIX, and OPG transcripts and mineralized nodules positive for Alizarin Red and immunocytochemical expression of osteocalcin, alkaline phosphatase, and collagen-I. In conclusion, we have demonstrated that fluorescently labelled hESC-OPs can maintain their GFP expression for the long term and their potential for osteogenic differentiation in vitro. In future, these fluorescently labelled hESC-OPs could be used for noninvasive assessment of bone regeneration, safety, and therapeutic efficacy.

  4. [Effect of overexpression of transcription factor Runx2 and Osterix on osteogenic differentiation of endothelial cells].

    Science.gov (United States)

    Yang, Guang-Zheng; Zhang, Wen-Jie; Ding, Xun; Zhang, Xiang-Kai; Jiang, Xin-Quan; Zhang, Zhi-Yuan

    2017-08-01

    To explore the effect of overexpression of Runx2 and Osterix (OSX) genes on osteogenic differentiation of human umbilical vein endothelial cells (HUVECs). Overexpressed Runx2 and OSX lentiviral vectors were transfected into HUVECs respectively. The osteogenic potential of transfected cells was identified by alkaline phosphatase (ALP) staining and ALP activity. Furthermore, real time-PCR, Western blot and immunofluorescence staining were performed to detect the expression of osteogenic genes and proteins in HUVECs. GraphPad Prism 6.01 software was used for statistical analysis. Overexpression of Runx2 gene was beneficial for osteogenic differentiation of HUVECs, while overexpression of osterix gene did not show osteogenic differential potential. Moreover, overexpression of Runx2 gene in HUVECs up-regulated the gene expression level of Runx2, OSX, ALP, bone sialoprotein (BSP), osteopontin (OPN), and osteocalcin (OCN), and up-regulated protein level of OPN and OCN. Overexpression of Runx2 could promote osteogenic differentiation of HUVECs.

  5. Serum miRNA Signatures Are Indicative of Skeletal Fractures in Postmenopausal Women With and Without Type 2 Diabetes and Influence Osteogenic and Adipogenic Differentiation of Adipose Tissue-Derived Mesenchymal Stem Cells In Vitro.

    Science.gov (United States)

    Heilmeier, Ursula; Hackl, Matthias; Skalicky, Susanna; Weilner, Sylvia; Schroeder, Fabian; Vierlinger, Klemens; Patsch, Janina M; Baum, Thomas; Oberbauer, Eleni; Lobach, Iryna; Burghardt, Andrew J; Schwartz, Ann V; Grillari, Johannes; Link, Thomas M

    2016-12-01

    Standard DXA measurements, including Fracture Risk Assessment Tool (FRAX) scores, have shown limitations in assessing fracture risk in Type 2 Diabetes (T2D), underscoring the need for novel biomarkers and suggesting that other pathomechanisms may drive diabetic bone fragility. MicroRNAs (miRNAs) are secreted into the circulation from cells of various tissues proportional to local disease severity and were recently found to be crucial to bone homeostasis and T2D. Here, we studied, if and which circulating miRNAs or combinations of miRNAs can discriminate best fracture status in a well-characterized study of diabetic bone disease and postmenopausal osteoporosis (n = 80 postmenopausal women). We then tested the most discriminative and most frequent miRNAs in vitro. Using miRNA-qPCR-arrays, we showed that 48 miRNAs can differentiate fracture status in T2D women and that several combinations of four miRNAs can discriminate diabetes-related fractures with high specificity and sensitivity (area under the receiver-operating characteristic curve values [AUCs], 0.92 to 0.96; 95% CI, 0.88 to 0.98). For the osteoporotic study arm, 23 miRNAs were fracture-indicative and potential combinations of four miRNAs showed AUCs from 0.97 to 1.00 (95% CI, 0.93 to 1.00). Because a role in bone homeostasis for those miRNAs that were most discriminative and most present among all miRNA combinations had not been described, we performed in vitro functional studies in human adipose tissue-derived mesenchymal stem cells to investigate the effect of miR-550a-5p, miR-188-3p, and miR-382-3p on osteogenesis, adipogenesis, and cell proliferation. We found that miR-382-3p significantly enhanced osteogenic differentiation (p women and should be further investigated for their diagnostic potential and their detailed function in diabetic bone. © 2016 American Society for Bone and Mineral Research. © 2016 American Society for Bone and Mineral Research.

  6. Aged human mesenchymal stem cells: the duration of bone morphogenetic protein-2 stimulation determines induction or inhibition of osteogenic differentiation

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    Jostein Heggebö

    2014-06-01

    Full Text Available Bone morphogenetic protein 2 (BMP-2 is a potent osteoinductive cytokine and a growing number of in vitro studies analyze its effects on human mesenchymal stem cells (hMSC derived from aged or osteoporotic donors. In these studies the exact quantification of osteogenic differentiation capacity is of fundamental interest. Nevertheless, the experimental conditions for osteogenic differentiation of aged hMSC have not been evaluated systematically and vary to a considerable extend. Aim of the study was to assess the influence of cell density, osteogenic differentiation media (ODM change intervals and duration of BMP-2 stimulation on osteoinduction. Furthermore, time series were carried out for osteogenic differentiation and BMP-2 concentration in ODM/BMP-2 cell culture supernatants. The experiments were performed using hMSC isolated from femoral heads of aged patients undergoing hip joint replacement. ODM change intervals of 96 hours resulted in significantly higher calcium deposition compared to shorter intervals. A cell density of 80% prior to stimulation led to stronger osteoinduction compared to higher cell densities. In ODM, aged hMSC showed a significant induction of calcium deposition after 9 days. Added to ODM, BMP-2 showed a stable concentration in the cell culture supernatants for at least 96 hours. Addition of BMP-2 to ODM for the initial 4 days led to a significantly higher induction of osteogenic differentiation compared to ODM alone. On the other hand, addition of BMP-2 for 21 days almost abrogated the osteoinductive effect of ODM. We could demonstrate that the factors investigated have a substantial impact on the extent of osteogenic differentiation of aged hMSC. Consequently, it is of upmost importance to standardize the experimental conditions in order to enable comparability between different studies. We here define standard conditions for osteogenic differentiation in regard to the specific features of aged hMSC. The finding that

  7. Aged Human Mesenchymal Stem Cells: The Duration of Bone Morphogenetic Protein-2 Stimulation Determines Induction or Inhibition of Osteogenic Differentiation

    Science.gov (United States)

    Heggebö, Jostein; Haasters, Florian; Polzer, Hans; Schwarz, Christina; Saller, Maximilian Michael; Mutschler, Wolf; Schieker, Matthias; Prall, Wolf Christian

    2014-01-01

    Bone morphogenetic protein 2 (BMP-2) is a potent osteoinductive cytokine and a growing number of in vitro studies analyze its effects on human mesenchymal stem cells (hMSC) derived from aged or osteoporotic donors. In these studies the exact quantification of osteogenic differentiation capacity is of fundamental interest. Nevertheless, the experimental conditions for osteogenic differentiation of aged hMSC have not been evaluated systematically and vary to a considerable extend. Aim of the study was to assess the influence of cell density, osteogenic differentiation media (ODM) change intervals and duration of BMP-2 stimulation on osteoinduction. Furthermore, time series were carried out for osteogenic differentiation and BMP-2 concentration in ODM/BMP-2 cell culture supernatants. The experiments were performed using hMSC isolated from femoral heads of aged patients undergoing hip joint replacement. ODM change intervals of 96 hours resulted in significantly higher calcium deposition compared to shorter intervals. A cell density of 80% prior to stimulation led to stronger osteoinduction compared to higher cell densities. In ODM, aged hMSC showed a significant induction of calcium deposition after 9 days. Added to ODM, BMP-2 showed a stable concentration in the cell culture supernatants for at least 96 hours. Addition of BMP-2 to ODM for the initial 4 days led to a significantly higher induction of osteogenic differentiation compared to ODM alone. On the other hand, addition of BMP-2 for 21 days almost abrogated the osteoinductive effect of ODM. We could demonstrate that the factors investigated have a substantial impact on the extent of osteogenic differentiation of aged hMSC. Consequently, it is of upmost importance to standardize the experimental conditions in order to enable comparability between different studies. We here define standard conditions for osteogenic differentiation in regard to the specific features of aged hMSC. The finding that BMP-2 induces or

  8. Proliferation and osteogenic differentiation of mesenchymal stromal cells in a novel porous hydroxyapatite scaffold.

    Science.gov (United States)

    Krishnamurithy, Genasan; Murali, Malliga Raman; Hamdi, Mohd; Abbas, Azlina Amir; Raghavendran, Hanumantharao Balaji; Kamarul, Tunku

    2015-01-01

    To compare the effect of bovine bone derived porous hydroxyapatite (BDHA) scaffold on proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells (hMSCs) compared with commercial hydroxyapatite (CHA) scaffold. The porosity and pore size were analyzed using micro-CT. The biocompatibility was demonstrated by alamar blue assay, and cell attachment through SEM and Hoechst staining. The osteogenic differentiation was demonstrated using biochemical assay and osteogenic gene expression. BDHA and CHA scaffolds showed porosity of 76.6 ± 0.6 and 64.3 ± 0.3% and pore size diameter of 0.04-0.25 and 0.1-2.6 mm, respectively. hMSCs proliferation, ALP activity, osteocalcin secretion and osteogenic gene expression are comparable in both the scaffolds. These results demonstrated that BDHA is biocompatible, supports cell adhesion and promotes proliferation and osteogenic differentiation.

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

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    Shu-Jem Su

    2013-01-01

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

  10. Rigid matrix supports osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHED).

    Science.gov (United States)

    Viale-Bouroncle, Sandra; Gosau, Martin; Küpper, Kevin; Möhl, Christoph; Brockhoff, Gero; Reichert, Torsten E; Schmalz, Gottfried; Ettl, Tobias; Morsczeck, Christian

    2012-12-01

    Stem cell fate can be induced by the grade of stiffness of the extracellular matrix, depending on the developed tissue or complex tissues. For example, a rigid extracellular matrix induces the osteogenic differentiation in bone marrow derived mesenchymal stem cells (MSCs), while a softer surface induces the osteogenic differentiation in dental follicle cells (DFCs). To determine whether differentiation of ectomesenchymal dental precursor cells is supported by similar grades of extracellular matrices (ECMs) stiffness, we examined the influence of the surface stiffness on the proliferation and osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHED). Cell proliferation of SHED was significantly decreased on cell culture surfaces with a muscle-like stiffness. A dexamethasone-based differentiation medium induced the osteogenic differentiation of SHED on substrates of varying mechanical stiffness. Here, the hardest surface improved the induction of osteogenic differentiation in comparison to that with the softest stiffness. In conclusion, our study showed that the osteogenic differentiation of ectomesenchymal dental precursor cells SHED and DFCs are not supported by similar grades of ECM stiffness. Copyright © 2012 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Sarah Strauß

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  13. Effect of canine cortical bone demineralization on osteogenic differentiation of adipose-derived mesenchymal stromal cells

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    Kwangrae Jo

    2017-08-01

    Full Text Available Demineralized bone allografts and mesenchymal stromal cells have been used to promote bone regeneration. However, the degree to which cortical bone should be demineralized for use in combination with adipose-derived mesenchymal stromal cells (Ad-MSCs remains to be clarified. In this study, the in vitro osteogenic ability of Ad-MSCs on allografts was investigated in relation to the extent of demineralization. Three treatment groups were established by varying exposure time to 0.6 N HCL: partially demineralized (PDB; 12 h, fully demineralized (FDB; 48 h, and non-demineralized bone (NDB; 0 h, as a control. Allografts were prepared as discs 6 mm in diameter for in vitro evaluation, and their demineralization and structure were evaluated by micro-computed tomography and scanning electron microscopy. Ad-MSC adhesion and proliferation were measured by MTS assay, and osteogenesis-related gene expression was assessed by quantitative reverse transcription polymerase chain reaction. PDB and FDB demineralization rates were 57.13 and 92.30%, respectively. Moreover, Ad-MSC adhesion rates on NDB, PDB, and FDB were 53.41, 60.65, and 61.32%, respectively. Proliferation of these cells on FDB increased significantly after 2 days of culture compared to the other groups (P < 0.05. Furthermore, expression of the osteogenic genes ALP, BMP-7, and TGF-β in the FDB group on culture day 3 was significantly elevated in comparison to the other treatments. Given its biocompatibility and promotion of the osteogenic differentiation of Ad-MSCs, our results suggest that FDB may be a suitable scaffold for use in the repair of bone defects.

  14. Low-intensity pulsed ultrasound stimulation facilitates osteogenic differentiation of human periodontal ligament cells.

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    Bo Hu

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

  15. Quantitative Membrane Proteomics in a Human Mesenchymal Stem Cell Line Undergoing Osteogenic Differentiation

    DEFF Research Database (Denmark)

    Christiansen, Helle

    devoid of cell senescence as encountered in primary cultures. The cell line used in this study has been shown capable of forming bone in vivo and osteogenic differentiation in vitro. We quantified a total number of 972 proteins of which 80 % fall within the categories of: transmembrane, membrane......  Mesenchymal stem cells (MSCs) (or marrow stromal cells) represent a population of cells located in the bone marrow that are capable of differentiation into several mesodermal cell types including osteoblasts. MSCs cannot presently be isolated based on protein markers, as no specific markers exist....... Mesenchymal stem cells are generally isolated based on physical-chemical characteristics such as adherence to plastic, isolating the monocyte fraction. The resultant cultures are often heterogeneous and can contain other cell types, providing a currently poorly defined basis for future clinical use...

  16. [Canonical Wnt signaling pathway of the osteogenic differentiation of human periodontal ligament stem cells induced by advanced glycation end products].

    Science.gov (United States)

    Yan, Wu; Chao, Deng; Kun, Yang; Xiaoxia, Cui; Qi, Liu; Yan, Jin

    2015-12-01

    The effect of advanced glycation end products (AGEs) on the osteogenic differentiation of humanperiodontal ligament stem cells(hPDLSCs) was discussed. Changes in the Wnt signaling pathway during glycation were also determined. In vitro tissue explanting method was primarily applied. Limiting diluted clone was cultured to obtain hPDLSCs in vitro. The subjects were divided into two groups: the healthy group (N-hPDLSCs) and the AGEs-stimulating group (A-hPDLSCs). Osteoblast mineralization was induced in the experimental groups. The following processes were performed: alizarin red staining; alkaline phosphatase (ALP) staining; real time polymerase chain reaction (real time PCR) for detecting osteogenic genes and Wnt classical pathway-related factors, DKK-1 and β-catenin; Western blot analysis. Bone protein and β-catenin were correlated in the nuclear expression. The cells were osteogenically induced. ALP staining showed that the N-hPDLSCs displayed the deepest color. Alizarin red staining indicated that the A-hPDLSCs group had less calcified nodules than the N-hPDLSCs group. The real time PCR results suggested that the expression of relative osteogenic genes in A-hPDLSCs was quite low. Statistically significant differences in differentiation were found between groups (P < 0.05). The Western blot result was similar to that of real time PCR. Classical Wnt signaling pathway-related factor β-catenin was higher in A-hPDLSCs than in N-hPDLSCs. By contrast, DKK-1, which is an inhibitor in the Wnt pathway, had a significantly lower expression rate in A-hPDLSCs than in N-hPDLSCs. The Western blot result also showed that β-catenin expression in the nucleoprotein in A-hPDLSCs was notably higher than in N-hPDLSCs. AGEs can inhibit hPDLSCs osteogenic differentiation. AGEs induce changes in the normal periodontal ligament stem cells classical Wnt pathway. Canonical Wnt pathway is reactivated because of AGEs stimulation.

  17. The role of dentin matrix protein 1 (DMP1) in regulation of osteogenic differentiation of rat dental follicle stem cells (DFSCs).

    Science.gov (United States)

    Rezai Rad, Maryam; Liu, Dawen; He, Hongzhi; Brooks, Hunter; Xiao, Mei; Wise, Gary E; Yao, Shaomian

    2015-04-01

    Primary isolated dental follicle stem cells (DFSCs) possess a strong osteogenesis capability, and such capability is reduced during in vitro culture. Because dentin matrix protein 1 (DMP1) is essential in the maturation of osteoblasts, our objectives were to determine (1) the expression of DMP1 in the DFSCs, (2) the correlation between DMP1 expression and osteogenic capability of DFSCs, and (3) the ability of DMP1 to promote osteogenic differentiation of DFSCs. DFSCs and their non-stem cell counterpart dental follicle cells (DFC) were established from postnatal rat pups. Expression of DMP1 in the DFSCs and DFC was determined using real-time RT-PCR and western blotting. Different passages of DFSCs were subjected to osteogenic induction. The correlation between osteogenesis and DMP1 expression was analyzed. Then, expression of DMP1 in the DFSCs was knocked-down using siRNA, followed by osteogenic induction to evaluate the effect of DMP1-knockdown. Finally, the late passage DFSCs with reduced DMP1 expression and osteogenic capability were cultured in osteogenic induction medium containing mouse recombinant DMP1 (mrDMP1) to determine if DMP1 can restore osteogenesis of DFSCs. DFSCs expressed much higher levels of DMP1 than did DFC. DMP1 expression was correlated with the osteogenic capability of DFSCs. Knockdown of DMP1 expression markedly decreased the osteogenesis and osteogenic gene expression in the DFSCs whereas adding mrDMP1 protein to the osteogenic induction medium enhanced osteogenesis. DMP1 is highly expressed in the DFSCs, but minimally expressed in non-stem cell DFC. DMP1 appears to play an important role for osteogenic differentiation of the DFSCs. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Isolation, characterization and osteogenic differentiation of adipose-derived stem cells: from small to large animal models.

    Science.gov (United States)

    Arrigoni, Elena; Lopa, Silvia; de Girolamo, Laura; Stanco, Deborah; Brini, Anna T

    2009-12-01

    One of the most important issues in orthopaedic surgery is the loss of bone resulting from trauma, infections, tumours or congenital deficiency. In view of the hypothetical future application of mesenchymal stem cells isolated from human adipose tissue in regenerative medicine, we have analysed and characterized adipose-derived stem cells (ASCs) isolated from adipose tissue of rat, rabbit and pig. We have compared their in vitro osteogenic differentiation abilities for exploitation in the repair of critical osteochondral defects in autologous pre-clinical models. The number of pluripotent cells per millilitre of adipose tissue is variable and the yield of rabbit ASCs is lower than that in rat and pig. However, all ASCs populations show both a stable doubling time during culture and a marked clonogenic ability. After exposure to osteogenic stimuli, ASCs from rat, rabbit and pig exhibit a significant increase in the expression of osteogenic markers such as alkaline phosphatase, extracellular calcium deposition, osteocalcin and osteonectin. However, differences have been observed depending on the animal species and/or differentiation period. Rabbit and porcine ASCs have been differentiated on granules of clinical grade hydroxyapatite (HA) towards osteoblast-like cells. These cells grow and adhere to the scaffold, with no inhibitory effect of HA during osteo-differentiation. Such in vitro studies are necessary in order to select suitable pre-clinical models to validate the use of autologous ASCs, alone or in association with proper biomaterials, for the repair of critical bone defects.

  19. The hedgehog-signaling pathway is repressed during the osteogenic differentiation of dental follicle cells

    DEFF Research Database (Denmark)

    Morsczeck, Christian; Reck, A; Beck, H C

    2017-01-01

    Dental follicle stem cells (DFCs) are precursor cells of alveolar osteoblasts, and previous studies have shown that the growth factor bone morphogenetic protein (BMP)2 induces the osteogenic differentiation of DFCs. However, the molecular mechanism down-stream of the induction of the osteogenic...... of repressors of the hedgehog-signaling pathway such as Patched 1 (PTCH1), Suppressor of Fused (SUFU), and Parathyroid Hormone-Related Peptide (PTHrP). Previous studies suggested that hedgehog proteins induce the osteogenic differentiation of mesenchymal stem cells via a paracrine pathway. Indian hedgehog (IHH...

  20. An engineered cell-imprinted substrate directs osteogenic differentiation in stem cells

    DEFF Research Database (Denmark)

    Kamguyan, Khorshid; Katbab, Ali Asghar; Mahmoudi, Morteza

    2018-01-01

    A cell-imprinted poly(dimethylsiloxane)/hydroxyapatite nanocomposite substrate was fabricated to engage topographical, mechanical, and chemical signals to stimulate and boost stem cell osteogenic differentiation. The physicochemical properties of the fabricated substrates, with nanoscale resoluti....... Besides the physical and mechanical effects, we found that the chemical signaling of osteoinductive hydroxyapatite nanoparticles, embedded in the nanocomposite substrates, could further improve and optimize stem cell osteogenic differentiation....

  1. The In Vitro and In Vivo Osteogenic Capability of the Extraction Socket-Derived Early Healing Tissue.

    Science.gov (United States)

    Luo, Jia; Xu, Jing; Cai, Jun; Wang, Limei; Sun, Qinfeng; Yang, Pishan

    2016-09-01

    Healed extraction socket is one autologous bone source. Extraction socket-derived early healing tissue (ESEHT) contains stem cells, osteoblasts, and growth factors, suggesting that it should have an osteogenic potential. The objective of this preliminary study is to explore the in vitro and in vivo osteogenic ability of ESEHT. ESEHT from 2-week healing extraction sockets and proper alveolar bone (PAB) from interdental septa or surrounding socket walls were acquired from beagle dogs. For in vitro experiments, ESEHT and PAB were separately cocultured with mouse bone marrow-derived stromal cell lines (st2 cells) using a transwell system. The effect of ESEHT or PAB on migration, proliferation, and osteogenic differentiation of st2 cells was investigated. For in vivo study, 36 inflammatory Class II furcation defects in the bilateral mandibles of dogs were surgically created, and then ESEHT and PAB from the maxilla of the same dogs were implanted into defects. Histologic observation and histometric analysis were performed after an 8-week healing period. The in vitro results indicated that ESEHT and PAB significantly promoted cellular migration, proliferation, alkaline phosphatase activity, expressions of bone sialoprotein, and Runt-related transcription factor 2 in messenger RNA and protein levels and, moreover, that ESEHT showed stronger activities than PAB except in chemotactic activity. The in vivo tests showed that ESEHT and PAB had a similar function in enhancing percentages of regenerated cementum and regenerated bone, which were significantly higher than those in blank control groups. Results showed that ESEHT possesses better effects on migration, proliferation, and osteogenic differentiation of mesenchymal stem cells in vitro but similar promotion effect on periodontal regeneration in vivo compared with PAB, suggesting that ESEHT may be one of the most effective graft materials for periodontal regeneration.

  2. [Putrescine Promotes Human Marrow Mesenchymal Stem Cells to Differentiate along Osteogenic Pathway].

    Science.gov (United States)

    Chen, Jing-Li; Bi, Xiao-Yun; Zhang, Hui; Wang, Fang; Wang, Yan; Guo, Zi-Kuan

    2015-06-01

    To investigate the effects of putrescine on the growth and differentiation of human bone marrow mesenchymal stem cells (MSC) to develop a new inductive medium mixture for their osteogenic differentiation. Human bone marrow MSC were collected from three healthy donors and were used to observe the growth-promoting activity of putrescine with MTT test. Experiments were divided into 3 groups: (1) putrescine group, (2) positive control group (presence of dexamethasone, ascorbate, and glycerol phosphate) and negative group (d-alpha with 5% FCS). The cellular expression level of Runx-2 was detected by PCR assay after the culture was maintained for 1 week. After 2 weeks, the intracellular activity of alkaline phosphatase was revealed by histochemistry staining, the phosphatase activity, and the protein concentration in the cell lysates were also detected. Furthermore, MSC were cultured in the presence of putrescine for 2 weeks and Oil-red O staining was performed to reveal the differentiated adipocytes; the cells induced by the standard agent cocktail were used as the positive control. Putrescine promoted the proliferation of human marrow MSC in a dose-dependent manner. MSC exposed to putrescine at a concentration of 100 µmol/L for 1 week expressed greatly higher level of Runx-2, compared with the negative control. Alkaline phosphatase activity was evidently observed after MSC were maintained in the presence of putrescine for 2 weeks. The phosphatase activity contrasted to the protein content in putrescine-treated MSC was significantly higher than that of the control cells (0.87±0.012 vs 0.52±0.010) (Pputrescine did not differentiate into adipoblasts. Putrescine can promote the proliferation and osteogenic differentiation of MSC, suggesting the potential application of putrescine as a novel inductive agent for in vitro osteogenesis of MSC.

  3. HIF-1α as a Regulator of BMP2-Induced Chondrogenic Differentiation, Osteogenic Differentiation, and Endochondral Ossification in Stem Cells

    Directory of Open Access Journals (Sweden)

    Nian Zhou

    2015-04-01

    Full Text Available Background/Aims: Joint cartilage defects are difficult to treat due to the limited self-repair capacities of cartilage. Cartilage tissue engineering based on stem cells and gene enhancement is a potential alternative for cartilage repair. Bone morphogenetic protein 2 (BMP2 has been shown to induce chondrogenic differentiation in mesenchymal stem cells (MSCs; however, maintaining the phenotypes of MSCs during cartilage repair since differentiation occurs along the endochondral ossification pathway. In this study, hypoxia inducible factor, or (HIF-1α, was determined to be a regulator of BMP2-induced chondrogenic differentiation, osteogenic differentiation, and endochondral bone formation. Methods: BMP2 was used to induce chondrogenic and osteogenic differentiation in stem cells and fetal limb development. After HIF-1α was added to the inducing system, any changes in the differentiation markers were assessed. Results: HIF-1α was found to potentiate BMP2-induced Sox9 and the expression of chondrogenesis by downstream markers, and inhibit Runx2 and the expression of osteogenesis by downstream markers in vitro. In subcutaneous stem cell implantation studies, HIF-1α was shown to potentiate BMP2-induced cartilage formation and inhibit endochondral ossification during ectopic bone/cartilage formation. In the fetal limb culture, HIF-1α and BMP2 synergistically promoted the expansion of the proliferating chondrocyte zone and inhibited chondrocyte hypertrophy and endochondral ossification. Conclusion: The results of this study indicated that, when combined with BMP2, HIF-1α induced MSC differentiation could become a new method of maintaining cartilage phenotypes during cartilage tissue engineering.

  4. The effect of human platelet-rich plasma on adipose-derived stem cell proliferation and osteogenic differentiation.

    Science.gov (United States)

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

    2014-07-01

    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. 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. Treatment with hPRP resulted in a statistically significant increase in cell proliferation compare to the negative control group (Pinvestigation.

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

    Science.gov (United States)

    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.

  6. Msx1-modulated muscle satellite cells retain a primitive state and exhibit an enhanced capacity for osteogenic differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Ke, E-mail: dingke@med.uestc.edu.cn [Department of Pediatric Surgery, School of medicine, University of Electronic Science and Technology of China, Chengdu 610072 (China); Sichuan Academy of Medical Sciences & Sichuan Provincial People' s Hospital, Chengdu 610072 (China); Department of Orthopaedics, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Liu, Wen-ying; Zeng, Qiang; Hou, Fang [Department of Pediatric Surgery, School of medicine, University of Electronic Science and Technology of China, Chengdu 610072 (China); Sichuan Academy of Medical Sciences & Sichuan Provincial People' s Hospital, Chengdu 610072 (China); Xu, Jian-zhong, E-mail: xjzspine@163.com [Department of Orthopaedics, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Yang, Zhong, E-mail: zyang1999@163.com [Department of Clinical Hematology, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China)

    2017-03-01

    Multipotent muscle satellite cells (MuSCs) have been identified as potential seed cells for bone tissue engineering. However, MuSCs exhibit a rapid loss of stemness after in vitro culturing, thereby compromising their therapeutic efficiency. Muscle segment homeobox gene 1 (msx1) has been found to induce the dedifferentiation of committed progenitor cells, as well as terminally differentiated myotubes. In this study, a Tet-off retroviral gene delivery system was used to modulate msx1 expression. After ten passages, MuSCs that did not express msx-1 (e.g., the non-msx1 group) were compared with MuSCs with induced msx-1 expression (e.g., the msx1 group). The latter group exhibited a more juvenile morphology, it contained a significantly lower percentage of senescent cells characterized by positive β-galactosidase staining, and it exhibited increased proliferation and a higher proliferation index. Immunocytochemical stainings further detected a more primitive gene expression profile for the msx1 group, while osteogenic differentiation assays and ectopic bone formation assays demonstrated an improved capacity for the msx1 group to undergo osteogenic differentiation. These results suggest that transient expression of msx1 in MuSCs can retain a primitive state, thereby enhancing their capacity for osteogenic differentiation and restoring the potential for MuSCs to serve as seed cells for bone tissue engineering.

  7. Effect of growth factors (BMP-4/7 & bFGF on proliferation & osteogenic differentiation of bone marrow stromal cells

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    Shaohui Yuan

    2013-01-01

    Full Text Available Background & objectives: BMP (bone morphogenetic protein-4/7 and bFGF (basic fibroblast growth factor significantly promote the osteogenic activity and the proliferation of rabbit BMSCs (bone marrow stromal cells, respectively. However, their synergistic effects on the proliferation and the differentiation of BMSCs remain unclear. In the present study, the effects of bFGF and BMP-4/7 were investigated on the proliferation and the differentiation of rat BMSCs in vitro. Methods: BMSCs were isolated from New Zealand white rabbits and cultured to the third passage. The samples were divided into five groups according to the material implanted: (A 80 ng/ml BMP-4/7; (B 80 ng/ml bFGF; (C 30 ng/ml BMP-4/7 and 30 ng/ml bFGF; (D 50 ng/ml BMP-4/7 and 50 ng/ml bFGF; and (E 80 ng/ml BMP-4/7 and 80 ng/ml bFGF. Cell proliferation was analyzed using methyl thiazolyl tetrazolium (MTT assay. Alkaline phosphatase activity and osteocalcin (OC dynamics were also measured. Results: BMP-4/7 alone significantly (P<0.05 promoted the proliferation of BMSCs. At the same time, it also promoted or inhibited the osteogenic differentiation of BMSCs. The synergistic effects of BMP-4/7 and bFGF significantly promoted both the proliferation and the osteogenic differentiation of BMSCs. The treatment of the synergistic effects was dose and time dependent. Interpretation & conclusions: A rational combination of BMP-4/7 and bFGF can promote the proliferation and the osteogenic differentiation of BMSCs. In addition, the synergistic functions are effective.

  8. MicroRNA-24 Regulates Osteogenic Differentiation via Targeting T-Cell Factor-1

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    Weigong Zhao

    2015-05-01

    Full Text Available MicroRNAs (miRNAs have been reported to have diverse biological roles in regulating many biological processes, including osteogenic differentiation. In the present study, we identified that miR-24 was a critical regulator during osteogenic differentiation. We found that overexpression of miR-24 significantly inhibited osteogenic differentiation, which decreased alkaline phosphatase activity, matrix mineralization and the expression of osteogenic differentiation markers. In contrast, inhibition of miR-24 exhibited an opposite effect. Furthermore, we delineated that miR-24 regulates post-transcriptionals of T-cell factor-1 (Tcf-1 via targeting the 3'-untranslated region (UTR of Tcf-1 mRNA. MiR-24 was further found to regulate the protein expression of Tcf-1 in the murine osteoprogenitors cells and bone mesenchymal stem cells. Additionally, the positive effect of miR-24 suppression on osteoblast differentiation was apparently abrogated by Tcf-1 silencing. Taken together, our data suggest that miR-24 participates in osteogenic differentiation by targeting and regulating Tcf-1 expression in osteoblastic cells.

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

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

    2015-01-01

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

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

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    Fumiko Matsuoka

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

  11. Tunable osteogenic differentiation of hMPCs in tubular perfusion system bioreactor.

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

    2016-08-01

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

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

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    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.

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

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    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

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

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    Marita Westhrin

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

  15. Activation of Cannabinoid Receptor 2 Enhances Osteogenic Differentiation of Bone Marrow Derived Mesenchymal Stem Cells

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    Yong-Xin Sun

    2015-01-01

    Full Text Available Bone marrow derived mesenchymal stem cells (BM-MSCs are considered as the most promising cells source for bone engineering. Cannabinoid (CB receptors play important roles in bone mass turnover. The aim of this study is to test if activation of CB2 receptor by chemical agonist could enhance the osteogenic differentiation and mineralization in bone BM-MSCs. Alkaline phosphatase (ALP activity staining and real time PCR were performed to test the osteogenic differentiation. Alizarin red staining was carried out to examine the mineralization. Small interference RNA (siRNA was used to study the role of CB2 receptor in osteogenic differentiation. Results showed activation of CB2 receptor increased ALP activity, promoted expression of osteogenic genes, and enhanced deposition of calcium in extracellular matrix. Knockdown of CB2 receptor by siRNA inhibited ALP activity and mineralization. Results of immunofluorescent staining showed that phosphorylation of p38 MAP kinase is reduced by knocking down of CB2 receptor. Finally, bone marrow samples demonstrated that expression of CB2 receptor is much lower in osteoporotic patients than in healthy donors. Taken together, data from this study suggested that activation of CB2 receptor plays important role in osteogenic differentiation of BM-MSCs. Lack of CB2 receptor may be related to osteoporosis.

  16. [Effect of astragalus membranaceus on the proliferation, osteogenic capacity and structure of periodontal ligament cells in vitro].

    Science.gov (United States)

    Zhang, Chao-liang; Kong, Xiang-li; Chen, Si-xiu; Li, Xiao-yu

    2010-10-01

    To investigate the effect of Astragalus membranaceus (APS) on the proliferation, osteogenic capacity and structure of periodontal ligament cells (PDLCs) in vitro. PDLCs were cultured in vitro with APS of 0.08, 0.1, 0.2, 0.4 mg x mL(-1). Methyl thiazolyl tetrazolium (MTr), alkaline phosphatase (ALP) and cell structure were detected to determine the proliferation and differentiation of PDLCs proliferation and differentiation. When the APS was 0.2 mg x mL(-1), the absorbance of MTT and ALP exhibit significantly increased as compared to the control (P structure. APS with proper concentration in short-term culture may promote the proliferation and differentiation of PDLCs.

  17. [Effects of drynariae rhizoma water-extraction on the ability of osteogenic differentiation and it's mechanism].

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    Yang, Li; Zhu, Xiao-Feng; Wang, Pan-Pan; Zhang, Rong-Hua

    2013-08-01

    To investigate the effects of Drynariae Rhizoma water-extraction on the ability of osteogenic differentiation and the expression of TGF-beta1 and BMP-2 in inducing mesenchymal stem cells (MSCs) differentiation into osteoblast and its mechanism. MSCs were isolated and purified by differential time adherent method; The most effective concentration of the Drynariae Rhizoma water-extraction on the proliferation and ostogenic differentiation of MSCs was confirmed by MTT method and the activity and positive staining of alkaline phosphatase (ALP) respectively; According to different induced condition, MSCs were divided into 4 groups: control group, classic group (induced by the classic osteoblast-induced system), Drynariae Rhizoma water-extraction group (induced by the most effective concentration of Drynariae Rhizoma water-extraction on differentiation into osteoblast) and Drynariae Rhizoma water-extraction + classic group (induced by the combination of classic osteoblast-induced system and the most effective concentration of Drynariae Rhizoma water-extraction on differentiation into osteoblast). ALP,type I collagen (Col I), bone gla protein (BGP) and calcium nodes in each group were detected and compared to indicate the osteogenic differentiation of each group. TGF-beta1 and BMP-2 were detected by ELISA. The most effective concentration of the Drynariae Rhizoma water-extraction on the proliferation and osteogenic differentiation of MSCs was 5 microg/mL and 50 microg/mL, respectively. The classic group, Drynariae Rhizoma water-extraction group and Drynariae Rhizoma water-extraction + classic group could promote the osteogenic differentiation of MSCs and increase the expression of TGF-beta1 and BMP-2. The Drynariae Rhizoma water-extraction can promote the proliferation and osteogenic differentiation of MSCs. Epimedium water-extraction can improve the differentiation of MSC into osteoblast and its mechanism may be related to increasing the expression of TGF-beta1 and BMP-2.

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

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    Niall Logan

    2013-01-01

    Full Text Available 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.

  19. The Effects of BMP-2, miR-31, miR-106a, and miR-148a on Osteogenic Differentiation of MSCs Derived from Amnion in Comparison with MSCs Derived from the Bone Marrow

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    Sirikul Manochantr

    2017-01-01

    Full Text Available Mesenchymal stromal cells (MSCs offering valuable anticipations for the treatment of degenerative diseases. They can be found in many tissues including amnion. MSCs from amnion (AM-MSCs can differentiate into osteoblast similar to that of bone marrow-derived MSCs (BM-MSCs. However, the ability is not much efficient compared to BM-MSCs. This study aimed to examine the effects of BMP-2 and miRNAs on osteogenic differentiation of AM-MSCs compared to those of BM-MSCs. The osteogenic differentiation capacity after miRNA treatment was assessed by ALP expression, ALP activity, and osteogenic marker gene expression. The results showed that the osteogenic differentiation capacity increased after BMP-2 treatment both in AM-MSCs and BM-MSCs. MiR-31, miR-106a, and miR-148a were downregulated during the osteogenic differentiation. After transfection with anti-miRNAs, ALP activity and osteogenic genes were increased over the time of differentiation. The data lead to the potential for using AM-MSCs as an alternative source for bone regeneration. Moreover, the information of miRNA expression and function during osteogenic differentiation may be useful for the development of new therapeutics or enhanced an in vitro culture technique required for stem cell-based therapies in the bone regeneration.

  20. Multi-level factorial analysis of Ca2+/Pi supplementation as bio-instructive media for in vitro biomimetic engineering of three-dimensional osteogenic hybrids.

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    Chai, Yoke Chin; Roberts, Scott J; Van Bael, Simon; Chen, Yantian; Luyten, Frank P; Schrooten, Jan

    2012-02-01

    We report on the in vitro use of Ca(2+)/P(i) supplementation as a bio-instructive medium to drive human periosteum-derived cells (hPDCs) toward osteogenic differentiation on three-dimensional (3D) porous Ti6Al4V scaffolds. Through a multilevel factorial analysis, we have systematically investigated the biological effect and interactions of Ca(2+) or P(i) supplementation in three selected media preparations (i.e., basic growth medium, osteogenic medium [OM], and osteogenic medium without β-glycerophosphate [OM(-)]) and have identified specific conditions which induce proliferation and significant osteogenic differentiation of two-dimensional (2D) hPDC cultures. These findings were translated from 2D to 3D cultures conditions to instruct hPDCs to populate porous Ti6Al4V scaffolds and to differentiate into the osteoblast lineage with collagenous matrix production and subsequent matrix mineralization on the 3D structures. These osteogenic hybrids may potentially serve as a clinically relevant customizable bone reparative unit, providing a biomimetic template to more effectively mediate in vivo bone regeneration.

  1. Combined Effects of Mechanical Strain and Hydroxyapatite/Collagen Composite on Osteogenic Differentiation of Rat Bone Marrow Derived Mesenchymal Stem Cells

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    Yan Huang

    2013-01-01

    Full Text Available Mesenchymal stem cells (MSCs represent a promising source for bone repair and regeneration. Recent lines of evidence have shown that appropriate strain could regulate the osteogenic differentiation of MSCs. Our previous study demonstrated that hydroxyapatite/collagen (HA/Col composite also played an important role in the osteogenic differentiation of MSCs. The aim of this study is to investigate the effects of mechanical strain and HA/Col composite on the osteogenic differentiation of rat bone marrow derived MSCs (rBMSCs in vitro. rBMSCs were treated with cyclic strain generated by a self-designed stretching device with or without the presence of HA/Col composite. Osteogenic differentiation levels were evaluated using reverse transcription polymerase chain reaction (RT-PCR, alkaline phosphatase spectrophotometry, and western blotting. The results demonstrated that mechanical strain combined with HA/Col composite could obviously induce the differentiation of rBMSCs into osteoblasts, which had a better effect than only mechanical strain or HA/Col composite treatment. This provides a new avenue for mechanistic studies of stem cell differentiation and a novel approach to obtain more committed differentiated cells.

  2. MiR-615-3p inhibits the osteogenic differentiation of human lumbar ligamentum flavum cells via suppression of osteogenic regulators GDF5 and FOXO1.

    Science.gov (United States)

    Yin, Jichao; Zhuang, Guihua; Zhu, Yi; Hu, Xinglv; Zhao, Hongmou; Zhang, Rongqiang; Guo, Hao; Fan, Xiaochen; Cao, Yi

    2017-07-01

    Ossification of the ligamentum flavum (OLF) is a disease of heterotopic ossification in spinal ligaments. The key of the OLF pathogenesis is the differentiation of fibroblasts into osteoblasts. In this study, we explored the role of miR-615-3p in the osteogenic differentiation of human LF cells. The expression of miR-615-3p was detected during the osteogenic differentiation of hFOB1.19 human osteoblasts, human BMSCs, and human LF cells. The qPCR results showed that miR-615-3p was being decreased during the osteogenic differentiation of these cell lineages. Then, both gain- and loss-function experiments, respectively performed by single-strand miR-615-3p mimic and antagomir, revealed that miR-615-3p negatively regulated the osteogenesis of hLF cells, manifested by a lighter staining degree with Alizarin Red and a decreased level of osteogenic marker genes, including alkaline phosphatase (ALP), RUNX2, osterix (ostx), osteocalcin (OCN), and osteopontin (OPN). Subsequently, our data on bioinformatic analysis, 3'-UTR luciferase activity assay, and protein level detection indicated that miR-615-3p directly targeted and suppressed the expression of FOXO1 and GDF5. Furthermore, knockdown of either FOXO1 or GDF5 could inhibit the osteogenic differentiation of hLF cells, which displayed a similar effect with the miR-615-3p mimic. In conclusion, miR-615-3p negatively regulates the osteogenic differentiation of hLF cells through post-transcriptionally suppressing osteogenic regulators GDF5 and FOXO1. It can be regarded as a potential target for human OLF therapy. © 2017 International Federation for Cell Biology.

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

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    Arpornmaeklong, Premjit; Pressler, Michael J

    2018-01-01

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

  4. Optimizing the osteogenic differentiation of human mesenchymal stromal cells by the synergistic action of growth factors.

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    Açil, Yahya; Ghoniem, Amir-Alexander; Wiltfang, Jörg; Gierloff, Matthias

    2014-12-01

    A variety of different growth factors, most notably bone morphogenetic proteins (BMPs), have been shown to stimulate the osteogenic differentiation of mesenchymal stromal cells (MSCs) in vitro. Yet, due to the lack of comparative studies it remains unclear which protocol is the most effective in the induction of osteogenesis in MSC cultures. The aim of this study was to compare the most potent growth factors in regard to their osteoinductive potential. Human MSCs were cultured for 10 days in the presence of BMP-2, BMP-6, BMP-9 + IGF-2 and BMP-2, -6, -9 (day 1 + 2: 50 ng/ml; days 3-6: 100 ng/ml; days 7-10: 200 ng/ml). The formation of the osteoblast phenotype was assessed by quantification of osteoblast-related marker genes using reverse transcription polymerase chain reaction (RT-PCR) and alkaline phosphatase (ALP) staining. Matrix mineralization was assessed by alizarin red S and von Kossa staining. Statistical analysis was carried out using the one-way analysis of variance (ANOVA) followed by Scheffe's post hoc procedure. Among the tested growth factors the combination of BMP-2 + BMP-6 + BMP-9 most effectively induced the upregulation of collagen type I, collagen type V, osteocalcin, alkaline phosphatase, RUNX2, BMP-2, osteonectin and DLX5 (p human MSCs in vitro. Copyright © 2014 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

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

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    Wang, Shengchao [Department of Preventive Dentistry, School of Stomatology, The Fourth Military Medical University, 145 West Changle Road, 710032 Xi' an (China); Kawashima, Nobuyuki, E-mail: kawashima.n.endo@tmd.ac.jp [Department of Pulp Biology and Endodontics, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Sakamoto, Kei; Katsube, Ken-ichi [Department of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Umezawa, Akihiro [Department of Reproductive Biology and Pathology, National Institute for Child Health and Development, 2-10-4 Ohkura, Setagaya-ku, Tokyo 157-8535 (Japan); Suda, Hideaki [Department of Pulp Biology and Endodontics, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); GCOE Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan)

    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.

  6. In vitro performance of 13-93 bioactive glass fiber and trabecular scaffolds with MLO-A5 osteogenic cells.

    Science.gov (United States)

    Modglin, Vernon C; Brown, Roger F; Fu, Qiang; Rahaman, Mohamed N; Jung, Steven B; Day, Delbert E

    2012-10-01

    This in vitro study was performed to evaluate the ability of two types of porous bioactive glass scaffolds to support the growth and differentiation of an established osteogenic cell line. The two scaffold types tested included 13-93 glass fiber and trabecular-like scaffolds seeded with murine MLO-A5 cells and cultured for intervals of 2 to 12 days. Culture in MTT-containing medium showed metabolically active cells both on the surface and within the interior of the scaffolds. Scanning electron microscopy revealed well-attached cells on both types of scaffolds with a continual increase in cell density over a 6-day period. Protein measurements also showed a linear increase in cell density during the incubation. Activity of alkaline phosphatase, a key indicator of osteoblast differentiation, increased about 10-fold during the 6-day incubation with both scaffold types. The addition of mineralization media to MLO-A5 seeded scaffolds triggered extensive formation of alizarin red-positive mineralized extracellular material, additional evidence of cell differentiation and completion of the final step of bone formation on the constructs. Collectively, the results indicate that the 13-93 glass fiber and trabecular scaffolds promote the attachment, growth, and differentiation of MLO-A5 osteogenic cells and could potentially be used for bone tissue engineering applications. Copyright © 2012 Wiley Periodicals, Inc.

  7. The effect of PKC activation and inhibition on osteogenic differentiation of human mesenchymal stem cells

    NARCIS (Netherlands)

    Liu, J.; Someren, Eugene; Mentink, Anouk; Licht, R.; Dechering, Koen; van Blitterswijk, Clemens; de Boer, Jan

    2010-01-01

    Human mesenchymal stem cells (hMSCs) are being considered for several areas of clinical therapy, due to their multipotent nature. For instance, osteogenic hMSCs are applied in bone tissue engineering, but current differentiation protocols need further optimization before they can be clinically

  8. [Effect of polydimethylsiloxane matrix elasticity on osteogenic differentiation of rat marrow stromal cells].

    Science.gov (United States)

    Mu, Y; Zhao, J Z; Yang, C; Zu, Y; Li, Q

    2017-08-09

    Objective: To investigate the effect of polydimethylsiloxane (PDMS) matrix elasticity on osteogenic differentiation of rat marrow stromal cells (rBMSC). Methods: A series of PDMS composite substrates with different elastic modulus were constructed by adjusting the relative concentrations of cross-linking agent. The Young's modulus was used to describe the elasticity of PDMS after measurement by atomic force microscope (AFM). After surface modification, rBMSC was seeded on PDMS matrix, and 7 days after rBMSC was cultured on the five different Young's moduli matrix, the differences of osteogenic differentiation of rBMSC were observed by the method of real-time PCR, Western blotting, and alkaline phosphatase assay. Results: The PDMS was suitable for cell culture after surface modification, and by altering the concentration of cross-linking agent, PDMS could mimic the majority of the tissues' elasticity in vivo. The related osteogenic differentiation markers expression showed significant difference between the five matrixes (Pmatrix which could be used in the investigation of inducing rBMSCs into osteoblastic lineages. PDMS substrate stiffness has an obvious influence on rBMSC osteogenic differentiation.

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

    Science.gov (United States)

    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

  10. Electromagnetic fields and nanomagnetic particles increase the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells

    National Research Council Canada - National Science Library

    KIM, MIN-OK; JUNG, HYUN; KIM, SOO-CHAN; PARK, JUNG-KEUG; SEO, YOUNG-KWON

    .... Nanomagnetic particles (MPs) also promote the differentiation potential of stem cells. In the present study, we investigated the effects of EMFs and MPs on the osteogenic differentiation of hBM-MSCs...

  11. Control of proliferation and osteogenic differentiation of human dental-pulp-derived stem cells by distinct surface structures.

    Science.gov (United States)

    Kolind, K; Kraft, D; Bøggild, T; Duch, M; Lovmand, J; Pedersen, F S; Bindslev, D A; Bünger, C E; Foss, M; Besenbacher, F

    2014-02-01

    The ability to control the behavior of stem cells provides crucial benefits, for example, in tissue engineering and toxicity/drug screening, which utilize the stem cell's capacity to engineer new tissues for regenerative purposes and the testing of new drugs in vitro. Recently, surface topography has been shown to influence stem cell differentiation; however, general trends are often difficult to establish due to differences in length scales, surface chemistries and detailed surface topographies. Here we apply a highly versatile screening approach to analyze the interplay of surface topographical parameters on cell attachment, morphology, proliferation and osteogenic differentiation of human mesenchymal dental-pulp-derived stem cells (DPSCs) cultured with and without osteogenic differentiation factors in the medium (ODM). Increasing the inter-pillar gap size from 1 to 6 μm for surfaces with small pillar sizes of 1 and 2 μm resulted in decreased proliferation and in more elongated cells with long pseudopodial protrusions. The same alterations of pillar topography, up to an inter-pillar gap size of 4 μm, also resulted in enhanced mineralization of DPSCs cultured without ODM, while no significant trend was observed for DPSCs cultured with ODM. Generally, cells cultured without ODM had a larger deposition of osteogenic markers on structured surfaces relative to the unstructured surfaces than what was found when culturing with ODM. We conclude that the topographical design of biomaterials can be optimized for the regulation of DPSC differentiation and speculate that the inclusion of ODM alters the ability of the cells to sense surface topographical cues. These results are essential in order to transfer the use of this highly proliferative, easily accessible stem cell into the clinic for use in cell therapy and regenerative medicine. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    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

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

    DEFF Research Database (Denmark)

    Weilner, Sylvia; Schraml, Elisabeth; Wieser, Matthias

    2016-01-01

    . 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......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...

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

    Science.gov (United States)

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

    2014-08-01

    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. 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 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. 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.

  15. A Long-Acting BMP-2 Release System Based on Poly(3-hydroxybutyrate Nanoparticles Modified by Amphiphilic Phospholipid for Osteogenic Differentiation

    Directory of Open Access Journals (Sweden)

    Xiaochun Peng

    2016-01-01

    Full Text Available 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.

  16. Lim Mineralization Protein 3 Induces the Osteogenic Differentiation of Human Amniotic Fluid Stromal Cells through Kruppel-Like Factor-4 Downregulation and Further Bone-Specific Gene Expression

    Directory of Open Access Journals (Sweden)

    Marta Barba

    2012-01-01

    Full Text Available Multipotent mesenchymal stem cells with extensive self-renewal properties can be easily isolated and rapidly expanded in culture from small volumes of amniotic fluid. These cells, namely, amniotic fluid-stromal cells (AFSCs, can be regarded as an attractive source for tissue engineering purposes, being phenotypically and genetically stable, plus overcoming all the safety and ethical issues related to the use of embryonic/fetal cells. LMP3 is a novel osteoinductive molecule acting upstream to the main osteogenic pathways. This study is aimed at delineating the basic molecular events underlying LMP3-induced osteogenesis, using AFSCs as a cellular model to focus on the molecular features underlying the multipotency/differentiation switch. For this purpose, AFSCs were isolated and characterized in vitro and transfected with a defective adenoviral vector expressing the human LMP3. LMP3 induced the successful osteogenic differentiation of AFSC by inducing the expression of osteogenic markers and osteospecific transcription factors. Moreover, LMP3 induced an early repression of the kruppel-like factor-4, implicated in MSC stemness maintenance. KLF4 repression was released upon LMP3 silencing, indicating that this event could be reasonably considered among the basic molecular events that govern the proliferation/differentiation switch during LMP3-induced osteogenic differentiation of AFSC.

  17. Low dose effect of bisphosphonates on hMSCs osteogenic response to titanium surface in vitro

    Directory of Open Access Journals (Sweden)

    N.R. Alqhtani

    2017-06-01

    Full Text Available Since the 1980s, titanium (Ti implants have been routinely used to replace missing teeth. This success is mainly due to the good biocompatibility of Ti and the phenomenon of osseointegration, with very early events at implant placement being important in determining good osseointegration. However, enhancing implant performance with coatings such as hydroxyapatite (HA and calcium phosphate has proved largely unsuccessful. Human mesenchymal stem cells (hMSCs are the first osteogenic cells to colonise implant surfaces and offer a target for enhancing osseointegration. We previously reported that small doses of bisphosphonate (BP may play an integral role in enhancing hMSC proliferation and osteogenic differentiation. The aim of this study is to investigate whether small doses of bisphosphonates enhance proliferation and osteogenic differentiation of hMSCs on Ti surfaces, to enhance bone osseointegration and to accelerate wound healing around the implant surface. Our data suggests that treating cells with small doses of BP (100 nM & 10 nM induces significant hMSC stimulation of osteogenic markers including calcium, collagen type I and ALP compared to control group on titanium surfaces (P < 0.05. In addition, cell proliferation and migration were significantly enhanced on titanium surfaces (P < 0.05.

  18. The effect of equiaxial stretching on the osteogenic differentiation and mechanical properties of human adipose stem cells

    DEFF Research Database (Denmark)

    Virjula, Sanni; Zhao, Feihu; Leivo, Joni

    2017-01-01

    , osteogenic differentiation and mechanical properties of hASCs. Tailor-made, pneumatic cell stretching devices were used to expose hASCs to cyclic equiaxial stretching in osteogenic medium. Cell attachment and focal adhesions were visualised using immunocytochemical vinculin staining on days 3 and 6...

  19. [Study progress of growth differentiation factor 5 or osteogenic protein 1 injection into a degenerated disc].

    Science.gov (United States)

    Lin, Zhijun; Liu, Hangtao; Wang, Wanming

    2008-04-01

    To review the advance in the experimental studies and evaluate the potential therapeutic application of the growth differentiation factor 5(GDF-5) and osteogenic protein 1 (OP-1) in intervertebral disc degeneration. Methods Relevant literature at home and abroad published in recent years was searched and analyzed comprehensively. Results The growth factor was one of the most potential proteins in curing the intervertebral disc degeneration. In vitro, exogenous GDF-5 or OP-1 increased the deoxyribonucleic acid and proteoglycan contents of both nucleus pulposus and annlus fibrosis cells types significantly. GDF-5 at 200 ng/mL or OP-1 significantly stimulated proteoglycan synthesis and collagen synthesis. In vivo, the injection of GDF-5 (100 microg) or OP-1(100 microg in 10 microL 5% lactose) resulted in a restoration of disc height, improvement of magnetic resonance imaging scores, and histologic grading scores had statistical significance. A single injection of GDF-5 or OP-1 has a reparative capacity on intervertebral discs, presumably based on its effect to stimulate matrix metabolism of intervertebral disc cells and enhance extracellular matrix production. A single injection of exogenous GDF-5 or OP-1 in the degenerated disc shows a good prospect.

  20. Extracellular matrix mimicking scaffold promotes osteogenic stem cell differentiation: A new approach in osteoporosis research.

    Science.gov (United States)

    Moll, C W I; Schmiedinger, T; Moll, M A; Seppi, T; Pfaller, K; Hess, M W; Gutleben, K; Lindtner, R A; Blauth, M; Krumschnabel, G; Ebner, H L

    2017-01-01

    Osteoporosis is a common metabolic disease, with mesenchymal stem cells discussed to play an important role in its pathomechanism. For in vitro osteoporosis studies, selection of adequate culture conditions is mandatory so as to preserve cell properties as far as possible. A suitable cell culture surface would ideally provide reproducible experimental conditions by resembling those in-vivo. Generating an improved growth surface for osteogenic differentiation of human bone marrow derived mesenchymal stem cells (hBMSCs). We modified electrospun gelatine meshes with hydroxyapatite nanopowder. The potential beneficial impact of the ensuing culture conditions were evaluated by cultivating and comparing the growth of cells from osteoporotic and non-osteoporotic donors on either hydroxyapatite-gelatine (HA) meshes, pure gelatine meshes, or 2D standard tissue culture surfaces. After 21 days of differentiation, cells grown on pure or HA-gelatine meshes showed significantly higher mineralization levels compared to cells cultured in standard conditions. The amount of mineralization varied considerably in hBMSC cultures of individual patients but showed no significant difference between stem cells obtained from osteoporotic or non-osteoporotic donors. Overall, these results indicate that the use of HA-gelatine meshes as growth surfaces may serve as a valuable tool for cultivation and differentiation of mesenchymal stem cells along the osteogenic lineage, facilitating future research on osteoporosis and related issues.

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

    Science.gov (United States)

    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  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.

  2. Activation of JNKs is essential for BMP9-induced osteogenic differentiation of mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Yan-fang Zhao

    2013-08-01

    Full Text Available Although BMP9 is highly capable of promoting osteogenicdifferentiation of mesenchymal stem cell (MSCs, the molecularmechanism involved remains to be fully elucidated. Here, weexplore the possible involvement and detail role of JNKs (c-JunN-terminal kinases in BMP9-induced osteogenic differentiationof MSCs. It was found that BMP9 stimulated the activation ofJNKs in MSCs. BMP9-induced osteogenic differentiation ofMSCs was dramatically inhibited by JNKs inhibitor SP600125.Moreover, BMP9-activated Smads signaling was decreased bySP600125 treatment in MSCs. The effects of inhibitor arereproduced with adenoviruses expressing siRNA targeted JNKs.Taken together, our results revealed that JNKs was activated inBMP9-induced osteogenic differentiation of MSCs. What ismost noteworthy, however, is that inhibition of JNKs activityresulted in reduction of BMP9-induced osteogenic differentiationof MSCs, implying that activation of JNKs is essential forBMP9 osteoinductive activity. [BMB Reports 2013; 46(8:422-427

  3. Three-dimensional multipotent progenitor cell aggregates for expansion, osteogenic differentiation and 'in vivo' tracing with AAV vector serotype 6.

    Science.gov (United States)

    Ferreira, J R; Hirsch, M L; Zhang, L; Park, Y; Samulski, R J; Hu, W-S; Ko, C-C

    2013-02-01

    Multipotent adult progenitor cells (MAPCs) are bone marrow-derived stem cells with a high growth rate suitable for therapeutical applications as three-dimensional (3D) aggregates. Combined applications of osteogenically differentiated MAPC (OD-MAPC) aggregates and adeno-associated viral vectors (AAV) in bone bioengineering are still deferred until information with regard to expansion technologies, osteogenic potential, and AAV cytotoxicity and transduction efficiency is better understood. In this study, we tested whether self-complementary AAV (scAAV) can potentially be used as a gene delivery system in an OD-MAPC-based 'in vivo' bone formation model in the craniofacial region. Both expansion of rat MAPC (rMAPC) and osteogenic differentiation with dexamethasone were also tested in 3D aggregate culture systems 'in vitro' and 'vivo'. rMAPCs grew as undifferentiated aggregates for 4 days, with a population doubling time of 37 h. After expansion, constant levels of Oct4 transcripts, and Oct4 and CD31 surface markers were observed, which constitute a hallmark of undifferentiated stage of rMAPCs. Dexamethasone effectively mediated rMAPC osteogenic differentiation by inducing the formation of a mineralized collagen type I network, and facilitated the activation of the wnt/β-catenin, a crucial pathway in skeletal development. To investigate the genetic modification of rMAPCs grown as 3D aggregates before implantation, scAAV serotypes 2, 3 and 6 were evaluated. scAAV6 packaged with the enhanced green fluorescent protein expression cassette efficiently mediated long-term transduction (10 days) 'in vitro' and 'vivo'. The reporter transduction event allowed the tracing of OD-rMAPC (induced by dexamethasone) aggregates following OD-rMAPC transfer into a macro-porous hydroxyapatite scaffold implanted in a rat calvaria model. Furthermore, the scAAV6-transduced OD-rMAPCs generated a bone-like matrix with a collagenous matrix rich in bone-specific proteins (osteocalcin and

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

    Directory of Open Access Journals (Sweden)

    Ikbale El-Ayachi

    2016-03-01

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

  5. AML-induced osteogenic differentiation in mesenchymal stromal cells supports leukemia growth.

    Science.gov (United States)

    Battula, V Lokesh; Le, Phuong M; Sun, Jeffrey C; Nguyen, Khoa; Yuan, Bin; Zhou, Ximin; Sonnylal, Sonali; McQueen, Teresa; Ruvolo, Vivian; Michel, Keith A; Ling, Xiaoyang; Jacamo, Rodrigo; Shpall, Elizabeth; Wang, Zhiqiang; Rao, Arvind; Al-Atrash, Gheath; Konopleva, Marina; Davis, R Eric; Harrington, Melvyn A; Cahill, Catherine W; Bueso-Ramos, Carlos; Andreeff, Michael

    2017-07-06

    Genotypic and phenotypic alterations in the bone marrow (BM) microenvironment, in particular in osteoprogenitor cells, have been shown to support leukemogenesis. However, it is unclear how leukemia cells alter the BM microenvironment to create a hospitable niche. Here, we report that acute myeloid leukemia (AML) cells, but not normal CD34+ or CD33+ cells, induce osteogenic differentiation in mesenchymal stromal cells (MSCs). In addition, AML cells inhibited adipogenic differentiation of MSCs. Mechanistic studies identified that AML-derived BMPs activate Smad1/5 signaling to induce osteogenic differentiation in MSCs. Gene expression array analysis revealed that AML cells induce connective tissue growth factor (CTGF) expression in BM-MSCs irrespective of AML type. Overexpression of CTGF in a transgenic mouse model greatly enhanced leukemia engraftment in vivo. Together, our data suggest that AML cells induce a preosteoblast-rich niche in the BM that in turn enhances AML expansion.

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

    Science.gov (United States)

    Ohata, Yasuhisa; Ozono, Keiichi

    2014-04-01

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

  7. Preparation and Evaluation of Dexamethasone (DEX/Growth and Differentiation Factor-5 (GDF-5 Surface-Modified Titanium Using β-Cyclodextrin-Conjugated Heparin (CD-Hep for Enhanced Osteogenic Activity In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Dae Hyeok Yang

    2017-08-01

    Full Text Available The most ideal implant models in the dental and orthopedic fields to minimize the failure rate of implantation involve the improvement of osseointegration with host bone. Therefore, a focus of this study is the preparation of surface-modified titanium (Ti samples of disc and screw types using dexamethasone (DEX and/or growth and differentiation factor-5 (GDF-5, as well as the evaluation of their efficacies on bone formation in vitro and in vivo. X-ray photoelectron spectroscopy (XPS, scanning electron microscopy (SEM and contact angle measurement were used to evaluate the surface chemical composition, surface morphology and wettability, respectively. The results showed that implant surfaces were successfully modified with DEX and/or GDF-5, and had rough surfaces along with hydrophilicity. DEX, GDF-5 or DEX/GDF-5 on the surface-modified samples were rapidly released within one day and released for 28 days in a sustained manner. The proliferation and bone formation of MC3T3-E1 cells cultured on pristine and surface-modified implants in vitro were examined by cell counting kit-8 (CCK-8 assay, as well as the measurements of alkaline phosphatase (ALP activity and calcium deposition, respectively. MC3T3-E1 cells cultured on DEX/GDF-5–Ti showed noticeable ALP activity and calcium deposition in vitro. Active bone formation and strong osseointegration occurred at the interface between DEX/GDF-5–Ti and host bone, as evaluated by micro computed-tomography (micro CT analysis. Surface modification using DEX/GDF-5 could be a good method for advanced implants for orthopaedic and dental applications.

  8. PS1/γ-Secretase-Mediated Cadherin Cleavage Induces β-Catenin Nuclear Translocation and Osteogenic Differentiation of Human Bone Marrow Stromal Cells

    Science.gov (United States)

    Dias, Rhayra B.; Fortuna-Costa, Anneliese; Chicaybam, Leonardo; Lopes, Daiana V.; Dutra, Hélio S.; Borojevic, Radovan; Bonamino, Martin; Mermelstein, Claudia

    2016-01-01

    Bone marrow stromal cells (BMSCs) are considered a promising tool for bone bioengineering. However, the mechanisms controlling osteoblastic commitment are still unclear. Osteogenic differentiation of BMSCs requires the activation of β-catenin signaling, classically known to be regulated by the canonical Wnt pathway. However, BMSCs treatment with canonical Wnts in vitro does not always result in osteogenic differentiation and evidence indicates that a more complex signaling pathway, involving cadherins, would be required to induce β-catenin signaling in these cells. Here we showed that Wnt3a alone did not induce TCF activation in BMSCs, maintaining the cells at a proliferative state. On the other hand, we verified that, upon BMSCs osteoinduction with dexamethasone, cadherins were cleaved by the PS1/γ-secretase complex at the plasma membrane, and this event was associated with an enhanced β-catenin translocation to the nucleus and signaling. When PS1/γ-secretase activity was inhibited, the osteogenic process was impaired. Altogether, we provide evidence that PS1/γ-secretase-mediated cadherin cleavage has as an important role in controlling β-catenin signaling during the onset of BMSCs osteogenic differentiation, as part of a complex signaling pathway responsible for cell fate decision. A comprehensive map of these pathways might contribute to the development of strategies to improve bone repair. PMID:28053606

  9. PS1/γ-Secretase-Mediated Cadherin Cleavage Induces β-Catenin Nuclear Translocation and Osteogenic Differentiation of Human Bone Marrow Stromal Cells

    Directory of Open Access Journals (Sweden)

    Danielle C. Bonfim

    2016-01-01

    Full Text Available Bone marrow stromal cells (BMSCs are considered a promising tool for bone bioengineering. However, the mechanisms controlling osteoblastic commitment are still unclear. Osteogenic differentiation of BMSCs requires the activation of β-catenin signaling, classically known to be regulated by the canonical Wnt pathway. However, BMSCs treatment with canonical Wnts in vitro does not always result in osteogenic differentiation and evidence indicates that a more complex signaling pathway, involving cadherins, would be required to induce β-catenin signaling in these cells. Here we showed that Wnt3a alone did not induce TCF activation in BMSCs, maintaining the cells at a proliferative state. On the other hand, we verified that, upon BMSCs osteoinduction with dexamethasone, cadherins were cleaved by the PS1/γ-secretase complex at the plasma membrane, and this event was associated with an enhanced β-catenin translocation to the nucleus and signaling. When PS1/γ-secretase activity was inhibited, the osteogenic process was impaired. Altogether, we provide evidence that PS1/γ-secretase-mediated cadherin cleavage has as an important role in controlling β-catenin signaling during the onset of BMSCs osteogenic differentiation, as part of a complex signaling pathway responsible for cell fate decision. A comprehensive map of these pathways might contribute to the development of strategies to improve bone repair.

  10. Activation of PKA/CREB Signaling is Involved in BMP9-Induced Osteogenic Differentiation of Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Hongyu Zhang

    2015-09-01

    Full Text Available Background/Aims: BMP9 is highly capable of promoting osteogenic differentiation of mesenchymal stem cells (MSCs although the molecular mechanism involved is largely unknown. Here, we explored the detail role of PKA/CREB signaling in BMP9-induced osteogenic differentiation. Methods: Activation status of PKA/CREB signaling is assessed by nonradioactive assay and Western blot. Using PKA inhibitors and a dominant negative protein of CREB (A-CREB, we investigated the effect of PKA/CREB signaling on BMP9-induced osteogenic differentiation. Results: We found that BMP9 promotes PKA activity and enhances CREB phosphorylation in MSCs. BMP9 is shown to down-regulate protein kinase A inhibitor γ (PKIγ expression. We demonstrated that PKA inhibitors suppress BMP9-induced early osteogenic marker alkaline phosphatase (ALP activity in MSCs as well as late osteogenic markers osteopontin (OPN, osteocalcin (OCN and matrix mineralization. We found that PKA inhibitor reduces BMP9-induced Runx2 activation and p38 phosphorylation in MSCs. Lastly, interference of CREB function by A-CREB decreased BMP9-induced osteogenic differentiation as well. Conclusion: Our results revealed that BMP9 may activate PKA/CREB signaling in MSCs through suppression of PKIγ expression. It is noteworthy that inhibition of PKA/CREB signaling may impair BMP9-induced osteogenic differentiation of MSCs, implying that activation of PKA/CREB signaling is required for BMP9 osteoinductive activity.

  11. Morinda citrifolia Leaf Extract Enhances Osteogenic Differentiation Through Activation of Wnt/β-Catenin Signaling.

    Science.gov (United States)

    Gu, Hanna; Boonanantanasarn, Kanitsak; Kang, Moonkyu; Kim, Ikhwi; Woo, Kyung Mi; Ryoo, Hyun-Mo; Baek, Jeong-Hwa

    2017-10-05

    Morinda citrifolia (Noni) leaf is an herbal medicine with application in the domestic treatment of a broad range of conditions, including bone fracture and luxation. However, the basic mechanism underlying the stimulation of osteogenic differentiation by Noni leaf extract remains poorly understood. This study aimed to examine the effect of this extract on osteogenic differentiation and the mechanism by which Noni leaf extract enhances osteogenic differentiation. Aqueous extract of Noni leaves was prepared, and rutin and kaempferol-3-O-rutinoside were identified to be two of its major components. C2C12 and human periodontal ligament (hPDL) cells were used to study the effect of Noni. Noni did not show cytotoxicity at a concentration range of 0.015%-1.0% (w/v%) and significantly enhanced the activity of alkaline phosphatase (ALP) and expression levels of osteoblast differentiation markers, including Runx2, ALP, osterix, and osteocalcin, bone morphogenetic protein 2, Wnt3a, and β-catenin. In addition, Noni enhanced the matrix mineralization of hPDL cells. In the signaling pathways, Noni increased the phosphorylation levels of Akt and GSK3β and nuclear translocation and transcriptional activity of β-catenin, which were attenuated by the addition of Dkk-1, a Wnt inhibitor, or LY294002, a PI3K inhibitor. These results suggest that Noni leaf extract enhances osteogenic differentiation through the PI3K/Akt-dependent activation of Wnt/β-catenin signaling. Noni leaf extract might be a novel alternative medicine for bone and periodontal regeneration in patients with periodontal diseases.

  12. The Osteogenic Differentiation Effect of the FN Type 10-Peptide Amphiphile on PCL Fiber

    Directory of Open Access Journals (Sweden)

    Ye-Rang Yun

    2018-01-01

    Full Text Available The fibronectin type 10-peptide amphiphile (FNIII10-PA was previously genetically engineered and showed osteogenic differentiation activity on rat bone marrow stem cells (rBMSCs. In this study, we investigated whether FNIII10-PA demonstrated cellular activity on polycaprolactone (PCL fibers. FNIII10-PA significantly increased protein production and cell adhesion activity on PCL fibers in a dose-dependent manner. In cell proliferation results, there was no effect on cell proliferation activity by FNIII10-PA; however, FNIII10-PA induced the osteogenic differentiation of MC3T3-E1 cells via upregulation of bone sialoprotein (BSP, collagen type I (Col I, osteocalcin (OC, osteopontin (OPN, and runt-related transcription factor 2 (Runx2 mitochondrial RNA (mRNA levels; it did not increase the alkaline phosphatase (ALP mRNA level. These results indicate that FNIII10-PA has potential as a new biomaterial for bone tissue engineering applications.

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

    Science.gov (United States)

    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.

  14. Strontium-substituted bioactive glasses in vitro osteogenic and antibacterial effects.

    Science.gov (United States)

    Liu, Jie; Rawlinson, Simon C F; Hill, Robert G; Fortune, Farida

    2016-03-01

    Bioactive glass forms a bone mineral apatite interface and can be engineered to promote optimal bone regeneration. Strontium (Sr(2+)) stimulates osteoblast and inhibits osteoclast activities in vitro, and is used clinically as a treatment for osteoporosis. Dental bone defect repair requires rapid bone formation for early osseointegration but, can be subject to infection. The aim of this study was to investigate the osteogenic and antibacterial effects of strontium-substituted bioactive glasses in vitro. Strontium-substituted bioactive glasses were designed and produced. Then the osteogenic potential and antibacterial effects of bioactive glass particulates were explored. Alkaline phosphatase activity, cell number, Type I collagen and mineral nodule formation of MC3T3-E1 cells were significantly promoted by the 5% strontium-substituted glass (5Sr). Furthermore, after incubation with 0.001g and 0.01g glass particulates, the growth of sub-gingival bacteria, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis was significantly inhibited; the antibacterial activity being dependent on the percentage of strontium in the glasses. These results show that strontium-substituted bioactive glasses significantly promote osteogenic responses of MC3T3-E1 osteoblast-like cells and inhibit the growth of A. actinomycetemcomitans and P. gingivalis. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

  15. Cyclic arginine-glycine-aspartate peptides enhance three-dimensional stem cell osteogenic differentiation.

    Science.gov (United States)

    Hsiong, Susan X; Boontheekul, Tanyarut; Huebsch, Nathaniel; Mooney, David J

    2009-02-01

    The role of morphogens in bone regeneration has been widely studied, whereas the effect of matrix cues, particularly on stem cell differentiation, are less well understood. In this work, we investigated the effects of arginine-glycine-aspartate (RGD) ligand conformation (linear vs cyclic RGD) on primary human bone marrow stromal cell (hBMSC) and D1 stem cell osteogenic differentiation in three-dimensional (3D) culture and compared their response with that of committed MC3T3-E1 preosteoblasts to determine whether the stage of cell differentiation altered the response to the adhesion ligands. Linear RGD densities that promoted osteogenic differentiation of committed cells (MC3T3-E1 preosteoblasts) did not induce differentiation of hBMSCs or D1 stem cells, although matrices presenting the cyclic form of this adhesion ligand enhanced osteoprogenitor differentiation in 3D culture. This may be due to enhanced integrin-ligand binding. These studies indicate that biomaterial design parameters optimized for differentiated cell types may not directly translate to stem cell populations, because less-committed cells may require more instruction than differentiated cells. It is likely that design of synthetic extracellular matrices tailored to promote stem cell differentiation may enhance bone regeneration by transplanted cells.

  16. MicroRNA-4739 regulates osteogenic and adipocytic differentiation of immortalized human bone marrow stromal cells via targeting LRP3

    DEFF Research Database (Denmark)

    Elsafadi, Mona; Manikandan, Muthurangan; Alajez, Nehad M

    2017-01-01

    3 (LRP3) in regulating the osteogenic and adipogenic differentiation of immortalized hBMSCs. Gene expression profiling revealed significantly higher LRP3 levels in the highly osteogenic hBMSC clone imCL1 than in the less osteogenic clone imCL2, as well as a significant upregulation of LRP3 during......Understanding the regulatory networks underlying lineage differentiation and fate determination of human bone marrow stromal cells (hBMSC) is a prerequisite for their therapeutic use. The goal of the current study was to unravel the novel role of the low-density lipoprotein receptor-related protein...... the osteogenic induction of the imCL1 clone. Data from functional and gene expression assays demonstrated the role of LRP3 as a molecular switch promoting hBMSC lineage differentiation into osteoblasts and inhibiting differentiation into adipocytes. Interestingly, microRNA (miRNA) expression profiling identified...

  17. Dental pulp stem cells: osteogenic differentiation and gene expression

    National Research Council Canada - National Science Library

    Mori, Giorgio; Brunetti, Giacomina; Oranger, Angela; Carbone, Claudia; Ballini, Andrea; Muzio, Lorenzo Lo; Colucci, Silvia; Mori, Claudio; Grassi, Felice Roberto; Grano, Maria

    2011-01-01

    Dental pulp stem cells (DPSCs) are an adult stem cell population with high proliferative potential and the ability to differentiate in many cell types, and this has led scientists to consider these cells to be an...

  18. Construction of a Luciferase Reporter System to Monitor Osteogenic Differentiation of Mesenchymal Stem Cells by Using a Mammalian Artificial Chromosome Vector.

    Science.gov (United States)

    Narai, Takashi; Katoh, Motonobu; Inoue, Toshiaki; Taniguchi, Makoto; Kazuki, Kanako; Kazuki, Yasuhiro; Sato, Kenzo; Kodani, Isamu; Ryoke, Kazuo; Oshimura, Mitsuo

    2015-03-01

    Mesenchymal stem cells (MSCs) hold promise for application in adult stem cell-mediated regenerative medicine in bone remodeling and fracture repair. MSCs in vitro can be directed to osteogenic lineage by dexamethasone (DEX); however, the use of DEX is not practical in clinical settings because of adverse side effects such as glucocorticoid-induced osteoporosis. For identifying substances that facilitate osteogenesis, a monitoring system, which detects the osteogenic differentiation stage of MSCs accurately and easily, is required. By focusing on the human osteocalcin (OC) gene whose expression profile is described along with osteogenic differentiation, we constructed the luciferase (Luc) reporter gene driven by the enhancer/promoter sequence of the human OC gene (OC-Luc) utilizing a mammalian artificial chromosome. Mammalian artificial chromosome is a suitable platform for loading reporter constructs, because of its stable episomal maintenance in host cells, transferability into any cell and assurance of long-term physiological transgene expression. We loaded the OC-Luc on a mammalian artificial chromosome vector engineered from mouse chromosome (designated as mouse artificial chromosome, MAC) in Chinese hamster ovary cells (OC-Luc/MAC) and transferred this into human MSC cells via chromosome transfer. OC-Luc/MAC in human MSC cells are responsive to positive and negative stimulation by 1 alpha,25-dihydroxyvitamin D3 and DEX in differentiation stage of MSCs to osteoblasts, reflecting the manner of physiological expression. The OC-Luc/MAC reporter system may contribute not only to monitoring the osteogenic differentiation stage from MSC but also to identify novel osteogenic drugs.

  19. Photostimulation of osteogenic differentiation on silk scaffolds by plasma arc light source.

    Science.gov (United States)

    Çakmak, Anıl Sera; Çakmak, Soner; Vatansever, H Seda; Gümüşderelioğlu, Menemşe

    2017-12-18

    Low-level laser therapy (LLLT) has been used for more than 30 years to heal wounds. In recent years, LLLT or photostimulation has been indicated as an effective tool for regenerative and dental medicine by using monochromatic light. The aim of this study is to indicate the usability of plasma arc light source for bone regeneration. This is why we used polychromatic light source providing effective wavelengths in the range of 590-1500 nm for cellular response and investigated photostimulation effects on osteogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on 3D silk scaffolds. Cellular responses were examined by using cell culture methods in terms of proliferation, differentiation, and morphological analyses. The results showed that photostimulation with a polychromatic light source (applied for 5 min from the 3rd day after seeding up to the 28th day in 2-day intervals with 92-mW/cm2 power from 10-cm distance to the cells) enhanced osteogenic differentiation of hMSCs according to higher alkaline phosphatase (ALP) activity, collagen and calcium content, osteogenic gene expressions, and matrix mineralization. In conclusion, we suggest that the plasma arc light source that was used here has a great potential for bone regeneration.

  20. YAP-mediated mechanotransduction regulates osteogenic and adipogenic differentiation of BMSCs on hierarchical structure.

    Science.gov (United States)

    Pan, Houhua; Xie, Youtao; Zhang, Zequan; Li, Kai; Hu, Dandan; Zheng, Xuebin; Fan, Qiming; Tang, Tingting

    2017-04-01

    Hierarchical structure mimicking the natural bone microenvironment has been considered as a promising platform to regulate cell functions. We have previously fabricated hierarchical macropore/nanowire structure and evidence has shown that it can better manipulate the cytoskeleton status and osteogenic performance of osteoblasts. However, how cues of hierarchical structure are translated and ultimately linked to BMSC lineage commitment have still remained elusive, which hinders the accurate knowledge and further development of the hierarchical structure. In this study, bone marrow-derived mesenchymal stem cells (BMSCs) fate on hierarchical structure was investigated as well as the detailed mechanisms. It was shown that well-developed cytoskeleton and focal adhesion were observed for BMSCs on hierarchical structure, which was accompanied by enhanced osteogenic and depressed adipogenic potential. Evidence of increased YAP activity and nuclear translocation were exhibited on hierarchical structure and YAP knockdown inhibited osteogenic differentiation and promoted adipogenic differentiation induced by hierarchical structure. Further remove of cytoskeleton tension inhibited YAP function, which confirmed the key role of YAP-mediated mechanotransduction in the BMSC differentiation. These results together provide information of the stem cell fate commitment on hierarchical structure and a promising approach to design advanced biomaterials by focusing on specific mechanotransduction process. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Expression of OCT-4 and SOX-2 in Bone Marrow-Derived Human Mesenchymal Stem Cells during Osteogenic Differentiation.

    Science.gov (United States)

    Matic, Igor; Antunovic, Maja; Brkic, Sime; Josipovic, Pavle; Mihalic, Katarina Caput; Karlak, Ivan; Ivkovic, Alan; Marijanovic, Inga

    2016-03-15

    Determine the levels of expression of pluripotency genes OCT-4 and SOX-2 before and after osteogenic differentiation of human mesenchymal stem cells (hMSCs). Human MSCs were derived from the bone marrow and differentiated into osteoblasts. The analyses were performed on days 0 and 14 of the cell culture. In vitro differentiation was evaluated due to bone markers - alkaline phosphatase (AP) activity and the messenger RNA (mRNA) expression of AP and bone sialoprotein (BSP). The OCT-4 and SOX-2 expression was evaluated at mRNA level by real-time qPCR and at protein level by immunocytochemistry. In vitro cultures on day 14 showed an increase in AP activity and upregulation of AP and BSP gene expression. OCT-4 and SOX-2 in undifferentiated hMSCs on day 0 is detectable and very low compared to tumor cell lines as a positive control. Immunocytochemistry detected OCT-4 in the cell nuclei prior (day 0) and post differentiation (day 14). On the same time points, cultures were negative for SOX-2 protein. Messenger RNA for pluripotency markers OCT-4 and SOX-2 isolated from hMSCs was less present, while OCT-4 protein was detected in cell nuclei prior and post differentiation into osteoblast lineage.

  2. Epigenetic Regulation of Mesenchymal Stem Cells: A Focus on Osteogenic and Adipogenic Differentiation

    Directory of Open Access Journals (Sweden)

    Chad M. Teven

    2011-01-01

    Full Text Available Stem cells are characterized by their capability to self-renew and terminally differentiate into multiple cell types. Somatic or adult stem cells have a finite self-renewal capacity and are lineage-restricted. The use of adult stem cells for therapeutic purposes has been a topic of recent interest given the ethical considerations associated with embryonic stem (ES cells. Mesenchymal stem cells (MSCs are adult stem cells that can differentiate into osteogenic, adipogenic, chondrogenic, or myogenic lineages. Owing to their ease of isolation and unique characteristics, MSCs have been widely regarded as potential candidates for tissue engineering and repair. While various signaling molecules important to MSC differentiation have been identified, our complete understanding of this process is lacking. Recent investigations focused on the role of epigenetic regulation in lineage-specific differentiation of MSCs have shown that unique patterns of DNA methylation and histone modifications play an important role in the induction of MSC differentiation toward specific lineages. Nevertheless, MSC epigenetic profiles reflect a more restricted differentiation potential as compared to ES cells. Here we review the effect of epigenetic modifications on MSC multipotency and differentiation, with a focus on osteogenic and adipogenic differentiation. We also highlight clinical applications of MSC epigenetics and nuclear reprogramming.

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

    Science.gov (United States)

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

    2015-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Huichao Wang

    2017-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Guo-yong Yu

    2016-01-01

    Full Text Available 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.

  6. GEORG-SCHMORL-PRIZE OF THE GERMAN SPINE SOCIETY (DWG) 2016: Comparison of in vitro osteogenic potential of iliac crest and degenerative facet joint bone autografts for intervertebral fusion in lumbar spinal stenosis.

    Science.gov (United States)

    Geurts, Jeroen; Ramp, Daniela; Schären, Stefan; Netzer, Cordula

    2017-05-01

    I collagen was enhanced during osteogenesis and significantly greater in iliac crest MSC. Correspondingly, COL1A2 mRNA expression was higher in osteogenically differentiated MSC from iliac crest. Adipocyte numbers showed significant differences between iliac crest (63 ± 60) and facet joint (18 ± 15) MSC under osteogenic conditions. Negative (GREM1) and positive (FABP4) adipogenic markers were not differentially expressed between sources. MSC from iliac crest and degenerative facet joints largely display similar clonogenic and osteogenic properties in vitro. Differences at the molecular level are not likely to impair the osteoinductive capacity of facet joint MSC. Bone autografts from facetectomy would be viable alternatives as bone autografts for intervertebral spinal fusion in lumbar spinal stenosis.

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

    Energy Technology Data Exchange (ETDEWEB)

    Gandhimathi, Chinnasamy [Cellular and Molecular Epigenetics Lab, Lee Kong Chian School of Medicine, Nanyang Technological University (Singapore); Venugopal, Jayarama Reddy [Center for Nanofibers and Nanotechnology, Nanoscience and Nanotechnology Initiative, National University of Singapore (Singapore); Tham, Allister Yingwei [Cellular and Molecular Epigenetics Lab, Lee Kong Chian School of Medicine, Nanyang Technological University (Singapore); Ramakrishna, Seeram [Center for Nanofibers and Nanotechnology, Nanoscience and Nanotechnology Initiative, National University of Singapore (Singapore); Kumar, Srinivasan Dinesh, E-mail: dineshkumar@ntu.edu.sg [Cellular and Molecular Epigenetics Lab, Lee Kong Chian School of Medicine, Nanyang Technological University (Singapore)

    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 ± 22 nm and 1.5–6.9 μm respectively. Resulting nanofibrous scaffolds are highly porous (87–94%) with ultimate tensile strength observed in the range of 1.51–4.86 MPa 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. - Highlights: • We fabricated and characterized hybrid porous nanofibrous scaffolds. • PLACL/SF/AA/TC/n-HA scaffolds promote cell differentiation and mineralization. • Porous nanofibrous scaffolds initiate MSC differentiation into osteogenic cells. • Biomimetic nanofibrous scaffolds have good potential for bone tissue engineering.

  8. Co(II)-mediated effects of plain and plasma immersion ion implanted cobalt-chromium alloys on the osteogenic differentiation of human mesenchymal stem cells.

    Science.gov (United States)

    Schröck, Kathleen; Lutz, Johanna; Mändl, Stephan; Hacker, Michael C; Kamprad, Manja; Schulz-Siegmund, Michaela

    2015-03-01

    Medical CoCr is one of the main alloys used for metal-on-metal prosthesis in patients with total hip arthroplasty. CoCr surfaces modified by nitrogen plasma immersion ion implantation (PIII) are characterized by improved wear resistance but also showed increased Co(II) ion release under in vitro conditions. For the first time, CoCr modified by nitrogen PIII was evaluated with regard to its effect on the osteogenic differentiation of MSC. The activity of alkaline phosphatase, the expression of the osteogenic genes Runt-related transcription factor 2, osteopontin as well as integrin-binding bone sialoprotein and the production of osteocalcin and hydroxyapatite were determined. The results of our study demonstrate that Co(II) ions released from the alloy affected the osteogenic differentiation of MSC. Distinct differences in differentiation markers were found between pristine and modified alloys for osteocalcin but not for integrin-binding sialoprotein and hydroxyapatite. Interestingly, osteopontin was upregulated in naive and differentiated MSC by Co(II) ions and modified CoCr, likely through the induction of a cellular hypoxic response. The findings of this study contribute to a better understanding of possible risk factors with regard to a clinical applicability of surface modified CoCr implant materials. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  9. Dental pulp-derived stromal cells exhibit a higher osteogenic potency than bone marrow-derived stromal cells in vitro and in a porcine critical-size bone defect model

    DEFF Research Database (Denmark)

    Jensen, Jonas; Tvedesøe, Claus; Rölfing, Jan Hendrik Duedal

    2016-01-01

    INTRODUCTION: The osteogenic differentiation of bone marrow-derived mesenchymal stromal cells (BMSCs) was compared with that of dental pulp-derived stromal cells (DPSCs) in vitro and in a pig calvaria critical-size bone defect model. METHODS: BMSCs and DPSCs were extracted from the tibia bone mar...

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

    Science.gov (United States)

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

    2016-03-09

    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. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. A minimal common osteochondrocytic differentiation medium for the osteogenic and chondrogenic differentiation of bone marrow stromal cells in the construction of osteochondral graft.

    Science.gov (United States)

    Li, Jian; Mareddy, Shobha; Tan, Dawn Meifang; Crawford, Ross; Long, Xing; Miao, Xigeng; Xiao, Yin

    2009-09-01

    To regenerate the complex tissue such as bone-cartilage construct using tissue engineering approach, controllable differentiation of bone marrow stromal cells (BMSCs) into chondrogenic and osteogenic lineages is crucially important. This study proposes to test a minimum common osteochondrocytic differentiation medium (MCDM) formulated by including common soluble supplements (dexamethasone and ascorbic acid) used to induce chondrogenic and osteogenic differentiation. The MCDM coupled with supplemented growth factors was tested for its ability to differentiate BMSCs into osteogenic and chondrogenic lineages in both two-dimensional and three-dimensional culture systems. When transforming growth factor beta3 was added to MCDM, BMSCs differentiated to chondrocyte-like cells, evidenced by the expression of glycosaminoglycans and type II collagen, whereas osteogenic differentiation was induced by supplementing osteogenic protein-1, resulting in detectable expression of osteopontin and osteocalcin. These chondrogenic and osteogenic differentiation markers were significantly enhanced in the three-dimensional cultures compared to the two-dimensional monolayer cultures. The results achieved in this study lay a foundation for future development of osteochondral graft, which could be engineered from bilayered scaffold with spatially loaded growth factors to control BMSC differentiation.

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

    Directory of Open Access Journals (Sweden)

    Xuehui Zhang

    2015-01-01

    Full Text Available 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.

  13. Electrospun Gelatin/β-TCP Composite Nanofibers Enhance Osteogenic Differentiation of BMSCs and In Vivo Bone Formation by Activating Ca (2+) -Sensing Receptor Signaling.

    Science.gov (United States)

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

    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 Ca(2+)-sensing receptor signaling.

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

    Directory of Open Access Journals (Sweden)

    Luis A Castillo Diaz

    2016-07-01

    Full Text Available 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.

  15. The effect of TRPM7 suppression on the proliferation, migration and osteogenic differentiation of human dental pulp stem cells.

    Science.gov (United States)

    Cui, L; Xu, S M; Ma, D D; Wu, B L

    2014-06-01

    To investigate the role of the Ca(2+) -Mg(2+) ion channel TRPM7 in the proliferation, migration and osteogenic differentiation of human dental pulp stem cells (hDPSCs). Immunohistochemistry was used to localize expression of TRPM7 in human dental pulp tissues and in cultured hDPSCs. Isolated hDPSCs were infected with recombinant lentiviruses expressing short hairpin RNA (shRNA) specific for TRPM7, or control shRNA, in order to suppress TRPM7 mRNA expression and investigate its functional role. The proliferation of the shRNA-infected hDPSCs was evaluated using both an MTT assay to measure viable cell numbers and cell cycle analysis. Cell migration was assessed using a transwell assay. The dynamic mRNA expression of TRPM7 during osteogenic differentiation of hDPSCs and the effect of shRNA specific for TRPM7 on hDPSC osteogenic differentiation were evaluated by real-time PCR. TRPM7 expression was widespread in human dental pulp tissue and was detected mainly in the cytomembrane and cytoplasm of hDPSCs. Suppression of TRPM7 inhibited both the proliferation and the migratory capacity of hDPSCs. TRPM7 mRNA expression was elevated during osteogenic differentiation of hDPSCs. TRPM7-specific shRNA inhibited osteogenic differentiation of hDPSCs, with downregulated mRNA expression of the osteogenic markers alkaline phosphatase (ALP), dentine sialophosphoprotein (DSPP), bone sialoprotein (BSP), runt-related transcription factor (RUNX2) and osterix (OSX). TRPM7 was involved in the regulation of hDPSC proliferation, migration and osteogenic differentiation and may play a role in the dental pulp repair process. © 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  16. Osteogenic differentiation of human periosteal-derived cells in a three-dimensional collagen scaffold.

    Science.gov (United States)

    Ryu, Young-Mo; Hah, Young-Sool; Park, Bong-Wook; Kim, Deok Ryong; Roh, Gu Seob; Kim, Jong-Ryoul; Kim, Uk-Kyu; Rho, Gyu-Jin; Maeng, Geun-Ho; Byun, June-Ho

    2011-06-01

    This study examined the osteogenic differentiation of cultured human periosteal-derived cells grown in a three dimensional collagen-based scaffold. Periosteal explants with the appropriate dimensions were harvested from the mandible during surgical extraction of lower impacted third molar. Periosteal-derived cells were introduced into cell culture. After passage 3, the cells were divided into two groups and cultured for 28 days. In one group, the cells were cultured in two-dimensional culture dishes with osteogenic inductive medium containing dexamethasone, ascorbic acid, and β-glycerophosphate. In the other group, the cells were seeded onto a three-dimensional collagen scaffold and cultured under the same conditions. We examined the bioactivity of alkaline phosphatase (ALP), the RT-PCR analysis for ALP and osteocalcin, and measurements of the calcium content in the periosteal-derived cells of two groups. Periosteal-derived cells were successfully differentiated into osteoblasts in the collagen-based scaffold. The ALP activity in the periosteal-derived cells was appreciably higher in the three-dimensional collagen scaffolds than in the two-dimensional culture dishes. The levels of ALP and osteocalcin mRNA in the periosteal-derived cells was also higher in the three-dimensional collagen scaffolds than in the two-dimensional culture dishes. The calcium level in the periosteal-derived cells seeded onto three-dimensional collagen scaffolds showed a 5.92-fold increase on day 7, 3.28-fold increase on day 14, 4.15-fold increase on day 21, and 2.91-fold increase on day 28, respectively, compared with that observed in two-dimensional culture dishes. These results suggest that periosteal-derived cells have good osteogenic capacity in a three-dimensional collagen scaffold, which provides a suitable environment for the osteoblastic differentiation of these cells.

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

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Wei [The Education Ministry Key Lab of Resource Chemistry and Shanghai Key Laboratory of Rare Earth Functional Materials, Shanghai Normal University, Shanghai 200234 (China); Tian, Bo [Shanghai Key Laboratory of Orthopedic Implant, Department of Orthopedic Surgery, Shanghai Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011 (China); Lei, Yong; Ke, Qin-Fei [The Education Ministry Key Lab of Resource Chemistry and Shanghai Key Laboratory of Rare Earth Functional Materials, Shanghai Normal University, Shanghai 200234 (China); Zhu, Zhen-An, E-mail: zhuzhenan2006@126.com [Shanghai Key Laboratory of Orthopedic Implant, Department of Orthopedic Surgery, Shanghai Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011 (China); Guo, Ya-Ping, E-mail: ypguo@shnu.edu.cn [The Education Ministry Key Lab of Resource Chemistry and Shanghai Key Laboratory of Rare Earth Functional Materials, Shanghai Normal University, Shanghai 200234 (China)

    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. - Highlights: • We prepare hydroxyapatite coatings with oriented nanoplate and nanorod arrays. • Hydroxyapatite coatings are in situ converted from bioglass coatings. • Hydroxyapatite coatings have good cytocompatibility and osteogenic differentiation. • Oriented hydroxyapatite coatings are used for orthopedic implants.

  18. In vivo osteogenic differentiation of stem cells inside compartmentalized capsules loaded with co-cultured endothelial cells.

    Science.gov (United States)

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

    2017-04-15

    Capsules coated with polyelectrolytes and co-encapsulating adipose stem (ASCs) and endothelial (ECs) cells with surface modified microparticles are developed. Microparticles and cells are freely dispersed in a liquified core, responsible to maximize the diffusion of essential molecules and allowing the geometrical freedom for the autonomous three-dimensional (3D) organization of cells. While the membrane wraps all the instructive cargo elements within a single structure, the microparticles provide a solid 3D substrate for the encapsulated cells. Our hypothesis is that inside this isolated biomimetic 3D environment, ECs would lead ASCs to differentiate into the osteogenic lineage to ultimately generate a mineralized tissue in vivo. For that, capsules encapsulating only ASCs (MONO capsules) or co-cultured with ECs (CO capsules) are subcutaneously implanted in nude mice up to 6weeks. Capsules implanted immediately after production or after 21days of in vitro osteogenic stimulation are tested. The most valuable outcome of the present study is the mineralized tissue in CO capsules without in vitro pre-differentiation, with similar levels compared to the pre-stimulated capsules in vitro. We believe that the proposed bioencapsulation strategy is a potent self-regulated system, which might find great applicability in bone tissue engineering. The diffusion efficiency of essential molecules for cell survival is a main issue in cell encapsulation. Former studies reported the superior biological outcome of encapsulated cells within liquified systems. However, most cells used in TE are anchorage-dependent, requiring a solid substrate to perform main cellular processes. We hypothesized that liquified capsules encapsulating microparticles are a promising attempt. Inspired by the multiphenotypic cellular environment of bone, we combine the concept of liquified capsules with co-cultures of stem and endothelial cells. After implantation, results show that co-cultured capsules

  19. Osteogenic potential of dualblocks cultured with human periodontal ligament stem cells: in vitro and synchrotron microtomography study.

    Science.gov (United States)

    Manescu, A; Giuliani, A; Mohammadi, S; Tromba, G; Mazzoni, S; Diomede, F; Zini, N; Piattelli, A; Trubiani, O

    2016-02-01

    In the present study, the early stages of in vitro bone formation in collagenated porcine scaffolds cultured with human periodontal ligament cells were investigated. The comparison between the osteogenic potential of this structure in basal and differentiating culture media was explored to predict the mechanism of its biological behavior as graft in human defect. Results were validated by synchrotron radiation X-Ray phase contrast computed microtomography (micro-CT). As the periodontal disease plays a key role in systemic and oral diseases, it is crucial to find advanced therapeutic clinical interventions to repair periodontal defects. This has been recently explored using cells and tissues developed in vitro that should ideally be immunologically, functionally, structurally and mechanically identical to the native tissue. In vitro cultures of human periodontal ligament cells, easily obtained by scraping of alveolar crestal and horizontal fibers of the periodontal ligament, were seeded on to collagenated porcine blocks constituted by natural cancellous and cortical bone. 3D images were obtained by synchrotron radiation micro-CT and processed with a phase-retrieval algorithm based on the transport of intensity equation. Starting from the second week of culture, newly formed mineralized bone was detected in all the scaffolds, both in basal and differentiating media. Bone mineralization was proved to occur preferentially in the trabecular portion and in differentiating media. The chosen method, supported by phase contrast micro-CT analysis, successfully and quantitatively monitored the early stages of bone formation and the rate of the bioscaffold resorption in basal and differentiating culture media. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo

    DEFF Research Database (Denmark)

    Eskildsen, Tilde; Taipaleenmäki, H.; Stenvang, Jan

    2011-01-01

    Elucidating the molecular mechanisms that regulate human stromal (mesenchymal) stem cell (hMSC) differentiation into osteogenic lineage is important for the development of anabolic therapies for treatment of osteoporosis. MicroRNAs (miRNAs) are short, noncoding RNAs that act as key regulators......, overexpression of miR-138 reduced ectopic bone formation in vivo by 85%, and conversely, in vivo bone formation was enhanced by 60% when miR-138 was antagonized. Target prediction analysis and experimental validation by luciferase 3' UTR reporter assay confirmed focal adhesion kinase, a kinase playing a central...

  1. FOXO1-suppressed miR-424 regulates the proliferation and osteogenic differentiation of MSCs by targeting FGF2 under oxidative stress

    Science.gov (United States)

    Li, Liangping; Qi, Qihua; Luo, Jiaquan; Huang, Sheng; Ling, Zemin; Gao, Manman; Zhou, Zhiyu; Stiehler, Maik; Zou, Xuenong

    2017-02-01

    Recently, microRNAs (miRNAs) have been identified as key regulators of the proliferation and differentiation of mesenchymal stem cells (MSCs). Our previous in vivo study and other in vitro studies using miRNA microarrays suggest that miR-424 is involved in the regulation of bone formation. However, the role and mechanism of miR-424 in bone formation still remain unknown. Here, we identified that the downregulation of miR-424 mediates bone formation under oxidative stress, and we explored its underlying mechanism. Our results showed that miR-424 was significantly downregulated in an anterior lumbar interbody fusion model of pigs and in a cell model of oxidative stress induced by H2O2. The overexpression of miR-424 inhibited proliferation and osteogenic differentiation shown by a decrease in alkaline phosphatase (ALP) activity, mineralization and osteogenic markers, including RUNX2 and ALP, whereas the knockdown of miR-424 led to the opposite results. Moreover, miR-424 exerts its effects by targeting FGF2. Furthermore, we found that FOXO1 suppressed miR-424 expression and bound to its promoter region. FOXO1 enhanced proliferation and osteogenic differentiation in part through the miR-424/FGF2 pathway. These results indicated that FOXO1-suppressed miR-424 regulates both the proliferation and osteogenic differentiation of MSCs via targeting FGF2, suggesting that miR-424 might be a potential novel therapeutic strategy for promoting bone formation.

  2. Ectopic expression of telomerase enhances osteopontin and osteocalcin expression during osteogenic differentiation of human mesenchymal stem cells from elder donors

    Directory of Open Access Journals (Sweden)

    Machado CB

    2009-01-01

    Full Text Available Age related bone loss is one of the most prevalent diseases in the elder population. The osteoblasts are the effectors cells of bone formation and regeneration. With the aging the osteoblasts become senescent reducing their ability to produce bone. Cellular replicative senescence is triggered by telomers shortening. Telomerase elongate the telomers length and maintain the cell proliferative capacity. Here, we demonstrated that the expression of human telomerase reverse transcriptase mediated by an adenovirus vector increases the levels of osteopontin and osteocalcin mRNA during the in vitro osteogenic differentiation of elderly human mesenchymal stem cells. Bone marrow human mesenchymal stem cells were obtained from old donors (>65 years and induced to differentiate into osteoblasts for 14 days. The levels of mRNA of human telomerase reverse transcriptase, osteopontin and osteocalcin during the differentiation were assessed by semi-quantitative PCR before and during the differentiation on days 7 and 14. Infected cells showed 1.5 fold increase in telomerase expression. Also telomerized cells exhibit 1.5 fold increase in osteopontin and 0.5 fold increase in osteocalcin expression compared to primary osteoblasts isolated from the same donors. The transformed cells were not able to form tumours in NUDE mice.

  3. Effects of strength training on osteogenic differentiation and bone strength in aging female Wistar rats

    Science.gov (United States)

    Singulani, Monique Patricio; Stringhetta-Garcia, Camila Tami; Santos, Leandro Figueiredo; Morais, Samuel Rodrigues Lourenço; Louzada, Mário Jefferson Quirino; Oliveira, Sandra Helena Penha; Chaves Neto, Antonio Hernandes; Dornelles, Rita Cássia Menegati

    2017-01-01

    The effects of strength training (ST) on the mechanical bone strength and osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs) from adult, aged and exercised aged rats were determined. The exercised aged animals displayed higher values of areal bone mineral density, compression test, alkaline phosphatase activity (ALP) and biological mineralization, while oil red O staining for adipocytes was lower. ST increased gene expression of runt-related transcription factor 2 (Runx2), osterix (Osx) as well as bone matrix protein expression, and reduced expression of peroxisome proliferator-activated receptor gamma (Pparγ). The production of pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α) was lower in BMSCs of the aged exercised group. The ST practice was able to improve the bone mechanical properties in aged female rats, increasing the potential for osteogenic differentiation of BMSCs, reducing the adipogenic differentiation and pro-inflammatory cytokine level. In summary, the data achieved in this study showed that strength training triggers physiological responses that result in changes in the bone microenvironment and bring benefits to biomechanical parameters of bone tissue, which could reduce the risk of fractures during senescent. PMID:28211481

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

    Directory of Open Access Journals (Sweden)

    Jingru Meng

    2016-04-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  6. Human Mesenchymal Stem Cells Growth and Osteogenic Differentiation on Piezoelectric Poly(vinylidene fluoride Microsphere Substrates

    Directory of Open Access Journals (Sweden)

    R. Sobreiro-Almeida

    2017-11-01

    Full Text Available The aim of this work was to determine the influence of the biomaterial environment on human mesenchymal stem cell (hMSC fate when cultured in supports with varying topography. Poly(vinylidene fluoride (PVDF culture supports were prepared with structures ranging between 2D and 3D, based on PVDF films on which PVDF microspheres were deposited with varying surface density. Maintenance of multipotentiality when cultured in expansion medium was studied by flow cytometry monitoring the expression of characteristic hMSCs markers, and revealed that cells were losing their characteristic surface markers on these supports. Cell morphology was assessed by scanning electron microscopy (SEM. Alkaline phosphatase activity was also assessed after seven days of culture on expansion medium. On the other hand, osteoblastic differentiation was monitored while culturing in osteogenic medium after cells reached confluence. Osteocalcin immunocytochemistry and alizarin red assays were performed. We show that flow cytometry is a suitable technique for the study of the differentiation of hMSC seeded onto biomaterials, giving a quantitative reliable analysis of hMSC-associated markers. We also show that electrosprayed piezoelectric poly(vinylidene fluoride is a suitable support for tissue engineering purposes, as hMSCs can proliferate, be viable and undergo osteogenic differentiation when chemically stimulated.

  7. Azadirachta indica triterpenoids promote osteoblast differentiation and mineralization in vitro and in vivo.

    Science.gov (United States)

    Kushwaha, Priyanka; Khedgikar, Vikram; Haldar, Saikat; Gautam, Jyoti; Mulani, Fayaj A; Thulasiram, Hirekodathakallu V; Trivedi, Ritu

    2016-08-01

    Terpenoids were isolated using chromatographic purification through solvent purification technique and identified as Azadirone (1), Epoxyazadiradione (2) Azadiradione (3) Gedunin (4) Nimbin (5) Salannin (6) Azadirachtin A (7) and Azadirachtin B (8) from Azadirachta indica. Out of eight compounds, only three compounds had osteogenic activity and enhanced osteoblast proliferation, differentiation and mineralization in osteoblast cells. Active compounds stimulated osteogenic genes ALP, RunX-2 and OCN expressions in vitro, but Azadirachtin A had a maximum ability to stimulate osteoblast differentiation and mineralization compared to other two active compounds. For in vivo study, Azadirachtin A injected subcutaneously in pups, which enhanced osteogenic gene expressions and promoted bone formation rate significantly. Here, we conclude that active compounds of Azadirachta indica have osteogenic activity and Azadirachtin A has a beneficial effects on bone. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Fransisca A. S. van Esterik

    2016-01-01

    Full Text Available 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.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Fangfang Wang

    Full Text Available 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

  11. Biomineralized matrices dominate soluble cues to direct osteogenic differentiation of human mesenchymal stem cells through adenosine signaling.

    Science.gov (United States)

    Kang, Heemin; Shih, Yu-Ru V; Varghese, Shyni

    2015-03-09

    Stem cell differentiation is determined by a repertoire of signals from its microenvironment, which includes the extracellular matrix (ECM) and soluble cues. The ability of mesenchymal stem cells (MSCs), a common precursor for the skeletal system, to differentiate into osteoblasts and adipocytes in response to their local cues plays an important role in skeletal tissue regeneration and homeostasis. In this study, we investigated whether a bone-specific calcium phosphate (CaP) mineral environment could induce osteogenic differentiation of human MSCs, while inhibiting their adipogenic differentiation, in the presence of adipogenic-inducing medium. We also examined the mechanism through which the mineralized matrix suppresses adipogenesis of hMSCs to promote their osteogenic differentiation. Our results show that hMSCs cultured on mineralized matrices underwent osteogenic differentiation despite being cultured in the presence of adipogenic medium, which indicates the dominance of matrix-based cues of the mineralized matrix in directing osteogenic commitment of stem cells. Furthermore, the mineralized matrix-driven attenuation of adipogenesis was reversed with the inhibition of A2b adenosine receptor (A2bR), implicating a role of adenosine signaling in mineralized environment-mediated inhibition of adipogenesis. Such synthetic matrices with an intrinsic ability to direct differentiation of multipotent adult stem cells toward a targeted phenotype while inhibiting their differentiation into other lineages not only will be a powerful tool in delineating the role of complex microenvironmental cues on stem cell commitment but also will contribute to functional tissue engineering and their translational applications.

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

    Energy Technology Data Exchange (ETDEWEB)

    Marędziak, Monika, E-mail: monika.maredziak@gmail.com [Faculty of Veterinary Medicine, University of Environmental and Life Sciences, Wrocław (Poland); Wroclaw Research Centre EIT+, Wrocław (Poland); Śmieszek, Agnieszka, E-mail: smieszek.agnieszka@gmail.com [Wroclaw Research Centre EIT+, Wrocław (Poland); Faculty of Biology, University of Environmental and Life Sciences, Wrocław (Poland); Tomaszewski, Krzysztof A., E-mail: krtomaszewski@gmail.com [Department of Anatomy, Jagiellonian University Medical College, Krakow (Poland); Lewandowski, Daniel, E-mail: daniel.lewandowski@pwr.wroc.pl [Institute of Materials Science and Applied Mechanics, Wroclaw University of Technology, Wroclaw (Poland); Marycz, Krzysztof, E-mail: krzysztofmarycz@interia.pl [Wroclaw Research Centre EIT+, Wrocław (Poland); Faculty of Biology, University of Environmental and Life Sciences, Wrocław (Poland)

    2016-01-15

    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. - Graphical abstract: Influence of static magnetic field on viability and differentiation properties of human adipose derived mesenchymal stem cells. Abbreviations: SMF – static magnetic field; hASCs – human adipose derived mesenchymal stem cells; PF – proliferation factor; PDT – population doubling time; CFU-E –> colony forming unit efficiency; OPN – osteopontin; OCL – osteocalcin; Col – collagen type I; BMP-2 – bone morphogenetic protein 2; Ca – calcium; P – phosphorus. - Highlights: • Effects of static

  13. The synergistic effects of fibroblast growth factor-2 and mineral trioxide aggregate on an osteogenic accelerator in vitro.

    Science.gov (United States)

    Liu, C-H; Huang, T-H; Hung, C-J; Lai, W-Y; Kao, C-T; Shie, M-Y

    2014-09-01

    To examine the effects of mineral trioxide aggregate (MTA)/fibroblast growth factor-2 (FGF-2) on material properties and in vitro human dental pulp cell (hDPCs) behaviour. The setting time and diametral tensile strength (DTS) of MTA and MTA/FGF-2 were measured. The structure of specimens before and after soaking in DMEM was examined under a scanning electron microscope. Alamar Blue was used for evaluating hDPCs proliferation. An enzyme-linked immunosorbent assay was employed to determine ALP and osteocalcin (OC) expression in hDPCs cultured on cements. The effect of small interfering RNA (siRNA) transfection targeting fibroblast growth factor receptor (FGFR) was also evaluated. One-way analysis of variance was used to evaluate the significance of the differences between the mean values. Setting time and DTS data were not found to be significant (P > 0.05) between MTA with and without FGF-2. Cell proliferation and differentiation increased significantly (P MTA. After siRNA transfection with FGFR, the proliferation and differentiation behaviour of the hDPCs appreciably decreased when cultured on an MTA/FGF-2 composite. In contrast, no significant amounts (P > 0.05) of ALP and OC were secreted by hDPCs seeded on MTA. Mineral trioxide aggregate with FGF-2 content enhanced the higher expression of hDPCs proliferation and osteogenic differentiation as compared to pure MTA cement. © 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd.

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

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    Powderly William G

    2008-03-01

    Full Text Available Abstract 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

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

    Science.gov (United States)

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

    2017-06-01

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

  16. Effect of Jagged-1 and Dll-1 on osteogenic differentiation by stem cells from human exfoliated deciduous teeth.

    Science.gov (United States)

    Sukarawan, Waleerat; Peetiakarawach, Karnnapas; Pavasant, Prasit; Osathanon, Thanaphum

    2016-05-01

    The aim of the present study was to determine the influence of Notch ligands, Jagged-1 and Dll-1, on osteogenic differentiation by stem cells from human exfoliated deciduous teeth. Notch ligands were immobilized on tissue culture surface using an indirect affinity immobilization technique. Cells from the remaining of dental pulp tissues from human deciduous teeth were isolated and characterized using flow cytometry and differentiation assay. Alkaline phosphatase (ALP) enzymatic activity, osteogenic marker gene expression, and mineralization were determined using ALP assay, real-time polymerase chain reaction, and alizarin red staining, respectively. The isolated cells exhibited CD44, CD90, and CD105 expression but lack of CD45 expression. Further, these cells were able to differentiate toward osteogenic lineage. The upregulation of HES-1 and HEY-1 was observed in those cells on Dll-1 and Jagged-1 coated surface. The significant increase of ALP activity and mineralization was noted in those cells seeded on Jagged-1 surface and these results were attenuated when cells were pretreated with gamma secretase inhibitor. The significant upregulation of ALP and collagen type I gene expression was also observed in those cells seeded on Jagged-1 surface. The inconsistent Dll-1 induced osteogenic differentiation was found and high Dll-1 immobilized dose (50 nM) slightly enhanced alkaline phosphatase enzymatic activity. However, the statistical significant difference was not obtained as compared to the hFc control. The surface immobilization of Notch ligands, Jagged-1 and Dll-1, likely to enhance osteogenic differentiation of SHEDs. However, Jagged-1 had more ability in enhancing osteogenic differentiation than Dll-1 in our model. Copyright © 2016. Published by Elsevier Ltd.

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

    Directory of Open Access Journals (Sweden)

    Fabian Duttenhoefer

    2015-01-01

    Full Text Available 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.

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

    Science.gov (United States)

    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. Copyright 2008 S. Karger AG, Basel.

  19. Standardized Sophora pachycarpa Root Extract Enhances Osteogenic Differentiation in Adipose-derived Human Mesenchymal Stem Cells.

    Science.gov (United States)

    Mollazadeh, Samaneh; Neshati, Vajiheh; Fazly Bazzaz, Bibi Sedigheh; Iranshahi, Mehrdad; Mojarrad, Majid; Naderi-Meshkin, Hojjat; Kerachian, Mohammad Amin

    2017-05-01

    Bone defect is an important topic in public health. Novel therapies are based on osteogenic induction by natural antiosteoporotic compounds including plant-derived estrogens. In the current study, the osteogenic potential of Sophora pachycarpa root extract (SPRE) was explored on human adipose-derived mesenchymal stem cells. Herein, adipose-derived mesenchymal stem cells were osteoinducted in the presence of increased concentrations of the extract for 21 days. Then, cell viability was evaluated by MTT assay, and the differentiated cells were stained by Alizarin Red S for calcium deposition and subjected to alkaline phosphatase (ALP) assay for enzymatic activity. To assess the expression of bone-related genes, treated cells were evaluated by real-time polymerase chain reaction. The MTT test demonstrated that SPRE had no toxic effects on the cell viability. Treating the cells with SPRE noticeably promoted ALP activity, mineralization, and mRNA expression of runt-related transcription factor 2 (RUNX2), bone gamma-carboxyglutamate protein (BGLAP), secreted phosphoprotein 1 (SPP1), and collagen type I alpha 1 (COL1A1). Additionally, cells subjected to 0.1 μg/mL SPRE showed the highest osteogenic effects. According to high-performance liquid chromatography fingerprinting of SPRE, the osteoprotective effects of SPRE is probably due to presence of phytochemicals with estrogen-like activity in the extract. Thus, SPRE might be a suitable therapeutic agent for bone defects therapy in the future research. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

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

    Energy Technology Data Exchange (ETDEWEB)

    Fiedler, Tomas, E-mail: tomas.fiedler@med.uni-rostock.de [Institute for Medical Microbiology, Virology, and Hygiene, Rostock University Medical Center, Schillingallee 70, D-18057 Rostock (Germany); Salamon, Achim; Adam, Stefanie; Herzmann, Nicole [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Taubenheim, Jan [Institute for Medical Microbiology, Virology, and Hygiene, Rostock University Medical Center, Schillingallee 70, D-18057 Rostock (Germany); Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Peters, Kirsten [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Keller, Vivian; Deiwick, Andrea [Laser Zentrum Hannover e.V., Department of Nanotechnology, Hollerithallee 8, D-30419 Hannover (Germany); Pflaum, Michael [Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover (Germany); Schlie-Wolter, Sabrina, E-mail: s.schlie@lzh.de [Laser Zentrum Hannover e.V., Department of Nanotechnology, Hollerithallee 8, D-30419 Hannover (Germany); Institute of Quantum Optics, Leibniz University of Hannover, Welfengarten 1, D-30617 Hannover (Germany)

    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. - Highlights: • Interplay of ECM and cell shape guides osteogenic differentiation of hASCs. • ECM components only present a promotive but not stimulative effect. • No direct correlation between ECM-enhanced cell elongation and differentiation. • Suppression of differentiation depends on a specific actin polymerization blocking. • Fibronectin sustains cell elongation and differentiation in case of blocking actin.

  2. Enhanced early osteogenic differentiation by silicon-substituted hydroxyapatite ceramics fabricated via ultrasonic spray pyrolysis route.

    Science.gov (United States)

    Honda, Michiyo; Kikushima, Koichi; Kawanobe, Yusuke; Konishi, Toshiisa; Mizumoto, Minori; Aizawa, Mamoru

    2012-12-01

    The influence of silicon-substituted hydroxyapatite (Si-HAp) on osteogenic differentiation was assessed by biological analysis. Si-HAp was prepared by ultrasonic spray pyrolysis (USSP) technique using various amounts of Si (0, 0.8, and 1.6 mass%). Chemical analysis revealed that Si was incorporated into the hydroxyapatite (HAp) lattice with no other crystalline phase and which caused the change of crystal structure. Biological analyses showed that the Si contents affected the cell proliferation and morphology, suggesting that there is an optimal Si content for cell culture. As for differentiation, alkaline phosphatase activity and osteocalcin production of Si-HAp were higher than those of HAp. Gene expression profiles also revealed that substitution of Si (0.8 mass%) up-regulated the expression levels of osteocalcin and especially Runx2, a master gene for osteoblast development. These results suggest that incorporating Si into the HAp lattice may enhance the bioactivity, particularly during early osteoblast development.

  3. Effects of Recombinant Overexpression of Bcl2 on the Proliferation, Apoptosis, and Osteogenic/Odontogenic Differentiation Potential of Dental Pulp Stem Cells.

    Science.gov (United States)

    Heng, Boon Chin; Ye, Xin; Liu, Yuan; Dissanayaka, Waruna Lakmal; Cheung, Gary Shun Pan; Zhang, Chengfei

    2016-04-01

    The therapeutic usefulness of dental pulp stem cells (DPSCs) is severely limited by low survivability upon transplantation in situ because of the presence of various proapoptotic factors within damaged/diseased tissues (ie, hypoxia and inflammation). One strategy to enhance the survivability of grafted DPSCs could be recombinant overexpression of antiapoptotic genes, such as the B-cell lymphoma 2 gene (Bcl2). DPSCs were transfected with the Bcl2 and/or GFP gene. Cell density and mitotic activity of transfected DPSCs within in vitro culture were evaluated with the water soluble tetrazolium salt-8 (WST-8) and bromodeoxyuridine assay, respectively, whereas apoptosis was evaluated through the detection of cytoplasmic histone-associated DNA fragments. The osteogenic/odontogenic differentiation potential of these cells was evaluated with quantitative real-time polymerase chain reaction, alkaline phosphatase, and alizarin red staining. Bcl2-transfected DPSCs exhibited consistently higher cell densities than the GFP-transfected control within in vitro culture, and this was not because of the higher mitotic rate but was instead attributed to enhanced cell survivability because of the inhibition of apoptosis by Bcl2. Recombinant overexpression of Bcl2 inhibited the osteogenic/odontogenic potential of DPSCs, as indicated by lower levels of alkaline phosphatase activity and mineralized calcium deposition, together with the down-regulated expression of several key osteogenic/odontogenic gene markers including collagen I, osteocalcin, dentin matrix protein-1, bone sialoprotein, and alkaline phosphatase. The results place a "caveat" or limitation on the use of recombinant Bcl2 overexpression as a therapeutic strategy for improving the survivability of grafted DPSCs in that the osteogenic/odontogenic potential of these cells may be compromised despite enhanced survival within the host. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights

  4. The role of the adaptive immune system in burn-induced heterotopic ossification and mesenchymal cell osteogenic differentiation.

    Science.gov (United States)

    Ranganathan, Kavitha; Agarwal, Shailesh; Cholok, David; Loder, Shawn; Li, Jonathan; Sung Hsieh, Hsiao Hsin; Wang, Stewart C; Buchman, Steven R; Levi, Benjamin

    2016-11-01

    Heterotopic ossification (HO) is the pathologic process of extraskeletal bone formation. Although the exact etiology remains unknown, inflammation appears to catalyze disease progression. The goal of this study is to determine the impact of the adaptive immune system on HO. HO was induced in 8-wk-old control C57BL/6 and immunocompromised Rag1tm1Mom (Rag1 KO) male mice deficient in B- and T-lymphocytes via combined Achilles tenotomy and burn injury. Microcomputed tomography quantified the extent of HO formation at the tenotomy site. Adipose-derived mesenchymal stem cells were harvested to evaluate osteogenic differentiation potential. Areas of developing HO demonstrated substantial enrichment of CD45 + leukocytes at 3 wk after injury. HO from Rag1 KO mice was substantially less mature with foci of cartilage and disorganized trabecular bone present 12 wk after injury. Rag1 KO mice formed 60% less bone compared to immunocompetent controls (4.67 ± 1.5 mm versus 7.76 ± 0.65 mm; P = 0.001). Tartrate-resistant acid phosphatase staining and immunofluorescent analysis of osteoprotegerin and nuclear factor kappa-light-chain-enhancer of activated B cells demonstrated no appreciable difference in osteoclast number or activation. Alizarin red staining in vitro demonstrated a significant decrease in osteogenic potential in immunocompromised mice compared to controls (29.1 ± 0.54 mm versus 12.1 ± 0.14 mm; P < 0.001). We demonstrate a prominent role for the adaptive immune system in the development of HO. In the absence of mature B- and T-lymphocytes, HO growth and development are attenuated. Furthermore, we demonstrate that mesenchymal populations from B- and T-cell deficient mice are inherently less osteogenic. This study identifies a potential therapeutic role for modulation of the adaptive immune system in the treatment of HO. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Enhanced Stem Cell Osteogenic Differentiation by Bioactive Glass Functionalized Graphene Oxide Substrates

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    Xiaoju Mo

    2016-01-01

    Full Text Available An unmet need in engineered bone regeneration is to develop scaffolds capable of manipulating stem cells osteogenesis. Graphene oxide (GO has been widely used as a biomaterial for various biomedical applications. However, it remains challenging to functionalize GO as ideal platform for specifically directing stem cell osteogenesis. Herein, we report facile functionalization of GO with dopamine and subsequent bioactive glass (BG to enhance stem cell adhesion, spreading, and osteogenic differentiation. On the basis of graphene, we obtained dopamine functionalized graphene oxide/bioactive glass (DGO/BG hybrid scaffolds containing different content of DGO by loading BG nanoparticles on graphene oxide surface using sol-gel method. To enhance the dispersion stability and facilitate subsequent nucleation of BG in GO, firstly, dopamine (DA was used to modify GO. Then, the modified GO was functionalized with bioactive glass (BG using sol-gel method. The adhesion, spreading, and osteoinductive effects of DGO/BG scaffold on rat bone marrow mesenchymal stem cells (rBMSCs were evaluated. DGO/BG hybrid scaffolds with different content of DGO could influence rBMSCs’ behavior. The highest expression level of osteogenic markers suggests that the DGO/BG hybrid scaffolds have great potential or elicit desired bone reparative outcome.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Shani Samuel

    2016-09-01

    Full Text Available 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

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

    Science.gov (United States)

    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.

  9. Effects of TGF-β1 and VEGF-A transgenes on the osteogenic potential of bone marrow stromal cells in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Shinji Kuroda

    2012-12-01

    Full Text Available An exogenous supply of growth factors and bioreplaceable scaffolds may help bone regeneration. The aim of this study was to examine the effects of TGF-β1 and VEGF-A transgenes on the osteogenic potential of bone marrow stromal cells. Rat bone marrow stromal cells were transfected with plasmids encoding mouse TGF-β1 and/or VEGF-A complementary DNAs and cultured for up to 28 days. Furthermore, collagen scaffolds carrying combinations of the plasmids-transfected cells were implanted subcutaneously in rats. The transgenes increased alkaline phosphatase activity, enhanced mineralized nodule formation, and elevated osteogenic gene expressions in vitro. In vivo, messenger RNA expression of osteogenic genes such as BMPs and Runx2 elevated higher by the transgenes. The data indicate that exogenous TGF-β1 and VEGF-A acted synergistically and could induce osteoblastic differentiation of bone marrow stromal cells in both cell culture and an animal model. The results may provide valuable information to optimize protocols for transgene-and-cell-based tissue engineering.

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

    Science.gov (United States)

    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.

  11. Preparation and investigation of polylactic acid, calcium carbonate and polyvinylalcohol nanofibrous scaffolds for osteogenic differentiation of mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    A. Doustgani

    2016-04-01

    Full Text Available Objective(s: In this study, the effect of electrospun fiber orientation on proliferation and differentiation of mesenchymal stem cells (MSCs was evaluated. Materials and Methods: Aligned and random nanocomposite nanofibrous scaffolds were electrospun from polylactic acid (PLA, poly (vinyl alcohol (PVA and calcium carbonate nanoparticles (nCaP. The surface morphology of prepared nanofibrous scaffolds with and without cell was examined using scanning electron microscopy. Mechanical properties of electrospun nanofibrous scaffolds were determined with a  universal testing machine. The in vitro properties of fabricated scaffolds was also investigated by the MTT assay and alkaline phosphatase activity (ALP.Results: The average fiber diameter for aligned and random nanofibers were 82 ± 12 nm and 124 ± 25 nm, respectively. The mechanical testing indicated the higher tensile strength and elastic modulus of aligned nanofibers. MTT and ALP results showed that alignment of nanofiber increased the osteogenic differentiation of stem cells.Conclusion: Aligned nanofibrous nanocomposite scaffolds of PLA/nCaP/PVA could be an excellent substrate for MSCs and represents a potential bone-filling material.

  12. Evaluation of the osteogenic differentiation of gingiva-derived stem cells grown on culture plates or in stem cell spheroids: Comparison of two- and three-dimensional cultures.

    Science.gov (United States)

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

    2017-09-01

    Three-dimensional cell culture systems provide a convenient in vitro model for the study of complex cell-cell and cell-matrix interactions in the absence of exogenous substrates. The current study aimed to evaluate the osteogenic differentiation potential of gingiva-derived stem cells cultured in two-dimensional or three-dimensional systems. To the best of our knowledge, the present study is the first to compare the growth of gingiva-derived stem cells in monolayer culture to a three-dimensional culture system with microwells. For three-dimensional culture, gingiva-derived stem cells were isolated and seeded into polydimethylsiloxane-based concave micromolds. Alkaline phosphatase activity and alizarin red S staining assays were then performed to evaluate osteogenesis and the degree of mineralization, respectively. Stem cell spheroids had a significantly increased level of alkaline phosphatase activity and mineralization compared with cells from the two-dimensional culture. In addition, an increase in mineralized deposits was observed with an increase in the loading cell number. The results of present study indicate that gingiva-derived stem cell spheroids exhibit an increased osteogenic potential compared with stem cells from two-dimensional culture. This highlights the potential of three-dimensional culture systems using gingiva-derived stem cells for regenerative medicine applications requiring stem cells with osteogenic potential.

  13. WNT5A induces osteogenic differentiation of human adipose stem cells via rho-associated kinase Rock

    NARCIS (Netherlands)

    Santos, A.; Bakker, A.D.; de Blieck-Hogervorst, J.M.A.; Klein-Nulend, J.

    2010-01-01

    Background aims. Human (h) adipose tissue-derived mesenchymal stromal cells (ASC) constitute an interesting cellular source for bone tissue engineering applications. Wnts, for example Wnt5a, are probably important regulators of osteogenic differentiation of stem cells, but the role of Wnt5a in hASC

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

    Science.gov (United States)

    Lv, Hao; Sun, Yujie; Zhang, Yuchen

    2015-05-27

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Qu, Bo [Department of Orthopaedics, Chengdu Military General Hospital, Chengdu 610083 (China); Ma, Yuan [Department of Neurosurgery, Chengdu Military General Hospital, Chengdu 610083 (China); Yan, Ming [Department of Orthopaedics, Xijing Hospital of The Fourth Military Medical University, Xi’an 710032 (China); Gong, Kai; Liang, Feng; Deng, Shaolin; Jiang, Kai; Ma, Zehui [Department of Orthopaedics, Chengdu Military General Hospital, Chengdu 610083 (China); Pan, Xianming, E-mail: xianmingpanxj@163.com [Department of Orthopaedics, Chengdu Military General Hospital, Chengdu 610083 (China)

    2016-09-09

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

  16. [Effect of sonicated extracts of Porphyromonas gingivalis on osteogenic differentiation of mouse osteoblasts].

    Science.gov (United States)

    Zhang, Jian-ying; Yu, Shao-jie; Fu, Yun

    2013-07-01

    To investigate the effects of sonicated extracts of Porphyromonas gingivalis (Pg) on osteogenic differentiation of mouse osteoblast cell line MC3T3-E1. PgW83 was cultured under standard anaerobic conditions and extracted by sonication. Mouse osteoblast cell line MC3T3-E1 was cultured with various concentrations of the extraction (0, 10, 100, 1000 mg/L). Western blotting was applied to investigate the expression of osteocalcin (OC), bone sialoprotein (BSP), osteopontin (OPN) and osteonectin (ON). The activity of alkaline phosphatase (ALP) was detected by microplate reader after 14 days. Mineralization nodule formation was measured by alizarin red staining after 21 days. Compared with the control group, the extracts of Pg decreased OC and ON expression in a dose-dependent manner (OC relative expression:1.000 ± 0.000,0.852 ± 0.110,0.625 ± 0.451,0.213 ± 0.053), (ON relative expression: 1.000 ± 0.000, 1.035 ± 0.133,0.141 ± 0.023,0.020 ± 0.003) (P extraction (0.572 ± 0.162) compared with control group, 10 and 100 mg/L (1.000 ± 0.000, 1.029 ± 0.135, 1.199 ± 0.337) (P extraction (BSP relative expression:1.000 ± 0.000,0.831 ± 0.182,0.897 ± 0.115,0.778 ± 0.235) (P > 0.05). Meanwhile, the extracts of Pg decreased ALP activity [control group:(0.0275 ± 0.0014) U/gprot, 10 mg/L: (0.0140 ± 0.0011) U/gprot, 100 mg/L: (0.0057 ± 0.0013) U/gprot, 1000 mg/L: (0.0020 ± 0.0008) U/gprot] (P < 0.05) and reduced mineralization nodule formation. The results suggest that Pg may inhibit osteoblasts'osteogenic function by down-regulation of osteogenic differentiation related proteins.

  17. Cyclic mechanical stretch enhances BMP9-induced osteogenic differentiation of mesenchymal stem cells.

    Science.gov (United States)

    Song, Yang; Tang, Yinhong; Song, Jinlin; Lei, Mingxing; Liang, Panpan; Fu, Tiwei; Su, Xudong; Zhou, Pengfei; Yang, Li; Huang, Enyi

    2018-02-10

    The purpose of this study was to investigate whether mechanical stretch can enhance the bone morphogenetic protein 9 (BMP9)-induced osteogenic differentiation in MSCs. Recombinant adenoviruses were used to overexpress the BMP9 in C3H10T1/2 MSCs. Cells were seeded onto six-well BioFlex collagen I-coated plates and subjected to cyclic mechanical stretch [6% elongation at 60 cycles/minute (1 Hz)] in a Flexercell FX-4000 strain unit for up to 12 hours. Immunostaining and confocal microscope were used to detect cytoskeleton organization. Cell cycle progression was checked by flow cytometry. Alkaline phosphatase activity was measured with a Chemiluminescence Assay Kit and was quantified with a histochemical staining assay. Matrix mineralization was examined by Alizarin Red S Staining. Mechanical stretch induces cytoskeleton reorganization and inhibits cell proliferation by preventing cells entry into S phase of the cell cycle. Although mechanical stretch alone does not induce the osteogenic differentiation of C3H10T1/2 MSCs, co-stimulation with mechanical stretch and BMP9 enhances alkaline phosphatase activity. The expression of key lineage-specific regulators (e.g., osteocalcin (OCN), SRY-related HMG-box 9, and runt-related transcription factor 2) is also increased after the co-stimulation, compared to the mechanical stretch stimulation along. Furthermore, mechanical stretch augments the BMP9-mediated bone matrix mineralization of C3H10T1/2 MSCs. Our results suggest that mechanical stretch enhances BMP9-induced osteoblastic lineage specification in C3H10T1/2 MSCs.

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

    Science.gov (United States)

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

    2015-10-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Gong, Kai; Qu, Bo; Liao, Dongfa; Liu, Da; Wang, Cairu; Zhou, Jingsong; Pan, Xianming, E-mail: xianmingpanxj@163.com

    2016-09-09

    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

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

    Science.gov (United States)

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

    2017-02-21

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

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

    Science.gov (United States)

    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 (pCells were encapsulated in 0.2% PuraMatrix(TM) at cell densities of 10x104, 20x104, 40x10 4 and 80x104. Cell viability with WST and proliferation with Live-Dead cell assays showed

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Structural and biochemical modification of a collagen scaffold to selectively enhance MSC tenogenic, chondrogenic, and osteogenic differentiation.

    Science.gov (United States)

    Caliari, Steven R; Harley, Brendan A C

    2014-07-01

    Biomaterial approaches for engineering orthopedic interfaces such as the tendon-bone junction (TBJ) are limited by a lack of understanding of how insoluble (microstructure, composition) and soluble regulators of stem cell fate work in concert to promote bioactivity and differentiation. One strategy for regenerating the interface is to design biomaterials containing spatially graded structural properties sufficient to induce divergent mesenchymal stem cell (MSC) differentiation into multiple interface-specific phenotypes. This work explores the hypothesis that selective structural modification to a 3D collagen-glycosaminoglycan (CG) scaffold combined with biochemical supplementation can drive human bone-marrow-derived MSC differentiation down tenogenic, osteogenic, and chondrogenic lineages. Tenogenic differentiation is enhanced in geometrically anisotropic scaffolds versus a standard isotropic control. Notably, blebbistatin treatment abrogates this microstructurally driven effect. Further, enhanced osteogenic differentiation and new mineral synthesis are achieved by incorporation of a calcium phosphate mineral phase within the CG scaffold along with the use of osteogenic induction media. Finally, chondrogenic differentiation is optimally driven by combining chondrogenic induction media with a reduced density scaffold that promotes increased cellular condensation, significantly higher expression of chondrogenic genes, and increased GAG deposition. Together these data provide critical insight regarding design rules for elements of an integrated biomaterial platform for orthopedic interface regeneration. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Evaluation of early stage human bone marrow stromal proliferation, cell migration and osteogenic differentiation on μ-MIM structured stainless steel surfaces.

    Science.gov (United States)

    Bitar, Malak; Benini, Fausta; Brose, Claudia; Friederici, Vera; Imgrund, Philipp; Bruinink, Arie

    2013-05-01

    It is well established that surface topography greatly affect cell-surface interactions. In a recent study we showed that microstructured stainless steel surfaces characterized by the presence of defined hexagonally arranged hemisphere-like structures significantly affected cell architecture (shape and focal adhesion size) of primary human bone mesenchymal stromal cells. This study aimed at further investigating the influence these microstructures (microcline protruding hemispheres) on critical aspects of cell behaviour namely; proliferation, migration and osteogenic differentiation. As with previously reported data, we used primary human bone mesenchymal stromal cells to investigate such effects at an early stage in vitro. Cells of different patients were utilised for cell migration studies. Our data showed that an increase in cell proliferation was exhibited as a function of surface topography (hemispheres). Cell migration velocity also varied as a function of surface topography on patient specific basis and seems to relate to the differentiated state of the seeded cell population (as demonstrated by bALP positivity). Osteogenic differentiation, however, did not exhibit significant variations (both up and down-regulation) as a function of both surface topography and time in culture.

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

    Energy Technology Data Exchange (ETDEWEB)

    Li, Huihua [Biomaterial Research Laboratory, Department of Material Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou 510632 (China); Luo, Chuang [Engineering Research Center of Artificial Organs and Materials, Ministry of Education, Guangzhou 510632 (China); Luo, Binghong, E-mail: tluobh@jnu.edu.cn [Biomaterial Research Laboratory, Department of Material Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou 510632 (China); Engineering Research Center of Artificial Organs and Materials, Ministry of Education, Guangzhou 510632 (China); Wen, Wei [Biomaterial Research Laboratory, Department of Material Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou 510632 (China); Engineering Research Center of Artificial Organs and Materials, Ministry of Education, Guangzhou 510632 (China); Wang, Xiaoying [Department of Biomedical Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632 (China); Ding, Shan [Biomaterial Research Laboratory, Department of Material Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou 510632 (China); Engineering Research Center of Artificial Organs and Materials, Ministry of Education, Guangzhou 510632 (China); Zhou, Changren, E-mail: tcrz9@jnu.edu.cn [Biomaterial Research Laboratory, Department of Material Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou 510632 (China); Engineering Research Center of Artificial Organs and Materials, Ministry of Education, Guangzhou 510632 (China)

    2016-01-01

    Graphical abstract: - Highlights: • COS was conveniently immobilized on PDLLA membrane based on PDOPA adhesive layer. • The hydrophilicity of PDLLA membrane was improved by modified with PDOPA and COS. • COS-functionalized PDLLA membrane is favorable to cell adhesion and proliferation. • COS-coated PDLLA membrane notably promote osteogenic differentiation of MC3T3-E1. - Abstract: 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.

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

    Science.gov (United States)

    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. © Georg Thieme Verlag KG Stuttgart · New York.

  7. Cytotoxicity assessment of modified bioactive glasses with MLO-A5 osteogenic cells in vitro.

    Science.gov (United States)

    Modglin, Vernon C; Brown, Roger F; Jung, Steven B; Day, Delbert E

    2013-05-01

    The primary objective of this study was to evaluate in vitro responses of MLO-A5 osteogenic cells to two modifications of the bioactive glass 13-93. The modified glasses, which were designed for use as cell support scaffolds and contained added boron to form the glasses 13-93 B1 and 13-93 B3, were made to accelerate formation of a bioactive hydroxyapatite surface layer and possibly enhance tissue growth. Quantitative MTT cytotoxicity tests revealed no inhibition of growth of MLO-A5 cells incubated with 13-93 glass extracts up to 10 mg/ml, moderate inhibition of growth with 13-93 B1 glass extracts, and noticeable inhibition of growth with 13-93 B3 glass extracts. A morphology-based biocompatibility test was also performed and yielded qualitative assessments of the relative biocompatibilities of glass extracts that agree with those obtained by the quantitative MTT test. However, as a proof of concept experiment, when MLO-A5 cells were seeded onto 13-93 B3 scaffolds in a dynamic in vitro environment, cell proliferation occurred as evidenced by qualitative and quantitative MTT labeling of scaffolds. Together these results demonstrate the in vitro toxicity of released borate ion in static experiments; however borate ion release can be mitigated in a dynamic environment similar to the human body where microvasculature is present. Here we argue that despite toxicity in static environments, boron-containing 13-93 compositions may warrant further study for use in tissue engineering applications.

  8. The Macrophage Polarization Regulates MSC Osteoblast Differentiation in vitro.

    Science.gov (United States)

    Gong, Lei; Zhao, Yan; Zhang, Yi; Ruan, Zhi

    2016-01-01

    Bone repair is a complex yet highly organized process involving interactions between various cell types and the extracellular environment. Macrophages are not only activated in inflammation during early phases of repair processes, but they are also present in bone throughout the whole bone repair process. Bone marrow derived mesenchymal stem cells (MSCs) represent an attractive therapeutic for bone fracture with their expansion potential, osteogenic capability, and potential for injury. However, less is known about the interaction between macrophage and MSC during bone repair and regeneration. This study was aimed to investigate whether macrophages in different statuses can regulate MSC osteoblast differentiation in vitro. Using in vitro cell coculture of macrophage and MSC, it was shown that macrophage polarization can regulate MSC osteoblast differentiation. This was evidenced by increased alkaline phosphatase (ALP), osteogenic markers, and bone mineralization in M2 macrophage cocultured MSC but decreased in M1 counterpart. These results might be mediated by pro-regenerative cytokines, such as TGF-β, VEGF, and IFG-1, produced by M2 macrophages and detrimental inflammation cytokines, such as IL-6, IL-12, and TNF-α, produced by M1 macrophages. Taken together, this shows that macrophage polarization could be crucial for maintaining bone homeostasis and promoting bone repair by regulating the MSC osteoblast differentiation. © 2016 by the Association of Clinical Scientists, Inc.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  11. Evaluation of Osteogenic and Cementogenic Potential of Periodontal Ligament Fibroblast Spheroids Using a Three-Dimensional In Vitro Model of Periodontium

    Directory of Open Access Journals (Sweden)

    Zurairah Berahim

    2015-01-01

    Full Text Available The aim of this study was to develop a three-dimensional in vitro model of periodontium to investigate the osteogenic and cementogenic differentiation potential of the periodontal ligament fibroblast (PDLF spheroids within a dentin-membrane complex. PDLFs were cultured in both spheroid forms and monolayers and were seeded onto two biological collagen-based and synthetic membranes. Cell-membrane composites were then transferred onto dentin slices with fibroblasts facing the dentin surface and further cultured for 20 days. The composites were then processed for histology and immunohistochemical analyses for osteocalcin, Runx2, periostin, and cementum attachment protein (CAP. Both membranes seeded with PDLF-derived cells adhered to dentin and fibroblasts were present at the dentin interface and spread within both membranes. All membrane-cell-dentine composites showed positive staining for osteocalcin, Runx2, and periostin. However, CAP was not expressed by any of the tissue composites. It can be concluded that PDLFs exhibited some osteogenic potential when cultured in a 3D matrix in the presence of dentin as shown by the expression of osteocalcin. However the interaction of cells and dentin in this study was unable to stimulate cementum formation. The type of membrane did not have a significant effect upon differentiation, but fibroblast seeded-PGA membrane demonstrated better attachment to dentin than the collagen membrane.

  12. Osteogenic differentiation of bone mesenchymal stem cells regulated by osteoblasts under EMF exposure in a co-culture system.

    Science.gov (United States)

    Yu, Ji-zhe; Wu, Hua; Yang, Yong; Liu, Chao-xu; Liu, Yang; Song, Ming-yu

    2014-04-01

    This study examined the osteogenic effect of electromagnetic fields (EMF) under the simulated in vivo conditions. Rat bone marrow mesenchymal stem cells (BMSCs) and rat osteoblasts were co-cultured and exposed to 50 Hz, 1.0 mT EMF for different terms. Unexposed single-cultured BMSCs and osteoblasts were set as controls. Cell proliferation features of single-cultured BMSCs and osteoblasts were studied by using a cell counting kit (CCK-8). For the co-culture system, cells in each group were randomly chosen for alkaline phosphatase (ALP) staining on the day 7. When EMF exposure lasted for 14 days, dishes in each group were randomly chosen for total RNA extraction and von Kossa staining. The mRNA expression of osteogenic markers was detected by using real-time PCR. Our study showed that short-term EMF exposure (2 h/day) could obviously promote proliferation of BMSCs and osteoblasts, while long-term EMF (8 h/day) could promote osteogenic differentiation significantly under co-cultured conditions. Under EMF exposure, osteogenesis-related mRNA expression changed obviously in co-cultured and single-cultured cells. It was noteworthy that most osteogenic indices in osteoblasts were increased markedly after co-culture except Bmp2, which was increased gradually when cells were exposed to EMF. Compared to other indices, the expression of Bmp2 in BMSCs was increased sharply in both single-cultured and co-cultured groups when they were exposed to EMF. The mRNA expression of Bmp2 in BMSCs was approximately four times higher in 8-h EMF group than that in the unexposed group. Our results suggest that Bmp2-mediated cellular interaction induced by EMF exposure might play an important role in the osteogenic differentiation of BMSCs.

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

    Energy Technology Data Exchange (ETDEWEB)

    Su, Wen-Ta, E-mail: f10549@ntut.edu.tw [Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, Taipei, Taiwan (China); Wu, Pai-Shuen [Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, Taipei, Taiwan (China); Huang, Te-Yang [Department of Orthopedic Surgery, Mackay Memorial Hospital, Taipei, Taiwan (China)

    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. - Highlights: • SHEDs have been considered as alternative sources of adult stem cells in tissue engineering. • Strontium phosphate into nanofibers provides a potential material for bone tissue engineering. • Nanofibers coated or blended with strontium phosphate for the osteogenic differentiation of SHEDs.

  14. Effects of Canonical NF-κB Signaling Pathway on the Proliferation and Odonto/Osteogenic Differentiation of Human Stem Cells from Apical Papilla

    Directory of Open Access Journals (Sweden)

    Junjun Li

    2014-01-01

    Full Text Available Background Information. NF-κB signaling pathway plays a complicated role in the biological functions of mesenchymal stem cells. However, the effects of NF-κB pathway on the odonto/osteogenic differentiation of stem cells from apical papilla (SCAPs remain unclear. The present study was designed to evaluate the effects of canonical NF-κB pathway on the osteo/odontogenic capacity of SCAPs in vitro. Results. Western blot results demonstrated that NF-κB pathway in SCAPs was successfully activated by TNF-α or blocked by BMS-345541. NF-κB pathway-activated SCAPs presented a higher proliferation activity compared with control groups, as indicated by dimethyl-thiazol-diphenyl tetrazolium bromide assay (MTT and flow cytometry assay (FCM. Wound scratch assay revealed that NF-κB pathway-activated SCAPs presented an improved migration capacity, enhanced alkaline phosphatase (ALP activity, and upregulated mineralization capacity of SCAPs, as compared with control groups. Meanwhile, the odonto/osteogenic markers (ALP/ALP, RUNX2/RUNX2, OSX/OSX, OCN/OCN, OPN/OPN, BSP/BSP, DSPP/DSP, and DMP-1/DMP-1 in NF-κB pathway-activated SCAPs were also significantly upregulated as compared with control groups at both protein and mRNA levels. However, NF-κB pathway-inhibited SCAPs exhibited a lower proliferation/migration capacity, and decreased odonto/osteogenic ability in comparison with control groups. Conclusion. Our findings suggest that classical NF-κB pathway plays a paramount role in the proliferation and committed differentiation of SCAPs.

  15. Inhibition of the osteogenic differentiation of mesenchymal stem cells derived from the offspring of rats treated with caffeine during pregnancy and lactation.

    Science.gov (United States)

    Reis, Amanda Maria Sena; Ocarino, Natália de Melo; Boeloni, Jankerle Neves; Gomes, Dawidson Assis; Goes, Alfredo Miranda; Ferreira, Andrea da Fonseca; Serakides, Rogéria

    2016-01-01

    Caffeine is an alkaloid that is widely consumed due to its presence in drugs, coffee, tea, and chocolate. This compound passes to offspring through the placenta and milk; can cause teratogenic mutations; and reduces the formation, growth, and mass of bone. Because mesenchymal stem cells (MSCs) are responsible for generating the entire skeleton, we hypothesized that these cells are targets of caffeine. This study evaluated the osteogenic differentiation of MSCs derived from the offspring of rats treated with caffeine during pregnancy and lactation. Twenty-four adult Wistar rats were randomly divided into four groups, including one control group and three experimental groups treated with 25, 50, or 100 mg/kg of caffeine. At weaning, three 21-day-old pups from each dam in each group were euthanized for extraction of bone marrow cells for in vitro tests. Caffeine doses of 50 and 100 mg/kg significantly reduced the activity of alkaline phosphatase at 7, 14, and 21 days and the expression of collagen I at 21 days. However, the expression of gene transcripts for alkaline phosphatase, Runx-2, and bone sialoprotein, as well as the synthesis of mineralization nodules, decreased significantly in all groups treated with caffeine. The expression of osteocalcin was significantly reduced only in the group treated with 50 mg/kg caffeine. The caffeine that passes from the mother to the offspring during pregnancy and lactation reduces the osteogenic differentiation of MSCs. We propose that this reduction in the osteogenic potential of MSCs may be involved in the pathogenesis of osteopenia resulting from caffeine consumption.

  16. Biomimetic nanocomposites to control osteogenic differentiation of human mesenchymal stem cells.

    Science.gov (United States)

    Liao, Susan; Nguyen, Luong T H; Ngiam, Michelle; Wang, Charlene; Cheng, Ziyuan; Chan, Casey K; Ramakrishna, Seeram

    2014-05-01

    The design of biomimetic nanomaterials that can directly influence the behavior of cells and facilitate the regeneration of tissues and organs has become an active area of research. Here, the production of materials based on nano-hydroxyapatite composites in scaffolds with nanofibrous and nanoporous topographies, designed to mimic the native bone matrix for applications in bone tissue engineering, is reported. Human mesenchymal stem cells grown on these nanocomposites are stimulated to rapidly produce bone minerals in situ, even in the absence of osteogenic supplements in the cell-culture medium. Nanocomposites comprising type I collagen and nano-hydroxyapatite are found to be especially efficient at inducing mineralization. When subcutaneously implanted into nude mice, this biomimetic nanocomposite is able to form a new bone matrix within only two weeks. Furthermore, when the nanocomposite is enriched with human mesenchymal stem cells before implantation, development of the bone matrix is accelerated to within one week. To the best of the authors' knowledge, this study provides the first clear in vitro and in vivo demonstration of osteoinduction controlled by the material characteristics of a biomimetic nanocomposite. This approach can potentially facilitate the translation of de novo bone-formation technologies to the clinic. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Cytokines TNF-α, IL-6, IL-17F, and IL-4 differentially affect osteogenic differentiation of human adipose stem cells

    NARCIS (Netherlands)

    Bastidas-Coral, A.P.; Bakker, A.D.; Zandieh-Doulabi, B.; Kleverlaan, C.J.; Bravenboer, N.; Forouzanfar, T.; Klein-Nulend, J.

    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

  18. A Novel Human TGF-β1 Fusion Protein in Combination with rhBMP-2 Increases Chondro-Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Silvia Claros

    2014-06-01

    Full Text Available Transforming growth factor-beta (TGF-β is involved in processes related to the differentiation and maturation of osteoprogenitor cells into osteoblasts. Rat bone marrow (BM cells were cultured in a collagen-gel containing 0.5% fetal bovine serum (FBS for 10 days in the presence of rhTGF (recombinant human TGF-β1-F2, a fusion protein engineered to include a high-affinity collagen-binding decapeptide derived from von Willebrand factor. Subsequently, cells were moderately expanded in medium with 10% FBS for 4 days and treated with a short pulse of rhBMP (recombinant human bone morphogenetic protein-2 for 4 h. During the last 2 days, dexamethasone and β-glycerophosphate were added to potentiate osteoinduction. Concomitant with an up-regulation of cell proliferation, DNA synthesis levels were determined. Polymerase chain reaction was performed to reveal the possible stemness of these cells. Osteogenic differentiation was evaluated in terms of alkaline phosphatase activity and mineralized matrix formation as well as by mRNA expression of osteogenic marker genes. Moreover, cells were placed inside diffusion chambers and implanted subcutaneously into the backs of adult rats for 4 weeks. Histological study provided evidence of cartilage and bone-like tissue formation. This experimental procedure is capable of selecting cell populations from BM that, in the presence of rhTGF-β1-F2 and rhBMP-2, achieve skeletogenic potential in vitro and in vivo.

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

    Science.gov (United States)

    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.

  20. TiO{sub 2}-enriched polymeric powder coatings support human mesenchymal cell spreading and osteogenic differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Mozumder, Mohammad Sayem; Zhu, Jesse [Department of Chemical and Biochemical Engineering, University of Western Ontario, London, Ontario, N6A 5B9 (Canada); Perinpanayagam, Hiran, E-mail: Hiran.Perinpanayagam@schulich.uwo.ca [Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, N6A 5C1 (Canada)

    2011-06-15

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

  1. [PDGFRα Participates in Basic Fibroblast Growth Factor-mediated Recovery of Human Bone Marrow Mesenchymal Stem Cell Proliferation and Osteogenic Differentiation after Irradiation].

    Science.gov (United States)

    Dai, Kai; Yang, Zhi; Xu, Shuang-Nian; Zhang, Jian-Min; Chen, Jie-Ping

    2015-12-01

    To explore the effects of basic fibroblast growth factor (bFGF) on human bone marrow mesenchymal stem cell (hBMMSC) damaged by irradiation and its underlying mechanisms. hBMMSC was irradiated with 0, 6, 12 Gy X ray, then flow cytometry, cell counting kit-8 (CCK-8), Western blot and alizarin red staining were used to detect the effects of X ray on apoptosis, proliferation and osteogenic differentiation of hBMMSC; 0, 1, 5, 10, 20 ng/ml bFGF was added to hBMMSC irradiated with X ray for selecting the suitable bFGF reaction concentration; then the Western blot was used to detect the expression of PDGFRα so as to evaluate whether the expression of PDGFRα participated in bFGF-mediated recovery of hBMMSC proliferation and osteogenic differentiation after irradiation. The proliferation and osteogenic differentiation of hBMMSC decreased remarkably after irradiation. bFGF promoted the recovery of proliferation and osteogenic differentiation of irradiated hBMMSC compared with untreated irradiated hBMMSC (P recovery of hBMMSC proliferation and osteogenic differentiation. The damage of hBMMSC proliferation and osteogenic differentiation associates with downregulation of PDGFRα expression induced by irrediation. PDGFRα involves in repairing effect of bFGF on irradiation damage of hBMMSC.

  2. Crosstalk between Wnt/β-catenin and estrogen receptor signaling synergistically promotes osteogenic differentiation of mesenchymal progenitor cells.

    Directory of Open Access Journals (Sweden)

    Yanhong Gao

    Full Text Available 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Tong; Wu, Yu-wei; Lu, Hui; Guo, Yuan [Second Dental Center, Peking University School and Hospital of Stomatology, Beijing (China); National Engineering Laboratory for Digital and Material Technology of Stomatology, Peking University School and Hospital of Stomatology, Beijing (China); Tang, Zhi-hui, E-mail: tang_zhihui@live.cn [Second Dental Center, Peking University School and Hospital of Stomatology, Beijing (China); National Engineering Laboratory for Digital and Material Technology of Stomatology, Peking University School and Hospital of Stomatology, Beijing (China)

    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

  4. Incorporation of Human-Platelet-Derived Growth Factor-BB Encapsulated Poly(lactic-co-glycolic acid) Microspheres into 3D CORAGRAF Enhances Osteogenic Differentiation of Mesenchymal Stromal Cells

    DEFF Research Database (Denmark)

    Mohan, Saktiswaren; Raghavendran, Hanumantharao Balaji; Karunanithi, Puvanan

    2017-01-01

    of growth factors has been demonstrated to produce severe side effects on the surrounding tissues. In this study, poly(lactic-co-glycolic acid) (PLGA) microspheres (MS) incorporated three-dimensional (3D) CORAGRAF scaffolds were engineered to achieve controlled release of platelet-derived growth factor-BB...... (PDGF-BB) for the differentiation of stem cells within the 3D polymer network. Fourier transform infrared spectroscopy, energy-dispersive X-ray spectroscopy, scanning electron microscopy, and microtomography were applied to characterize the fabricated scaffolds. In vitro study revealed that the CORAGRAF-PLGA-PDGF-BB...... scaffold system enhanced the release of PDGF-BB for the regulation of cell behavior. Stromal cell attachment, viability, release of osteogenic differentiation markers such as osteocalcin, and upregulation of osteogenic gene expression exhibited positive response. Overall, the developed scaffold system...

  5. Bone-forming peptide-3 induces osteogenic differentiation of bone marrow stromal cells via regulation of the ERK1/2 and Smad1/5/8 pathways

    Directory of Open Access Journals (Sweden)

    Jun Sik Lee

    2018-01-01

    Full Text Available A bone-remodeling imbalance induced by increased bone resorption and osteoclast formation causes skeletal diseases such as osteoporosis. Induction of osteogenic differentiation of bone marrow stromal cells (BMSCs leads to bone regeneration. Many researchers have tried to develop new adjuvants as specific stimulators of bone regeneration for therapeutic use in patients with bone resorption. We tried to develop a new adjuvant that has stronger osteogenic differentiation-promoting activity than bone morphogenetic proteins (BMPs. In this study, we identified a new peptide, which we called bone-forming peptide (BFP-3, derived from the immature precursor of BMP-7. Upon osteogenic differentiation, BMSCs treated with BFP-3 exhibited higher alkaline phosphatase (ALP activity and mineralization ability and significantly up-regulated expression of osteogenic genes such as ALP, osteocalcin (OC, Osterix, and Runx2 compared with control BMSCs. Furthermore, fluorescence-activated cell sorting (FACS and immunofluorescence analyses demonstrated that BFP-3 treatment up-regulated CD44 expression. Interestingly, extracellular signal-regulated kinase 1/2 (ERK1/2 and Smad1/5/8 phosphorylation was increased by BFP-3 treatment during osteogenic differentiation. Furthermore, BFP-3-induced osteogenic differentiation was significantly decreased by treatment with ERK1/2- and Smad-specific inhibitors. These results suggest that BFP-3 plays an important role in regulating osteogenic differentiation of BMSCs through increasing levels of osteogenic-inducing factors and regulating the ERK1/2 and Smad1/5/8 signaling pathways. Our finding indicates that BFP-3 may be a potential new therapeutic target for promoting bone formation.

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

    Science.gov (United States)

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

    2015-08-01

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

  7. The Osteogenic Potential of Mesoporous Bioglasses/Silk and Non-Mesoporous Bioglasses/Silk Scaffolds in Ovariectomized Rats: In vitro and In vivo Evaluation

    Science.gov (United States)

    Zhang, Yufeng; Shi, Bin

    2013-01-01

    Silk-based scaffolds have been introduced to bone tissue regeneration for years, however, their local therapeutic efficency in bone metabolic disease condition has been seldom reported. According to our previous report, mesoporous bioactive glass (MBG)/silk scaffolds exhibits superior in vitro bioactivity and in vivo osteogenic properties compared to non-mesoporous bioactive glass (BG)/silk scaffolds, but no information could be found about their efficiency in osteoporotic (OVX) environment. This study investigated a biomaterial-based approach for improving MSCs behavior in vitro, and accelerating OVX defect healing by using 3D BG/silk and MBG/silk scaffolds, and pure silk scaffolds as control. The results of SEM, CCK-8 assay and quantitative ALP activity showed that MBG/silk scaffolds can improve attachment, proliferation and osteogenic differentiation of both O-MSCs and sham control. In vivo therapeutic efficiency was evaluated by μCT analysis, hematoxylin and eosin staining, safranin O staining and tartrate-resistant acid phosphatase, indicating accelerated bone formation with compatible scaffold degradation and reduced osteoclastic response of defect healing in OVX rats after 2 and 4 weeks treatment, with a rank order of MBG/silk > BG/silk > silk group. Immunohistochemical markers of COL I, OPN, BSP and OCN also revealed that MBG/silk scaffolds can better induce accelerated collagen and non-collagen matrix production. The findings of this study suggest that MBG/silk scaffolds provide a better environment for cell attachment, proliferation and differentiation, and act as potential substitute for treating local osteoporotic defects. PMID:24265840

  8. 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

    Science.gov (United States)

    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 dental implants.

  9. Effects of carbon doping on the microstructural, micro/nano-mechanical, and mesenchymal stromal cells biocompatibility and osteogenic differentiation properties of alumina

    DEFF Research Database (Denmark)

    Krishnamurithy, Genasan; Yahya, Noor Azlin; Mehrali, Mehdi

    2016-01-01

    It has been demonstrated that carbon (C) doped aluminium oxide (Al2O3) nanocomposite (C −0.012wt%) had greater wear resistance and lower surface grains pull out percentage when compared with monolithic Al2O3. In the present study, we investigated the physicochemical, micro- and nanomechanical, cell...... attachment, in vitro biocompatibility and osteogenic differentiation properties of Al2O3 doped carbon (0.012wt%) nanocomposite (Al2O3/C). Data were compared to values obtained for monolithic alumina (Al2O3). The calcined Al2O3/C nanocomposite was densified using cold isostatic pressing and followed...... and contact angle investigations demonstrated highly contoured micro-surface topography. The micro and nano-hardness indicate an improved wear resistance of the Al2O3/C when compared with monolithic Al2O3. SEM, confocal images and alamar blue reduction assay suggested good cell attachment and proliferation...

  10. Shear stress induced by an interstitial level of slow flow increases the osteogenic differentiation of mesenchymal stem cells through TAZ activation.

    Directory of Open Access Journals (Sweden)

    Kyung Min Kim

    Full Text Available Shear stress activates cellular signaling involved in cellular proliferation, differentiation, and migration. However, the mechanisms of mesenchymal stem cell (MSC differentiation under interstitial flow are not fully understood. Here, we show the increased osteogenic differentiation of MSCs under exposure to constant, extremely low shear stress created by osmotic pressure-induced flow in a microfluidic chip. The interstitial level of shear stress in the proposed microfluidic system stimulated nuclear localization of TAZ (transcriptional coactivator with PDZ-binding motif, a transcriptional modulator of MSCs, activated TAZ target genes such as CTGF and Cyr61, and induced osteogenic differentiation. TAZ-depleted cells showed defects in shear stress-induced osteogenic differentiation. In shear stress induced cellular signaling, Rho signaling pathway was important forthe nuclear localization of TAZ. Taken together, these results suggest that TAZ is an important mediator of interstitial flow-driven shear stress signaling in osteoblast differentiation of MSCs.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

    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

  13. Effects of Titanium Surface Microtopography and Simvastatin on Growth and Osteogenic Differentiation of Human Mesenchymal Stem Cells in Estrogen-Deprived Cell Culture.

    Science.gov (United States)

    Arpornmaeklong, Premjit; Pripatnanont, Prisana; Chookiatsiri, Chonticha; Tangtrakulwanich, Boonsin

    This study aimed to investigate the effects of titanium surface topography and simvastatin on growth and osteogenic differentiation of human bone marrow stromal cells (hBMSCs) in estrogen-deprived (ED) cell culture. Human BMSCs were seeded on cell culture plates, smooth-surface titanium (Ti) disks, and sandblasted with large grits and acid etched (SLA)-surface Ti disks; and subsequently cultured in regular (fetal bovine serum [FBS]), ED, and ED-with 100 nM simvastatin (ED-SIM) culture media for 14 to 21 days. Live/dead cell staining, scanning electron microscope examination, and cell viability assay were performed to determine cell attachment, morphology, and growth. Expression levels of osteoblast-associated genes, Runx2 and bone sialoprotein and levels of alkaline phosphatase (ALP) activity, calcium content, and osteocalcin in culture media were measured to determine osteoblastic differentiation. Expression levels of bone morphogenetic protein-2 (BMP-2) were investigated to examine stimulating effects of simvastatin (n = 4 to 5, mean ± SD). In vitro mineralization was verified by calcein staining. Human BMSCs exhibited different attachment and shapes on smooth and SLA titanium surfaces. Estrogen-deprived cell culture decreased cell attachment and growth, particularly on the SLA titanium surface, but cells were able to grow to reach confluence on day 21 in the ED-osteogenic (OS) culture medium. Promoting effects of the SLA titanium surface in ED-OS were significantly decreased. Simvastatin significantly increased osteogenic differentiation of human BMSCs on the SLA titanium surface in the ED-OS medium, and the promoting effects of simvastatin corresponded with the increasing of BMP-2 gene expression on the SLA titanium surface in ED-OS-SIM culture medium. The ED cell culture model provided a well-defined platform for investigating the effects of hormones and growth factors on cells and titanium surface interaction. Titanium, the SLA surface, and simvastatin

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

    Science.gov (United States)

    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.

  15. In vitro Osteogenic impulse effect of Dexamethasone on periodontal ligament stem cells

    Science.gov (United States)

    Roozegar, Mohamad Ali; Mohammadi, Tayebeh Malek; Havasian, Mohamad Reza; Panahi, Jafar; Hashemian, Amirreza; Amraei, Mansur; Hoshmand, Behzad

    2015-01-01

    Periodontium is a complex organ composed of mineralized epithelial and connective tissue. Dexamethasone could stimulate proliferation of osteoblast and fibroblasts. This study aimed to assess the osteogenic effect of dexamethasone on periodental ligament (PDL) stem cells. PDL stem cells were collected from periodontal ligament tissue of root of extracted premolar of young and healthy people. The stem cells were cultured in α-MEM Medium in three groups, one group with basic medium contains (α- MEM and FBS 10 % and 50 mmol of β_ gelisrophosphat and L_ ascorbic acid µg/ml), the second group: basic medium with dexamethasone and the third one: basic medium without any osteogenic stimulant. Mineralization of cellular layer was analyzed with Alizarin red stain method. Osteogenic analysis was done by Alkaline phosphates and calcium test. These analysis indicated that the amount of intra-cellular calcium and alkaline phosphates in the Dexamethasone group was far more than the control and basic group (P<0.05). The results of Alizarin red stain indicated more mineralization of cultured cells in Dexamethasone group (P<0.05). The study results showed that Dexamethasone has significant osteogenic effect on PDL stem cells and further studies are recommended to evaluate its effect on treatment of bone disorders. PMID:25848170

  16. Addition of bone morphogenetic protein type 2 to ascorbate and β-glycerophosphate supplementation did not enhance osteogenic differentiation of human adipose-derived stem cells

    Directory of Open Access Journals (Sweden)

    Ariadne Cristiane Cabral Cruz

    2012-12-01

    Full Text Available Bone morphogenetic protein type 2 (BMP-2 is a potent local factor, which promotes bone formation and has been used as an osteogenic supplement for mesenchymal stem cells. OBJECTIVES: This study evaluated the effect of a recombinant BMP-2 as well as the endogenous BMP-4 and BMP-7 in the osteogenic differentiation of adipose-derived stem cells (ASCs in medium supplemented with ascorbate and β-glycerophosphate. MATERIAL AND METHODS: Human ASCs were treated with osteogenic medium in the presence (ASCs+OM+BMP-2 or absence (ASCs+OM of BMP-2. The alkaline phosphatase (ALP activity was determined and the extracellular matrix mineralization was evaluated by Von Kossa staining and calcium quantification. The expressions of BMP-4, BMP-7, Smad1, Smad4, and phosphorylated Smad1/5/8 were analyzed by western blotting. Relative mRNA expressions of Smad1, BMP receptor type II (BMPR-II, osteonectin, and osteocalcin were evaluated by qPCR. Results: ASCs+OM demonstrated the highest expression of BMP-4 and BMP-7 at days 21 and 7, respectively, the highest levels of BMPR-II mRNA expression at day 28, and the highest levels of Smad1 mRNA at days 14 and 28. ASCs+OM+BMP-2 demonstrated the highest levels of Smad1 mRNA expression at days 1, 7, and 21, the highest expression of Smad1 at day 7, the highest expression of Smad4 at day 14, the highest ALP activity at days 14 and 21, and expression of phosphorylated Smad1/5/8 at day 7. ASCs+OM and ASCs+OM+BMP2 showed similar ALP activity at days 7 and 28, similar osteonectin and osteocalcin mRNA expression at all time periods, and similar calcium depositions at all time periods. CONCLUSIONS: We concluded that human ASCs expressed endogenous BMP-4 and BMP-7. Moreover, the supplementation of ASCs with BMP-2 did not increase the level of osteogenic markers in the initial (ALP activity, intermediate (osteonectin and osteocalcin, or final (calcium deposition phases, suggesting that the exogenous addition of BMP-2 did not improve

  17. Stromal cell-derived factor-1 stimulates cell recruitment, vascularization and osteogenic differentiation

    NARCIS (Netherlands)

    Eman, Rhandy M; Oner, F Cumhur; Kruyt, Moyo C; Dhert, Wouter J A; Alblas, Jacqueline

    The use of growth factors in osteogenic constructs to promote recruitment of bone forming endogenous cells is not clear, while the advantage of circumventing cell seeding techniques before implantation is highly recognized. Therefore, the additive effect of the chemokine stromal cell-derived

  18. Role of gender in burn-induced heterotopic ossification and mesenchymal cell osteogenic differentiation.

    Science.gov (United States)

    Ranganathan, Kavitha; Peterson, Jonathan; Agarwal, Shailesh; Oluwatobi, Eboda; Loder, Shawn; Forsberg, Jonathan A; Davis, Thomas A; Buchman, Steven R; Wang, Stewart C; Levi, Benjamin

    2015-06-01

    Heterotopic ossification most commonly occurs after burn injury, joint arthroplasty, and trauma. Male gender has been identified as a risk factor for the development of heterotopic ossification. It remains unclear why adult male patients are more predisposed to this pathologic condition than adult female patients. In this study, the authors use their validated tenotomy/burn model to explore differences in heterotopic ossification between male and female mice. The authors used their Achilles tenotomy and burn model to evaluate the osteogenic potential of mesenchymal stem cells of male and female injured and noninjured mice. Groups consisted of injured male (n = 3), injured female (n = 3), noninjured male (n = 3), and noninjured female (n = 3) mice. The osteogenic potential of cells harvested from each group was assessed through RNA and protein levels and quantified using micro-computed tomographic scan. Histomorphometry was used to verify micro-computed tomographic findings, and immunohistochemistry was used to assess osteogenic signaling at the site of heterotopic ossification. Mesenchymal stem cells of male mice demonstrated greater osteogenic gene and protein expression than those of female mice (p ossification site in male mice. The authors demonstrate that male mice form quantitatively more bone compared with female mice using their burn/tenotomy model. These findings can be explained at least in part by differences in bone morphogenetic protein and insulin-like growth factor 1 signaling.

  19. Silk fibroin/chitosan thin film promotes osteogenic and adipogenic differentiation of rat bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Li, Da-Wei; He, Jin; He, Feng-Li; Liu, Ya-Li; Liu, Yang-Yang; Ye, Ya-Jing; Deng, Xudong; Yin, Da-Chuan

    2018-01-01

    As a biodegradable polymer thin film, silk fibroin/chitosan composite film overcomes the defects of pure silk fibroin and chitosan films, respectively, and shows remarkable biocompatibility, appropriate hydrophilicity and mechanical properties. Silk fibroin/chitosan thin film can be used not only as metal implant coating for bone injury repair, but also as tissue engineering scaffold for skin, cornea, adipose, and other soft tissue injury repair. However, the biocompatibility of silk fibroin/chitosan thin film for mesenchymal stem cells, a kind of important seed cell of tissue engineering and regenerative medicine, is rarely reported. In this study, silk fibroin/chitosan film was prepared by solvent casting method, and the rat bone marrow-derived mesenchymal stem cells were cultured on the silk fibroin/chitosan thin film. Osteogenic and adipogenic differentiation of rat bone marrow-derived mesenchymal stem cells were induced, respectively. The proliferation ability, osteogenic and adipogenic differentiation abilities of rat bone marrow-derived mesenchymal stem cells were systematically compared between silk fibroin/chitosan thin film and polystyrene tissue culture plates. The results showed that silk fibroin/chitosan thin film not only provided a comparable environment for the growth and proliferation of rat bone marrow-derived mesenchymal stem cells but also promoted their osteogenic and adipogenic differentiation. This work provided information of rat bone marrow-derived mesenchymal stem cells behavior on silk fibroin/chitosan thin film and extended the application of silk fibroin/chitosan thin film. Based on the results, we suggested that the silk fibroin/chitosan thin film could be a promising material for tissue engineering of bone, cartilage, adipose, and skin.

  20. Osteogenic Differentiation of Human Dental Pulp-derived Stem Cells under Various Ex-vivo Culture Conditions

    Directory of Open Access Journals (Sweden)

    Jana Karbanová

    2010-01-01

    Full Text Available Dental pulp stem cells (DPSCs can be easily isolated and cultured in low-serum containing medium supplemented with growth factors PDGF-BB and EGF while exhibiting multipotency and immature phenotypic characteristics. In the present study, we investigated their potential to differentiate towards osteogenic lineages using various culture conditions in order to optimize their therapeutic use. DPSCs were cultured either as a cell monolayer or as three-dimensional (3D micro-mass structures. Monolayers preincubated with bFGF and valproic acid for one week prior their differentiation were cultured in serum containing standard osteodifferentiation medium for four weeks, which resulted in multilayered nodule formation. Micro-mass structures were cultured for same period either in serum containing medium or under serum-free conditions supplemented with TGF-β3 with or without BMP-2. Histochemically, we detected massive collagen I and weak calcium phosphate depositions in multilayered nodules. When culture 3D-aggregates in either standard osteodifferentiation medium or serum-free medium containing TGF-β3, only small amount of collagen I fibres was observed and almost no deposits of calcium phosphate were detected. In contrast, in presence of both TGF-β3 and BMP-2 in the serum- free medium a significant amount of collagen I fibers/bundles and calcification were detected, which is in line with osteogenic effect of BMP-2. Thus, our data indicate that certain environmental cues can enhance differentiation process of DPSCs into osteogenic lineage, which suggest their possible utilization in tissue engineering.

  1. Genetic differences in osteogenic differentiation potency in the thoracic ossification of the ligamentum flavum under cyclic mechanical stress.

    Science.gov (United States)

    Ning, Shanglong; Chen, Zhongqiang; Fan, Dongwei; Sun, Chuiguo; Zhang, Chi; Zeng, Yan; Li, Weishi; Hou, Xiaofei; Qu, Xiaochen; Ma, Yunlong; Yu, Huilei

    2017-01-01

    Mechanical stress and genetic factors play important roles in the occurrence of thoracic ossification of ligament flavum (TOLF), which can occur at one, two, or multiple levels of the spine. It is unclear whether single- and multiple-level TOLF differ in terms of osteogenic differentiation potency and osteogenesis-related gene expression under cyclic mechanical stress. This was addressed in the present study using patients with non‑TOLF and single‑ and multiple‑level TOLF (n=8 per group). Primary ligament cells were cultured and osteogenesis was induced by application of cyclic mechanical stress. Osteogenic differentiation was assessed by evaluating alkaline phosphatase (ALP) activity and the mRNA and protein expression of osteogenesis‑related genes, including ALP, bone morphogenetic protein 2 (BMP2), Runt‑related transcription factor‑2 (Runx‑2), osterix, osteopontin (OPN) and osteocalcin. The application of cyclic mechanical stress resulted in higher ALP activity in the multiple‑level than in the single‑level TOLF group, whereas no changes were observed in the non‑TOLF group. The ALP, BMP2, OPN and osterix mRNA levels were higher in the multiple‑level as compared to the single‑level TOLF group, and the levels of all osteogenesis-related genes, apart from Runx2, were higher in the multiple‑level as compared to the non‑TOLF group. The osterix and ALP protein levels were higher in the multiple‑level TOLF group than in the other 2 groups, and were increased with the longer duration of stress. These results highlight the differences in osteogenic differentiation potency between single‑ and multiple‑level TOLF that may be related to the different pathogenesis and genetic background.

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

    Energy Technology Data Exchange (ETDEWEB)

    Zou, Yulong [Department of Orthopaedic; Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Qazvini, Nader Taheri [Institute for Molecular Engineering, The University of Chicago, Chicago, Illinois 60637, United States; Argonne National Laboratory, Argonne, Illinois 60439, United States; Zane, Kylie [Institute for Molecular Engineering, The University of Chicago, Chicago, Illinois 60637, United States; Sadati, Monirosadat [Institute for Molecular Engineering, The University of Chicago, Chicago, Illinois 60637, United States; Argonne National Laboratory, Argonne, Illinois 60439, United States; Wei, Qiang [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Liao, Junyi [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Fan, Jiaming [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Song, Dongzhe [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Department of Conservative Dentistry and Endodontics, West China School of Stomatology, Sichuan University, Chengdu 610041, China; Liu, Jianxiang [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Department; amp, Technology, Wuhan 430022, China; Ma, Chao [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Departments of Neurosurgery and Otolaryngology-Head; amp, Neck Surgery, The Affiliated Zhongnan Hospital of Wuhan University, Wuhan 430071, China; Qu, Xiangyang [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Chen, Liqun [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Yu, Xinyi [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Zhang, Zhicai [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Department; amp, Technology, Wuhan 430022, China; Zhao, Chen [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Zeng, Zongyue [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Zhang, Ruyi [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Yan, Shujuan [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Wu, Tingting [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Departments of Neurosurgery and Otolaryngology-Head; amp, Neck Surgery, The Affiliated Zhongnan Hospital of Wuhan University, Wuhan 430071, China; Wu, Xingye [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Shu, Yi [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Li, Yasha [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Zhang, Wenwen [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Department; Reid, Russell R. [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Department of Surgery, Section of Plastic; Lee, Michael J. [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Wolf, Jennifer Moritis [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Tirrell, Matthew [Institute for Molecular Engineering, The University of Chicago, Chicago, Illinois 60637, United States; Argonne National Laboratory, Argonne, Illinois 60439, United States; He, Tong-Chuan [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; de Pablo, Juan J. [Institute for Molecular Engineering, The University of Chicago, Chicago, Illinois 60637, United States; Argonne National Laboratory, Argonne, Illinois 60439, United States; Deng, Zhong-Liang [Department of Orthopaedic

    2017-05-04

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

  3. MicroRNA-320a Regulates the Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells by Targeting HOXA10

    Directory of Open Access Journals (Sweden)

    Jianhou Huang

    2016-01-01

    Full Text Available Background/Aims: Human bone marrow-derived mesenchymal stem cells (hMSCs are a promising cell source for bone engineering owing to their high potential to differentiate into osteoblasts. The bone morphogenetic protein-inducible gene homeobox a10 (HOXA10 is a critical regulator of osteogenesis. The objective of the present study was to identify microR-NAs (miRNAs targeting HOXA10 and examine the effects on the osteogenic differentiation of hMSCs. Methods: Based on in silico analysis, HOXA10-targeting miRNAs were selected and their regulatory roles in osteoblast differentiation were investigated. Results: Six HOXA10-targeting miRNAs were identifIed by computational analysis, of which miR-320a was selected for further analysis because it was downregulated during osteogenic induction. Overexpression of miR-320a downregulated HOXA10 and significantly inhibited osteogenesis in hMSCs, as determined by the downregulation of the osteogenic markers Runx2, ALP, and OC and the inhibition of ALP activity and matrix mineralization, whereas miR-320a inhibition had the opposite effects. Furthermore, ectopic expression of HOXA10 (not including 3′-UTR rescued the effects of miR-320a on osteogenic differentiation. Conclusion: These results suggest that miR-320a acts as a critical regulator of osteogenic differentiation of hMSCs by repressing its target HOXA10.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  6. Human platelet lysate supports ex vivo expansion and enhances osteogenic differentiation of human bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Xia, Wenjie; Li, Hui; Wang, Zhen; Xu, Ru; Fu, Yongshui; Zhang, Xiuming; Ye, Xin; Huang, Yingfeng; Xiang, Andy Peng; Yu, Weihua

    2011-06-01

    MSCs (mesenchymal stem cells) with their versatile growth and differentiation potential are ideal candidates for use in regenerative medicine and are currently making their way into clinical trials, which requires the development of xeno-free protocols for their culture. In this study, MSCs were cultured in 10% FCS or 7.5% HPL (human platelet lysate)-supplemented media. We found that both groups of MSCs showed a comparable morphology, phenotype and proliferation. The percentage of cells in the S- and G2-/M-phases, however, was slightly up-regulated (Pplatelet derived growth factor)-AB and IGF (insulin-like growth factor)-1. In addition, compared with FCS group, MSCs in HPL group showed an increase in osteogenic differentiation and a decrease in adipogenic differentiation. In conclusion, MSCs in HPL-supplemented media maintained similar growing potential and phenotype, while osteogenic potential was enhanced. HPL offers a promising alternative to FCS for MSC expansion for clinical application, especially in bone injury diseases.

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

    Science.gov (United States)

    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. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. In vitro Osteogenic impulse effect of Dexamethasone on periodontal ligament stem cells

    OpenAIRE

    Roozegar, Mohamad Ali; Mohammadi, Tayebeh Malek; Havasian, Mohamad Reza; Panahi, Jafar; Hashemian, Amirreza; Amraei, Mansur; Hoshmand, Behzad

    2015-01-01

    Periodontium is a complex organ composed of mineralized epithelial and connective tissue. Dexamethasone could stimulate proliferation of osteoblast and fibroblasts. This study aimed to assess the osteogenic effect of dexamethasone on periodental ligament (PDL) stem cells. PDL stem cells were collected from periodontal ligament tissue of root of extracted premolar of young and healthy people. The stem cells were cultured in ?-MEM Medium in three groups, one group with basic medium contains (?-...

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

    Science.gov (United States)

    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.

  10. Low-power GaAlAs laser irradiation promotes the proliferation and osteogenic differentiation of stem cells via IGF1 and BMP2.

    Directory of Open Access Journals (Sweden)

    Jyun-Yi Wu

    Full Text Available Low-power laser irradiation (LPLI has been found to induce various biological effects and cellular processes. Also, LPLI has been shown to promote fracture repair. Until now, it has been unclear how LPLI promotes bone formation and fracture healing. The aim of this study was to investigate the potential mechanism of LPLI-mediated enhancement of bone formation using mouse bone marrow mesenchymal stem cells (D1 cells. D1 cells were irradiated daily with a gallium-aluminum-arsenide (GaAlAs laser at dose of 0, 1, 2, or 4 J/cm(2. The lactate dehydrogenase (LDH assay showed no cytotoxic effects of LPLI on D1 cells, and instead, LPLI at 4 J/cm(2 significantly promoted D1 cell proliferation. LPLI also enhanced osteogenic differentiation in a dose-dependent manner and moderately increased expression of osteogenic markers. The neutralization experiments indicated that LPLI regulated insulin-like growth factor 1 (IGF1 and bone morphogenetic protein 2 (BMP2 signaling to promote cell proliferation and/or osteogenic differentiation. In conclusion, our study suggests that LPLI may induce IGF1 expression to promote both the proliferation and osteogenic differentiation of D1 cells, whereas it may induce BMP2 expression primarily to enhance osteogenic differentiation.

  11. Osteochondral defect repair using bilayered hydrogels encapsulating both chondrogenically and osteogenically pre-differentiated mesenchymal stem cells in a rabbit model

    NARCIS (Netherlands)

    Lam, J.; Lu, S.; Lee, E.J.; Trachtenberg, J.E.; Meretoja, V.V.; Dahlin, R.L.; van den Beucken, J.J.; Tabata, Y.; Wong, M.E.; Jansen, J.A.; Mikos, A.G.; Kasper, F.K.

    2014-01-01

    OBJECTIVE: To investigate the ability of cell-laden bilayered hydrogels encapsulating chondrogenically and osteogenically (OS) pre-differentiated mesenchymal stem cells (MSCs) to effect osteochondral defect repair in a rabbit model. By varying the period of chondrogenic pre-differentiation from 7

  12. N-CoR modulates osteogenic differentiation of rat mesenchymal stem cells through the PI3K/Akt-cell signaling pathway

    Directory of Open Access Journals (Sweden)

    Qin Yi

    2016-01-01

    Full Text Available The nuclear receptor corepressor (N-CoR is involved in the regulation of diverse transcription factors. We previously found that N-CoR could regulate adipogenic differentiation of rat mesenchymal stem cells (MSCs,but whether it modulated osteogenic differentiation of this type of cells was uncertain. Therefore, in the present study, we investigated the effect and mechanism of N-CoRon osteogenic differentiation of rat MSCs. The results showed that MSCs cultured in osteogenic medium successfully differentiated into osteogenic cells. Overexpression of N-CoR decreased cell proliferation, alkaline phosphatase (ALPactivity, calcium accumulation, mRNA expression of genes including bone sialoprotein (BSP, osteocalcin (OCN, osteopontin (OPN, Osterix and Runx2, and protein expression of phosphor-Akt(pAkt. Conversely, knocking down cellular N-CoR by small interfering RNA (siRNA promoted pAkt activity and cell differentiation. Overexpression or knockdown of N-CoRhad no significant influences on the protein expression of pyruvate dehydrogenase kinase isozyme 1 (PDK1, phosphatidylinositol 3-kinase (PI3K and total Akt, indicating that N-CoR regulated the changes in the PI3K/Akt signaling pathway by targeting pAkt. To further prove the function of the PI3K/Akt signaling in N-CoR-regulated osteogenic differentiation, we used the PI3K inhibitor (LY294002 to block the activation of the PI3K/Akt pathway and found that overexpression of N-CoR showed no effects on ALP activity, calcium level and mRNA expression of BSP, osteocalcin OCN, OPN, Osterix and Runx2 in rat MSCs following the inhibition of the PI3K/Akt pathway. These findings suggest that N-CoR regulates osteogenic differentiation of rat MSCs through suppression of the PI3K/Akt signaling pathway.

  13. Loss of the osteogenic differentiation potential during senescence is limited to bone progenitor cells and is dependent on p53.

    Science.gov (United States)

    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.

  14. Loss of the osteogenic differentiation potential during senescence is limited to bone progenitor cells and is dependent on p53.

    Directory of Open Access Journals (Sweden)

    Geneviève Despars

    Full Text Available 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.

  15. Dexamethasone in osteogenic medium strongly induces adipocyte differentiation of mouse bone marrow stromal cells and increases osteoblast differentiation

    NARCIS (Netherlands)

    O. Ghali (Olfa); O. Broux (Odile); G. Falgayrac (Guillaume); N. Haren (Nathalie); J.P.T.M. van Leeuwen (Hans); G. Penel (Guillaume); P. Hardouin (Pierre); C. Chauveau (Christophe)

    2015-01-01

    textabstractBackground: Osteoblasts and adipocytes share a common mesenchymal stem cell origin. Therefore, it has been suggested that the accumulation of marrow adipocytes observed in bone loss is caused by a shift in the commitment of mesenchymal stem cells from the osteogenic pathway to the

  16. Dexamethasone in osteogenic medium strongly induces adipocyte differentiation of mouse bone marrow stromal cells and increases osteoblast differentiation

    NARCIS (Netherlands)

    O. Ghali (Olfa); O. Broux (Odile); G. Falgayrac (Guillaume); N. Haren (Nathalie); J.P.T.M. van Leeuwen (Hans); G. Penel (Guillaume); P. Hardouin (Pierre); C. Chauveau (Christophe)

    2015-01-01

    textabstractBACKGROUND: Osteoblasts and adipocytes share a common mesenchymal stem cell origin. Therefore, it has been suggested that the accumulation of marrow adipocytes observed in bone loss is caused by a shift in the commitment of mesenchymal stem cells from the osteogenic pathway to the

  17. Two-photon FLIM of NAD(P)H and FAD in mesenchymal stem cells undergoing either osteogenic or chondrogenic differentiation.

    Science.gov (United States)

    Meleshina, Aleksandra V; Dudenkova, Varvara V; Bystrova, Alena S; Kuznetsova, Daria S; Shirmanova, Marina V; Zagaynova, Elena V

    2017-01-28

    Metabolic plasticity and the versatility of different lineages of stem cells as they satisfy their energy demands are not completely understood. In this study we investigated the metabolic changes in mesenchymal stem cells (MSCs) undergoing differentiation in two directions, osteogenic and chondrogenic, using two-photon fluorescence microscopy combined with FLIM. Differentiation was induced by incubating the human bone marrow MSCs in osteogenic or chondrogenic mediums. Cellular metabolism was examined on the basis of the fluorescence of the metabolic cofactors NAD(P)H and FAD. The optical redox ratio (FAD/NAD(P)H) and the fluorescence lifetimes of NAD(P)H and FAD were traced using two-photon fluorescence microscopy combined with FLIM. The cells were imaged before the induction of differentiation (day 0) and on days 7, 14, and 21 of osteogenic and chondrogenic differentiation. Based on the data for the FAD/NAD(P)H redox ratio and on the fluorescence lifetimes of protein-bound NAD(P)H, we registered a metabolic shift toward a more glycolytic status in the process of MSC differentiation. The difference was that, in osteogenic differentiation, an increase in oxidative phosphorylation preceded the shift to the glycolytic status in the process of such MSC differentiation. The fluorescence lifetime characteristics of FAD indicated the stimulation of an unknown metabolic pathway, where protein-bound FAD participates. In this study, probing of the metabolic status of MSCs during osteogenic and chondrogenic differentiation was implemented for the first time with the use of optical metabolic imaging of the two cofactors - NAD(P)H and FAD. Our data suggest that biosynthetic processes, associated, presumably, with the synthesis of collagen, drive energy metabolism in differentiating cells, and promote a metabolic shift from a more oxidative to a more glycolytic state.

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

    Energy Technology Data Exchange (ETDEWEB)

    Lima, João; Gonçalves, Ana I.; Rodrigues, Márcia T.; Reis, Rui L. [3Bs Research Group–Biomaterials, Biodegradables and Biomimetics, University of Minho, Guimarães (Portugal); ICVS/3Bs–PT Government Associate Laboratory, Braga/Guimarães (Portugal); Gomes, Manuela E., E-mail: megomes@dep.uminho.pt [3Bs Research Group–Biomaterials, Biodegradables and Biomimetics, University of Minho, Guimarães (Portugal); ICVS/3Bs–PT Government Associate Laboratory, Braga/Guimarães (Portugal)

    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. - Highlights: • Cellular viability was not negatively influenced by the nanoparticles. • Chondrogenic medium influences more the synthesis of cartilage-like ECM than MNPs. • Synergetic effect among

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

    Science.gov (United States)

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

    2017-07-01

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

  20. The synergistic effect on osteogenic differentiation of human mesenchymal stem cells by diode laser-treated stimulating human umbilical vein endothelial cells

    Science.gov (United States)

    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.

  1. Caffeine inhibits the viability and osteogenic differentiation of rat bone marrow-derived mesenchymal stromal cells.

    Science.gov (United States)

    Zhou, Y; Guan, X X; Zhu, Z L; Guo, J; Huang, Y C; Hou, W W; Yu, H Y

    2010-12-01

    Caffeine is consumed extensively in Europe and North America. As a risk factor for osteoporosis, epidemiological studies have observed that caffeine can decrease bone mineral density, adversely affect calcium absorption and increase the risk of bone fracture. However, the exact mechanisms have not been fully investigated. Here, we examined the effects of caffeine on the viability and osteogenesis of rat bone marrow-derived mesenchymal stromal cells (rBMSCs). Cell viability, apoptosis and necrosis were quantified using thymidine incorporation and flow cytometry. Sequential gene expressions in osteogenic process were measured by real-time PCR. cAMP, alkaline phosphatase and osteocalcin were assessed by immunoassay, spectrophotometry and radioimmunoassay, respectively. Mineralization was determined by calcium deposition. After treating BMSCs with high caffeine concentrations (0.1-1mM), their viability decreased in a concentration-dependent manner. This cell death was primarily due to necrosis and, to a small extent, apoptosis. Genes and protein sequentially expressed in osteogenesis, including Cbfa1/Runx2, collagen I, alkaline phosphatase and its protein, were significantly downregulated except for osteocalcin and its protein. Moreover, caffeine inhibited calcium deposition in a concentration- and time-dependent manner, but increased intracellular cAMP in a concentration-dependent manner. By suppressing the commitment of BMSCs to the osteogenic lineage and selectively inhibiting gene expression, caffeine downregulated some important events in osteogenesis and ultimately affected bone mass.

  2. Altered microRNA expression profile in exosomes during osteogenic differentiation of human bone marrow-derived mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Ji-Feng Xu

    Full Text Available The physiological role of microRNAs (miRNAs in osteoblast differentiation remains elusive. Exosomal miRNAs isolated from human bone marrow-derived mesenchymal stem cells (BMSCs culture were profiled using miRNA arrays containing probes for 894 human matured miRNAs. Seventy-nine miRNAs (∼8.84% could be detected in exosomes isolated from BMSC culture supernatants when normalized to endogenous control genes RNU44. Among them, nine exosomal miRNAs were up regulated and 4 miRNAs were under regulated significantly (Relative fold>2, p<0.05 when compared with the values at 0 day with maximum changes at 1 to 7 days. Five miRNAs (miR-199b, miR-218, miR-148a, miR-135b, and miR-221 were further validated and differentially expressed in the individual exosomal samples from hBMSCs cultured at different time points. Bioinformatic analysis by DIANA-mirPath demonstrated that RNA degradation, mRNA surveillance pathway, Wnt signaling pathway, RNA transport were the most prominent pathways enriched in quantiles with differential exosomal miRNA patterns related to osteogenic differentiation. These data demonstrated exosomal miRNA is a regulator of osteoblast differentiation.

  3. Comparative characteristics of porous bioceramics for an osteogenic response in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Hye-Rim Lee

    Full Text Available Porous calcium phosphate ceramics are used in orthopedic and craniofacial applications to treat bone loss, or in dental applications to replace missing teeth. The implantation of these materials, however, does not induce stem cell differentiation, so suitable additional materials such as porous calcium phosphate discs are needed to influence physicochemical responses or structural changes. Rabbit adipose-derived stem cells (ADSC and mouse osteoblastic cells (MC3T3-E1 were evaluated in vitro by the MTT assay, semi-quantitative RT-PCR, and immunoblotting using cells cultured in medium supplemented with extracts from bioceramics, including calcium metaphosphate (CMP, hydroxyapatite (HA and collagen-grafted HA (HA-col. In vivo evaluation of the bone forming capacity of these bioceramics in rat models using femur defects and intramuscular implants for 12 weeks was performed. Histological analysis showed that newly formed stromal-rich tissues were observed in all the implanted regions and that the implants showed positive immunoreaction against type I collagen and alkaline phosphatase (ALP. The intramuscular implant region, in particular, showed strong positive immunoreactivity for both type I collagen and ALP, which was further confirmed by mRNA expression and immunoblotting results, indicating that each bioceramic material enhanced osteogenesis stimulation. These results support our hypothesis that smart bioceramics can induce osteoconduction and osteoinduction in vivo, although mature bone formation, including lacunae, osteocytes, and mineralization, was not prominent until 12 weeks after implantation.

  4. Increased Osteogenic Differentiation of Periodontal Ligament Stem Cells on Polydopamine Film Occurs via Activation of Integrin and PI3K Signaling Pathways

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    Jeong Seok Lee

    2014-11-01

    Full Text Available Background/Aims: Mussel-inspired polydopamine (PDA is known to be an effective bioadhesive and bioactive material for controlling stem cell fate, which is important in stem cell-based regenerative medicine; however, the effect of PDA on osteogenic differentiation of periodontal ligament stem cells (PDLSCs is not fully understood. In this study, we investigated the osteoinductive effect of PDA on PDLSCs and examined how this phenomenon is encouraged. Methods: Osteogenic induction of PDLSCs was established by culturing cells on PDA film or on an uncoated polystyrene surface as a control. Osteogenic differentiation of PDLSCs was assessed by measurement of intracellular calcium levels and alkaline phosphatase (ALP activity as well as by evaluation of protein expression of osteocalcin (OCN, osterix (OSX, and runt-related transcription factor 2 (RUNX2. Results: The PDLSCs cultured on PDA film showed higher osteogenic activity than those on the control surface. Moreover, PDLSCs on PDA film expressed increased levels of the integrin adhesion receptors integrin α5 and β1 compared to control cells. Expression of one isoform of the intracellular signaling protein phosphatidylinositol-3-kinase (PI3K, p110γ, was increased in PDLSCs on PDA film in a PDA dose-dependent manner. This signaling protein was found to interact with integrin β1, demonstrating integrin-linked PI3K activation in response to PDA. Finally, the blockage of PI3K reduced the PDA-induced osteogenic activity of PDLSCs. Conclusion: our findings suggest that the bioadhesive PDA stimulates osteogenic differentiation of PDLSCs via activation of the integrin α5/β1 and PI3K signaling pathways.

  5. [Effect of microRNA-17 on osteogenic differentiation of advanced glycation end products-stimulated human periodontal ligament stem cells].

    Science.gov (United States)

    Chao, Deng; Yan, Wu; Kun, Yang; Xiaoxia, Cui; Qi, Liu; Yan, Jin

    2015-02-01

    This study aims to detect microRNA-17(mir-17) expression on the osteogenic differentiation of advanced glycation end products (AGEs)-stimulated hunman periodontal ligament stem cells (HPDLSCs) and to analyze the influence of these cells on this process. HPDLSCs were isolated using limited dilution technique. After osteogenic differentiation occurred, different time points of mir-17 expression in the experimental groups were detected by real time polymerase chain reaction (PCR). The mir-17 overexpression and inhibition were evaluated using cell transfection technique. Differences in gene expressions were detected by real time PCR; differences in protein expressions were analyzed by Western blot. The mir-17 expression was reduced after osteogenic differentiation occurred at 3, 7, and 14 d compared with that in the control group (P < 0.05). The expression levels of bone sialoprotein (BSP), Runt-related transcription factor-2 (Runx-2)and alkaline phosphatase (ALP) in the experimental groups were lower than those in the mimic control group when mir-17 expression increased. In addition, the protein expression levels of Runx-2 in the experimental groups were lower than those in the control group. The expression levels of BSP, Runx-2 and ALP in the experimental groups were higher than those in the inhibitor control group when mir-17 expression decreased. Likewise, the protein expression levels of Runx-2 in the experimental groups were higher than those in the control group. AGEs inhibit the osteogenic differentiation of HPDLSCs by affecting mir-17 expression.

  6. Enhancing osteogenic differentiation of MC3T3-E1 cells by immobilizing inorganic polyphosphate onto hyaluronic acid hydrogel.

    Science.gov (United States)

    Wu, Andy T H; Aoki, Teruo; Sakoda, Megumu; Ohta, Seiichi; Ichimura, Shigetoshi; Ito, Taichi; Ushida, Takashi; Furukawa, Katsuko S

    2015-01-12

    In tissue engineering, precise control of cues in the microenvironment is essential to stimulate cells to undergo bioactivities such as proliferation, differentiation, and matrix production. However, current approaches are inefficient in providing nondepleting cues. In this study, we have developed a novel bioactive hydrogel (HAX-PolyP) capable of enhancing tissue growth by conjugating inorganic polyphosphate chains onto hyaluronic acid macromers. The immobilized polyphosphates provided constant osteoconductive stimulation to the embedded murine osteoblast precursor cells, resulting in up-regulation of osteogenic marker genes and enhanced levels of ALP activity. The osteoconductive activity was significantly higher when compared to those stimulated with free-floating polyphosphates. Even at very low concentrations, immobilization of polyphosphates onto the scaffold allowed sufficient signaling leading to more effective osteoconduction. These results demonstrate the potential of our novel material as an injectable bioactive scaffold, which can be clinically useful for developing bone grafts and bone regeneration applications.

  7. Correlation of NADH fluorescence lifetime and oxidative phosphorylation metabolism in the osteogenic differentiation of human mesenchymal stem cell

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  9. Systematic comparison of biologically active foreign ions-codoped calcium phosphate microparticles on osteogenic differentiation in rat osteoporotic and normal mesenchymal stem cells.

    Science.gov (United States)

    Chen, Xiao-Yi; Xu, San-Zhong; Wang, Xuan-Wei; Yang, Xian-Yan; Ma, Liang; Zhang, Lei; Yang, Guo-Jing; Yang, Fan; Wang, Lin-Hong; Zhang, Xin-Li; Ting, Kang; Gao, Chang-You; Mou, Xiao-Zhou; Gou, Zhong-Ru; Zou, Hai

    2017-05-30

    Osteoporosis is a disease characterized by structural deterioration of bone tissue, leading to skeletal fragility with increased fracture risk. Calcium phosphates (CaPs) are widely used in bone tissue engineering strategies as they have similarities to bone apatite except for the absence of trace elements (TEs) in the CaPs. Bioactive glasses (BGs) have also been used successfully in clinic for craniomaxillofacial and dental applications during the last two decades due to their excellent potential for bonding with bone and inducing osteoblastic differentiation. In this study, we evaluated the osteogenic effects of the ionic dissolution products of the quaternary Si-Sr-Zn-Mg-codoped CaP (TEs-CaP) or 45S5 Bioglass® (45S5 BG), both as mixtures and separately, on rat bone marrow-derived mesenchymal stem cells (rOMSCs & rMSCs) from osteoporotic and normal animals, using an MTT test and Alizarin Red S staining. The materials enhanced cell proliferation and osteogenic differentiation, especially the combination of the BG and TEs-CaP. Analysis by quantitative PCR and ELISA indicated that the expression of osteogenic-specific genes and proteins were elevated. These investigations suggest that the TEs-CaP and 45S5 BG operate synergistically to create an extracellular environment that promotes proliferation and terminal osteogenic differentiation of both osteoporotic and normal rMSCs.

  10. Cellular localization of NRF2 determines the self-renewal and osteogenic differentiation potential of human MSCs via the P53-SIRT1 axis.

    Science.gov (United States)

    Yoon, D S; Choi, Y; Lee, J W

    2016-02-11

    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.

  11. Facile fabrication of bioactive ultra-small protein–hydroxyapatite nanoconjugates via liquid-phase laser ablation and their enhanced osteogenic differentiation activity

    KAUST Repository

    Rodio, Marina

    2016-11-24

    Hydroxyapatite bioactive complexes are being increasingly recognized as effective available means in regenerative medicine. Conventional technologies for their synthesis have drawbacks from a synthetic standpoint, mainly requiring high temperatures and multi-step processes. Here, we show that ultra-small hydroxyapatite conjugated-nanoparticles (Ha-CNPs) can be obtained at room temperature by Pulsed Laser Ablation (PLA) directly in protein solution using picosecond pulses at near infrared wavelengths. The results showed that the nanoparticle size was driven by the concentration of the protein. Using this approach, we obtained aqueous soluble and ultra-small crystalline nanoparticles of ≈3 nm diameter coated with protein molecules (surface coverage ≈ 5.5 pmol cm; zeta potential ≈-33.5 mV). These nanoparticles showed low cytotoxicity in vitro compared to chemically synthesized nanoparticles, and revealed proliferative and osteoinductive effects on human bone marrow mesenchymal stem cells (hMSCs). The resulting enhanced cell osteogenic differentiation suggested that our PLA-based synthetic approach might be exploited in novel applications of regenerative medicine.

  12. Hyaluronic Acid Gel-Based Scaffolds as Potential Carrier for Growth Factors: An In Vitro Bioassay on Its Osteogenic Potential

    Directory of Open Access Journals (Sweden)

    Masako Fujioka-Kobayashi

    2016-11-01

    Full Text Available Hyaluronic acid (HA has been utilized for a variety of regenerative medical procedures due to its widespread presence in connective tissue and perceived biocompatibility. The aim of the present study was to investigate HA in combination with recombinant human bone morphogenetic protein 9 (rhBMP9, one of the most osteogenic growth factors of the BMP family. HA was first combined with rhBMP9 and assessed for the adsorption and release of rhBMP9 over 10 days by ELISA. Thereafter, ST2 pre-osteoblasts were investigated by comparing (1 control tissue culture plastic, (2 HA alone, and (3 HA with rhBMP9 (100 ng/mL. Cellular proliferation was investigated by a MTS assay at one, three and five days and osteoblast differentiation was investigated by alkaline phosphatase (ALP activity at seven days, alizarin red staining at 14 days and real-time PCR for osteoblast differentiation markers. The results demonstrated that rhBMP9 adsorbed within HA scaffolds and was released over a 10-day period in a controlled manner. While HA and rhBMP9 had little effect on cell proliferation, a marked and pronounced effect was observed for cell differentiation. rhBMP9 significantly induced ALP activity, mRNA levels of collagen1α2, and ALP and osteocalcin (OCN at three or 14 days. HA also demonstrated some ability to induce osteoblast differentiation by increasing mRNA levels of OCN and increasing alizarin red staining at 14 days. In conclusion, the results from the present study demonstrate that (1 HA may serve as a potential carrier for various growth factors, and (2 rhBMP9 is a potent and promising inducer of osteoblast differentiation. Future animal studies are now necessary to investigate this combination approach in vivo.

  13. Donor-matched mesenchymal stem cells from knee infrapatellar and subcutaneous adipose tissue of osteoarthritic donors display differential chondrogenic and osteogenic commitment.

    Science.gov (United States)

    Lopa, S; Colombini, A; Stanco, D; de Girolamo, L; Sansone, V; Moretti, M

    2014-04-23

    Cell-based therapies have recently been proposed for the treatment of degenerative articular pathologies, such as early osteoarthritis, with an emphasis on autologous mesenchymal stem cells (MSCs), as an alternative to terminally differentiated cells. In this study, we performed a donor-matched comparison between infrapatellar fat pad MSCs (IFP-MSCs) and knee subcutaneous adipose tissue stem cells (ASCs), as appealing candidates for cell-based therapies that are easily accessible during surgery. IFP-MSCs and ASCs were obtained from 25 osteoarthritic patients undergoing total knee replacement and compared for their immunophenotype and differentiative potential. Undifferentiated IFP-MSCs and ASCs displayed the same immunophenotype, typical of MSCs (CD13+/CD29+/CD44+/CD73+/CD90+/CD105+/CD166+/CD31-/CD45-). IFP-MSCs and ASCs showed similar adipogenic potential, though undifferentiated ASCs had higher LEP expression compared to IFP-MSCs (p<0.01). Higher levels of calcified matrix (p<0.05) and alkaline phosphatase (p<0.05) in ASCs highlighted their superior osteogenic commitment compared to IFP-MSCs. Conversely, IFP-MSCs pellets showed greater amounts of glycosaminoglycans (p<0.01) and superior expression of ACAN (p<0.001), SOX9, COMP (p<0.001) and COL2A1 (p<0.05) compared to ASCs pellets, revealing a superior chondrogenic potential. This was also supported by lower COL10A1 (p<0.05) and COL1A1 (p<0.01) expression and lower alkaline phosphatase release (p<0.05) by IFP-MSCs compared to ASCs. The observed dissimilarities between IFP-MSCs and ASCs show that, despite expressing similar surface markers, MSCs deriving from different fat depots in the same surgical site possess specific features. Furthermore, the in vitro peculiar commitment of IFP-MSCs and ASCs from osteoarthritic donors towards the chondrogenic or osteogenic lineage may suggest a preferential use for cartilage and bone cell-based treatments, respectively.

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

    Science.gov (United States)

    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.

  15. Evaluation of a thermoresponsive polycaprolactone scaffold for in vitro three-dimensional stem cell differentiation.

    Science.gov (United States)

    Hruschka, Veronika; Saeed, Aram; Slezak, Paul; Cheikh Al Ghanami, Racha; Feichtinger, Georg Alexander; Alexander, Cameron; Redl, Heinz; Shakesheff, Kevin; Wolbank, Susanne

    2015-01-01

    Tissue engineering (TE) strategies aim at imitating the natural process of regeneration by using bioresorbable scaffolds that support cellular attachment, migration, proliferation, and differentiation. Based on the idea of combining a fully degradable polymer [poly(ɛ-caprolactone)] with a thermoresponsive polymer (polyethylene glycol methacrylate), a scaffold was developed, which liquefies below 20°C and solidifies at 37°C. In this study, this scaffold was evaluated for its ability to support C2C12 cells and human adipose-derived stem cells (ASCs) to generate an expandable three-dimensional (3D) construct for soft or bone TE. As a first step, biomaterial seeding was optimized and cellular attachment, survival, distribution, and persistence within the 3D material were characterized. C2C12 cells were differentiated toward the osteogenic as well as myogenic lineage, while ASCs were cultured in control, adipogenic, or osteogenic differentiation media. Differentiation was examined using quantitative real-time PCR for the expression of osteogenic, myogenic, and adipogenic markers and by enzyme activity and immunoassays. Both cell types attached and were found evenly distributed within the material. C2C12 cells and ASCs demonstrated the potential to differentiate in all tested lineages under 2D conditions. Under 3D osteogenic conditions for C2C12 cells, only osteocalcin expression (fold induction: 16.3±0.2) and alkaline phosphatase (ALP) activity (pdifferentiation of ASC was limited and donor dependent. Only one donor showed an increase in the osteogenic markers osteocalcin (p=0.027) and osteopontin (p=0.038). In contrast, differentiation toward the myogenic or adipogenic lineage showed expression of specific markers in 3D, at least at the level of the 2D culture. In 3D culture, strong induction of myogenin (pdifferentiation of one donor showed greater expression of peroxisome proliferative-activated receptor gamma (PPARγ) (p=0.004), fatty acid binding protein 4

  16. Isolation of Progenitors that Exhibit Myogenic/Osteogenic Bipotency In Vitro by Fluorescence-Activated Cell Sorting from Human Fetal Muscle

    Directory of Open Access Journals (Sweden)

    Alessandra Castiglioni

    2014-01-01

    Full Text Available Fluorescence-activated cell sorting (FACS strategies to purify distinct cell types from the pool of fetal human myofiber-associated (hMFA cells were developed. We demonstrate that cells expressing the satellite cell marker PAX7 are highly enriched within the subset of CD45−CD11b−GlyA−CD31−CD34−CD56intITGA7hi hMFA cells. These CD45−CD11b−GlyA−CD31−CD34−CD56intITGA7hi cells lack adipogenic capacity but exhibit robust, bipotent myogenic and osteogenic activity in vitro and engraft myofibers when transplanted into mouse muscle. In contrast, CD45−CD11b−GlyA−CD31−CD34+ fetal hMFA cells represent stromal constituents of muscle that do not express PAX7, lack myogenic function, and exhibit adipogenic and osteogenic capacity in vitro. Adult muscle likewise contains PAX7+ CD45−CD11b−GlyA−CD31−CD34−CD56intITGA7hi hMFA cells with in vitro myogenic and osteogenic activity, although these cells are present at lower frequency in comparison to their fetal counterparts. The ability to directly isolate functionally distinct progenitor cells from human muscle will enable novel insights into muscle lineage specification and homeostasis.

  17. High-throughput assay for the identification of compounds regulating osteogenic differentiation of human mesenchymal stromal cells.

    Directory of Open Access Journals (Sweden)

    Hugo Alves

    Full Text Available Human mesenchymal stromal cells are regarded as the golden standard for cell-based therapies. They present multilineage differentiation potential and trophic and immunosuppressive abilities, making them the best candidate for clinical applications. Several molecules have been described to increase bone formation and were mainly discovered by candidate approaches towards known signaling pathways controlling osteogenesis. However, their bone forming potential is still limited, making the search for novel molecules a necessity. High-throughput screening (HTS not only allows the screening of a large number of diverse chemical compounds, but also allows the discovery of unexpected signaling pathways and molecular mechanisms for a certain application, even without the prior knowledge of the full molecular pathway. Typically HTS is performed in cell lines, however, in this manuscript we have performed a phenotypical screen on more clinically relevant human mesenchymal stromal cells, as a proof of principle that HTS can be performed in those cells and can be used to find small molecules that impact stem cell fate. From a library of pharmacologically active small molecules, we were able to identify novel compounds with increased osteogenic activity. These compounds allowed achieving levels of bone-specific alkaline phosphatase higher than any other combination previously known. By combining biochemical techniques, we were able to demonstrate that a medium to high-throughput phenotypic assay can be performed in academic research laboratories allowing the discovery of novel molecules able to enhance stem cell differentiation.

  18. Osteogenic differentiation of bone marrow mesenchymal stem cells by magnetic nanoparticle composite scaffolds under a pulsed electromagnetic field

    Directory of Open Access Journals (Sweden)

    Jianghong Huang

    2017-05-01

    Full Text Available This study was conducted to investigate the effect of magnetic nanoparticle composite scaffold under a pulsed electromagnetic field on bone marrow mesenchymal stem cells (BMSCs, which was achieved by examining the biological behaviors of cell adhesion, proliferation and differentiation on the surface of the scaffolds. This may provide some experimental evidence for the use of magnetic nanoparticles in medical application. The magnetic nanoparticle composite scaffolds were evaluated and characterized by the following indexes: the cell proliferation was detected by the CCK-8 method, the alkaline phosphatase (ALP activity was examined by a detection kit, and the expression of type I collagen and osteocalcin gene were evaluated by RT-PCR. The CCK-8 test showed that there was no significant difference in Group A (BMSCs-seeded magnetic scaffolds under the electromagnetic field, B (BMSCs-seeded magnetic scaffolds and C (BMSCs cultured alone (P > 0.05. The value for the ALP activity in Group A was higher than the other two groups. In addition, the RT-PCR results showed that the expression of type I collagen gene in Group A was enhanced (P  0.05. To conclude, magnetic nanoparticles may induce the osteogenic differentiation with the action of the pulsed electromagnetic field.

  19. Calcium sensing receptor expression in ovine amniotic fluid mesenchymal stem cells and the potential role of R-568 during osteogenic differentiation.

    Directory of Open Access Journals (Sweden)

    Pamela Di Tomo

    Full Text Available Amniotic fluid-derived stem (AFS cells have been identified as a promising source for cell therapy applications in bone traumatic and degenerative damage. Calcium Sensing Receptor (CaSR, a G protein-coupled receptor able to bind calcium ions, plays a physiological role in regulating bone metabolism. It is expressed in different kinds of cells, as well as in some stem cells. The bone CaSR could potentially be targeted by allosteric modulators, in particular by agonists such as calcimimetic R-568, which may potentially be helpful for the treatment of bone disease. The aim of our study was first to investigate the presence of CaSR in ovine Amniotic Fluid Mesenchymal Stem Cells (oAFMSCs and then the potential role of calcimimetics in in vitro osteogenesis. oAFMSCs were isolated, characterized and analyzed to examine the possible presence of CaSR by western blotting and flow cytometry analysis. Once we had demonstrated CaSR expression, we worked out that 1 µM R-568 was the optimal and effective concentration by cell viability test (MTT, cell number, Alkaline Phosphatase (ALP and Alizarin Red S (ARS assays. Interestingly, we observed that basal diffuse CaSR expression in oAFMSCs increased at the membrane when cells were treated with R-568 (1 µM, potentially resulting in activation of the receptor. This was associated with significantly increased cell mineralization (ALP and ARS staining and augmented intracellular calcium and Inositol trisphosphate (IP3 levels, thus demonstrating a potential role for calcimimetics during osteogenic differentiation. Calhex-231, a CaSR allosteric inhibitor, totally reversed R-568 induced mineralization. Taken together, our results demonstrate for the first time that CaSR is expressed in oAFMSCs and that calcimimetic R-568, possibly through CaSR activation, can significantly improve the osteogenic process. Hence, our study may provide useful information on the mechanisms regulating osteogenesis in oAFMSCs, perhaps

  20. Mechanical stimulation promote the osteogenic differentiation of bone marrow stromal cells through epigenetic regulation of Sonic Hedgehog.

    Science.gov (United States)

    Wang, Chuandong; Shan, Shengzhou; Wang, Chenglong; Wang, Jing; Li, Jiao; Hu, Guoli; Dai, Kerong; Li, Qingfeng; Zhang, Xiaoling

    2017-03-15

    Mechanical unloading leads to bone loss and disuse osteoporosis partly due to impaired osteoblastogenesis of bone marrow stromal cells (BMSCs). However, the underlying molecular mechanisms of this phenomenon are not fully understood. In this study, we demonstrated that cyclic mechanical stretch (CMS) promotes osteoblastogenesis of BMSCs both in vivo and in vitro. Besides, we found that Hedgehog (Hh) signaling pathway was activated in this process. Inhibition of which by either knockdown of Sonic hedgehog (Shh) or treating BMSCs with Hh inhibitors attenuated the osteogenic effect of CMS on BMSCs, suggesting that Hh signaling pathway acts as an endogenous mediator of mechanical stimuli on BMSCs. Furthermore, we demonstrated that Shh expression level was regulated by DNA methylation mechanism. Chromatin Immunoprecipitation (ChIP) assay showed that DNA methyltransferase 3b (Dnmt3b) binds to Shh gene promoter, leading to DNA hypermethylation in mechanical unloading BMSCs. However, mechanical stimulation down-regulates the protein level of Dnmt3b, results in DNA demethylation and Shh expression. More importantly, we found that inhibition of Dnmt3b partly rescued bone loss in HU mice by mechanical unloading. Our results demonstrate, for the first time, that mechanical stimulation regulates osteoblastic genes expression via direct regulation of Dnmt3b, and the therapeutic inhibition of Dnmt3b may be an efficient strategy for enhancing bone formation under mechanical unloading. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Effect of scaffold microarchitecture on osteogenic differentiation of human mesenchymal stem cells

    National Research Council Canada - National Science Library

    Phadke, Ameya; Hwang, YongSung; Kim, Su Hee; Kim, Soo Hyun; Yamaguchi, Tomonori; Masuda, Koichi; Varghese, Shyni

    2013-01-01

    Design of macroporous synthetic grafts that can promote infiltration of cells, their differentiation, and synthesis of bone-specific extracellular matrix is a key determinant for in vivo bone tissue...

  2. Long non-coding RNA HOTAIR inhibits miR-17-5p to regulate osteogenic differentiation and proliferation in non-traumatic osteonecrosis of femoral head.

    Directory of Open Access Journals (Sweden)

    Biaofang Wei

    Full Text Available The biological functions of non-coding RNAs (ncRNAs have been widely identified in many human diseases. In the present study, the relationship between long non-coding RNA HOTAIR and microRNA-17-5p (miR-17-5p and their roles in osteogenic differentiation and proliferation in non-traumatic osteonecrosis of femoral head (ONFH were investigated.The expression levels of HOTAIR and miR-17-5p in the mesenchymal stem cells (MSCs derived from patients with non-traumatic ONFH and osteoarthritis (OA were examined by real-time PCR. BMP-2 induced human MSCs from bone marrow (hMSC-BM were used for osteogenic differentiation.It was observed that the expression level of miR-17-5p was lower and the level of HOTAIR was higher in samples of non-traumatic ONFH compared with OA. HOTAIR downregulation induced by si-HOTAIR led to the increase of miR-17-5p expression and the decrease of miR-17-5p target gene SMAD7 expression. The values of osteogenic differentiation markers, including RUNX2 and COL1A1 mRNA expression and ALP activity, were also elevated by si-HOTAIR. However, the increase of these values was canceled by miR-17-5p inhibitor or SMAD7 upregulation.HOTAIR played a role in regulating osteogenic differentiation and proliferation through modulating miR-17-5p and its target gene SMAD7 in non-traumatic ONFH.

  3. Proliferation and osteogenic differentiation of rat BMSCs on a novel Ti/SiC metal matrix nanocomposite modified by friction stir processing

    Science.gov (United States)

    Zhu, Chenyuan; Lv, Yuting; Qian, Chao; Qian, Haixin; Jiao, Ting; Wang, Liqiang; Zhang, Fuqiang

    2016-12-01

    The aims of this study were to fabricate a novel titanium/silicon carbide (Ti/SiC) metal matrix nanocomposite (MMNC) by friction stir processing (FSP) and to investigate its microstructure and mechanical properties. In addition, the adhesion, proliferation and osteogenic differentiation of rat bone marrow stromal cells (BMSCs) on the nanocomposite surface were investigated. The MMNC microstructure was observed by both scanning and transmission electron microscopy. Mechanical properties were characterized by nanoindentation and Vickers hardness testing. Integrin β1 immunofluorescence, cell adhesion, and MTT assays were used to evaluate the effects of the nanocomposite on cell adhesion and proliferation. Osteogenic and angiogenic differentiation were evaluated by alkaline phosphatase (ALP) staining, ALP activity, PCR and osteocalcin immunofluorescence. The observed microstructures and mechanical properties clearly indicated that FSP is a very effective technique for modifying Ti/SiC MMNC to contain uniformly distributed nanoparticles. In the interiors of recrystallized grains, characteristics including twins, fine recrystallized grains, and dislocations formed concurrently. Adhesion, proliferation, and osteogenic and angiogenic differentiation of rat BMSCs were all enhanced on the novel Ti/SiC MMNC surface. In conclusion, nanocomposites modified using FSP technology not only have superior mechanical properties under stress-bearing conditions but also provide improved surface and physicochemical properties for cell attachment and osseointegration.

  4. Oral bacterial extracts facilitate early osteogenic/dentinogenic differentiation in human dental pulp-derived cells.

    Science.gov (United States)

    Abe, Shu; Imaizumi, Mari; Mikami, Yoshikazu; Wada, Yoshiyuki; Tsuchiya, Shuhei; Irie, Seiko; Suzuki, Shinnosuke; Satomura, Kazuhito; Ishihara, Kazuyuki; Honda, Masaki J

    2010-01-01

    Bacterial metabolites demineralize dental hard tissues, and soluble factors lead to tertiary dentinogenesis in the area of the dentin-pulp complex. However, it is unclear whether the oral bacteria are directly involved in the differentiation of dental pulp cells. In this study, we evaluated the effect of oral bacterial extracts on cellular differentiation in human dental pulp-derived cells (hDPC). The hDPC were obtained from third molar teeth, and the cells were subcultured. The sonicated extracts were obtained from Porphyromonas gingivalis (gram-negative) and Streptococcus mutans (gram-positive). The effect of bacterial extracts on cellular growth and differentiation in hDPC were tested. Alkaline phosphatase activity and bone sialoprotein (BSP) gene expression were increased in hDPC exposed to low concentrations of both sonicated extracts, whereas the activity was decreased upon exposure to high concentrations of sonicated extracts from P. gingivalis. This is the first evidence that oral bacteria have a positive effect on cellular differentiation in hPDC. Copyright 2010 Mosby, Inc. All rights reserved.

  5. Surface modifications by gas plasma control osteogenic differentiation of MC3T3-E1 cells

    NARCIS (Netherlands)

    Barradas, A.M.C.; Lachmann, K.; Hlawacek, G.; Frielink, C.; Truckenmüller, R.K.; Boerman, O.C.; van Gastel, Raoul; Garritsen, H.S.P.; Garritsen, H.S.P.; Thomas, M.; Moroni, Lorenzo; van Blitterswijk, Clemens; de Boer, Jan

    2012-01-01

    Numerous studies have shown that the physicochemical properties of biomaterials can control cell activity. Cell adhesion, proliferation, differentiation as well as tissue formation in vivo can be tuned by properties such as the porosity, surface micro- and nanoscale topography and chemical

  6. Surface modifications by gas plasma control osteogenic differentiation of MC3T3-E1 cells.

    NARCIS (Netherlands)

    Barradas, A.M.; Lachmann, K.; Hlawacek, G.; Frielink, C.; Truckenmoller, R.; Boerman, O.C.; Gastel, R. van; Garritsen, H.; Thomas, M.; Moroni, L.; Blitterswijk, C. Van; Boer, J. den

    2012-01-01

    Numerous studies have shown that the physicochemical properties of biomaterials can control cell activity. Cell adhesion, proliferation, differentiation as well as tissue formation in vivo can be tuned by properties such as the porosity, surface micro- and nanoscale topography and chemical

  7. Effect of 1 mT sinusoidal electromagnetic fields on proliferation and osteogenic differentiation of rat bone marrow mesenchymal stromal cells.

    Science.gov (United States)

    Liu, Chaoxu; Yu, Jizhe; Yang, Yong; Tang, Xiangyu; Zhao, Dongming; Zhao, Wenchun; Wu, Hua

    2013-09-01

    Electromagnetic field (EMF) stimulation is clinically beneficial for fracture nonunion and a wide range of bone disorders. However, no consensus has been reached on the optimal parameters of the EMF. The exact mechanism by which EMFs enhance osteogenesis has also not been defined. In the present study, a sinusoidal 1 mT EMF at frequencies of 10, 30, 50, and 70 Hz were administered to rat bone marrow mesenchymal stromal cells (rBMSCs) in the cyclic mode of 2 h exposures followed by 4 h of culture without exposure. The cell viability, proliferation, expression of some osteogenic genes, and mineralization of the extracellular matrix were investigated. It was found that the cell viability was decreased by EMF exposures of 50 and 70 Hz. The proliferation of rBMSCs was elevated significantly in the 10 Hz EMF-treated group during the culture periods. The expression of alkaline phosphatase (ALP) and osteocalcin (OC), two early-phase osteogenic differentiation markers, was up-regulated by the 1 mT, 10 Hz EMF after 1 week. However, the expression of genes that marked the later-phase osteogenic differentiation and maturation of osteoblasts was elevated by the stimulation of 50 Hz EMFs after 2 weeks. In addition, it was observed that the mineralization of the extracellular matrix was enhanced by 50 Hz EMF exposure. These results indicated that the 1 mT EMF at different frequencies had disparate effects on the viability, proliferation and osteogenic differentiation of rBMSCs, and may be beneficial for developing novel therapeutic approaches in bone regenerative medicine. Copyright © 2013 Wiley Periodicals, Inc.

  8. Effect of surface modification of nanofibres with glutamic acid peptide on calcium phosphate nucleation and osteogenic differentiation of marrow stromal cells.

    Science.gov (United States)

    Karaman, Ozan; Kumar, Ankur; Moeinzadeh, Seyedsina; He, Xuezhong; Cui, Tong; Jabbari, Esmaiel

    2016-02-01

    Biomineralization is mediated by extracellular matrix (ECM) proteins with amino acid sequences rich in glutamic acid. The objective of this study was to investigate the effect of calcium phosphate deposition on aligned nanofibres surface-modified with a glutamic acid peptide on osteogenic differentiation of rat marrow stromal cells. Blend of EEGGC peptide (GLU) conjugated low molecular weight polylactide (PLA) and high molecular weight poly(lactide-co-glycolide) (PLGA) was electrospun to form aligned nanofibres (GLU-NF). The GLU-NF microsheets were incubated in a modified simulated body fluid for nucleation of calcium phosphate crystals on the fibre surface. To achieve a high calcium phosphate to fibre ratio, a layer-by-layer approach was used to improve diffusion of calcium and phosphate ions inside the microsheets. Based on dissipative particle dynamics simulation of PLGA/PLA-GLU fibres, > 80% of GLU peptide was localized to the fibre surface. Calcium phosphate to fibre ratios as high as 200%, between those of cancellous (160%) and cortical (310%) bone, was obtained with the layer-by-layer approach. The extent of osteogenic differentiation and mineralization of marrow stromal cells seeded on GLU-NF microsheets was directly related to the amount of calcium phosphate deposition on the fibres prior to cell seeding. Expression of osteogenic markers osteopontin, alkaline phosphatase (ALP), osteocalcin and type 1 collagen increased gradually with calcium phosphate deposition on GLU-NF microsheets. Results demonstrate that surface modification of aligned synthetic nanofibres with EEGGC peptide dramatically affects nucleation and growth of calcium phosphate crystals on the fibres leading to increased osteogenic differentiation of marrow stromal cells and mineralization. Copyright © 2013 John Wiley & Sons, Ltd.

  9. Endoplasmic reticulum (ER stress inducible factor cysteine-rich with EGF-like domains 2 (Creld2 is an important mediator of BMP9-regulated osteogenic differentiation of mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Jiye Zhang

    Full Text Available Mesenchymal stem cells (MSCs are multipotent progenitors that can undergo osteogenic differentiation under proper stimuli. We demonstrated that BMP9 is one of the most osteogenic BMPs. However, the molecular mechanism underlying BMP9-initiated osteogenic signaling in MSCs remains unclear. Through gene expression profiling analysis we identified several candidate mediators of BMP9 osteogenic signaling. Here, we focus on one such signaling mediator and investigate the functional role of cysteine-rich with EGF-like domains 2 (Creld2 in BMP9-initiated osteogenic signaling. Creld2 was originally identified as an ER stress-inducible factor localized in the ER-Golgi apparatus. Our genomewide expression profiling analysis indicates that Creld2 is among the top up-regulated genes in BMP9-stimulated MSCs. We confirm that Creld2 is up-regulated by BMP9 in MSCs. ChIP analysis indicates that Smad1/5/8 directly binds to the Creld2 promoter in a BMP9-dependent fashion. Exogenous expression of Creld2 in MSCs potentiates BMP9-induced early and late osteogenic markers, and matrix mineralization. Conversely, silencing Creld2 expression inhibits BMP9-induced osteogenic differentiation. In vivo stem cell implantation assay reveals that exogenous Creld2 promotes BMP9-induced ectopic bone formation and matrix mineralization, whereas silencing Creld2 expression diminishes BMP9-induced bone formation and matrix mineralization. We further show that Creld2 is localized in ER and the ER stress inducers potentiate BMP9-induced osteogenic differentiation. Our results strongly suggest that Creld2 may be directly regulated by BMP9 and ER stress response may play an important role in regulating osteogenic differentiation.

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

    Science.gov (United States)

    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

  11. Induction of Osteogenic Differentiation of Human Adipose-Derived Stem Cells by a Novel Self-Supporting Graphene Hydrogel Film and the Possible Underlying Mechanism.

    Science.gov (United States)

    Lyu, Cheng-Qi; Lu, Jia-Yu; Cao, Chun-Hua; Luo, Deng; Fu, Yin-Xin; He, Yu-Shi; Zou, De-Rong

    2015-09-16

    Graphene and its derivatives have received increasing attention from scientists in the field of biomedical sciences because of their unique physical properties, which are responsible for their interesting biological functions. With a range of extraordinary properties such as high surface area, high mechanical strength, and ease of functionalization, graphene is considered highly promising for application in bone tissue engineering. Here, we examined the effect of using a self-supporting graphene hydrogel (SGH) film to induce the osteogenic differentiation of human adipose-derived stem cells (hADSCs). In comparison to conventional graphene and carbon fiber films, the SGH film had higher mechanical strength and flexibility. Moreover, we found that the SGH film was nontoxic and biocompatible. Of particular interest is the fact that the film alone could stimulate the osteogenic differentiation of hADSCs, independent of additional chemical inducers. Such effects are stronger for the SGH film than for graphene or carbon fiber films, although the induction capacity of the SGH film is not as high as that of the osteogenic-induced medium. The excellent osteoinductivity of the SGH film is closely related to its remarkable physical properties that include specific nanostructures, surface morphology, strong cell adherence, reasonable surface hydrophilicity, and high protein absorption.

  12. Biomimetic hybrid scaffolds for osteo-chondral tissue repair: Design and osteogenic differentiation of human placenta-derived cells (hPDC).

    Science.gov (United States)

    Farè, Silvia; Bertoldi, Serena; Meskinfam, Masoumeh; Spoldi, Valentina; Tanzi, M Cristina; Parolini, Ornella

    2015-08-01

    A novel functionally-graded hybrid (FGHY) scaffold was designed and developed with a load-bearing structure represented by a PU foam loaded with a graded composition of CaPs (biomimetic component) and pectin gel as cell carrier. hPDC populations encapsulated in pectin gels and injected into the FGHY scaffolds demonstrated the ability to differentiate toward the osteogenic lineage. The ability of these biomimetic hybrid scaffolds to stimulate cell adhesion and proliferation and to support differentiation of hPDCs make these scaffolds excellent candidates for an use in bone regeneration.

  13. Dental pulp-derived stromal cells exhibit a higher osteogenic potency than bone marrow-derived stromal cells in vitro and in a porcine critical-size bone defect model

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    Jensen Jonas

    2016-01-01

    Full Text Available Introduction: The osteogenic differentiation of bone marrow-derived mesenchymal stromal cells (BMSCs was compared with that of dental pulp-derived stromal cells (DPSCs in vitro and in a pig calvaria critical-size bone defect model. Methods: BMSCs and DPSCs were extracted from the tibia bone marrow and the molar teeth of each pig, respectively. BMSCs and DPSCs were cultured in monolayer and on a three-dimensional (3D polycaprolactone (PCL – hyaluronic acid – tricalcium phosphate (HT-PCL scaffold. Population doubling (PD, alkaline phosphatase (ALP activity, and calcium deposition were measured in monolayer. In the 3D culture ALP activity, DNA content, and calcium deposition were evaluated. Six non-penetrating critical-size defects were made in each calvarium of 14 pigs. Three paired sub-studies were conducted: (1 empty defects vs. HT-PCL scaffolds; (2 PCL scaffolds vs. HT-PCL scaffolds; and (3 autologous BMSCs on HT-PCL scaffolds vs. autologous DPSCs on HT-PCL scaffolds. The observation time was five weeks. Bone volume fractions (BV/TV were assessed with micro-computed tomography (μCT and histomorphometry. Results and discussion: The results from the in vitro study revealed a higher ALP activity and calcium deposition of the DPSC cultures compared with BMSC cultures. Significantly more bone was present in the HT-PCL group than in both the pure PCL scaffold group and the empty defect group in vivo. DPSCs generated more bone than BMSCs when seeded on HT-PCL. In conclusion, DPSCs exhibited a higher osteogenic potential compared with BMSCs both in vitro and in vivo, making it a potential cell source for future bone tissue engineering.

  14. Synthesis and characterization of nanosized calcium phosphates by flame spray pyrolysis, and their effect on osteogenic differentiation of stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Ataol, Sibel; Tezcaner, Ayşen [Middle East Technical University, Department of Biomedical Engineering (Turkey); Duygulu, Ozgur [TUBITAK Marmara Research Center, Materials Institute (Turkey); Keskin, Dilek [Middle East Technical University, Department of Biomedical Engineering (Turkey); Machin, Nesrin E., E-mail: nesrinmachin@gmail.com [Kocaeli University, Department of Chemical Engineering (Turkey)

    2015-02-15

    The present study evaluates the synthesis of biocompatible osteoconductive and osteoinductive nano calcium phosphate (CaP) particles by industrially applied, aerosol-derived flame spray pyrolysis method for biomedical field. Calcium phosphate nanoparticles were produced in a range of calcium-to-phosphorus ratio, (1.20–2.19) in order to analyze the morphology and crystallinity changes, and to test the bioactivity of particles. The characterization results confirmed that nanometer-sized, spherical calcium phosphate particles were produced. The average primary particle size was determined as 23 nm by counting more than 500 particles in TEM pictures. XRD patterns, HRTEM, SAED, and SEM analyses revealed the amorphous nature of the as-prepared nano calcium phosphate particles at low Ca/P ratios. Increases in the specific surface area and crystallinity were observed with the increasing Ca/P ratio. TGA–DTA analysis showed that the thermally stable crystal phases formed after 700 °C. Cell culture studies were conducted with urine-derived stem cells that possess the characteristics of mesenchymal stem cells. Synthesized amorphous nanoparticles did not have cytotoxic effect at 5–50 μg/ml concentration range. Cells treated with the as-prepared nanoparticles had higher alkaline phosphatase (ALP) enzyme activity than control cells, indicating osteogenic differentiation of cells. A slight decrease in ALP activity of cells treated with two highest Ca:P ratios at 50 μg/ml concentration was observed at day 7. The findings suggest that calcium phosphate nanoparticles produced in this work have a potential to be used as biomaterials in biomedical applications.

  15. MGF E peptide pretreatment improves the proliferation and osteogenic differentiation of BMSCs via MEK-ERK1/2 and PI3K-Akt pathway under severe hypoxia.

    Science.gov (United States)

    Sha, Yongqiang; Lv, Yonggang; Xu, Zhiling; Yang, Li; Hao, Xiaoying; Afandi, Ruli

    2017-11-15

    Severe hypoxia always inhibits the cell proliferation, osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs), and hinders bone defect repair. Herein we explored the effects of mechano-growth factor (MGF) E peptide on the proliferation and osteogenic differentiation of BMSCs under severe hypoxia. CoCl2 was utilized to simulate severe hypoxia. MTS was used to detect cell viability. Cell proliferation was verified through flow cytometry and EdU assay. Osteogenic differentiation of BMSCs and osteoblast-specific genes were detected through alkaline phosphatase (ALP) and Alizarin Red S staining, and quantitative real-time PCR, respectively. Hypoxia-inducible factor 1α (HIF-1α), p-ERK1/2 and p-Akt expression levels were detected through western blotting and immunofluorescence. Severe hypoxia induced HIF-1α accumulation and transferring into the nucleus, and reduced cell proliferation and osteogenic differentiation of BMSCs. The expression levels of osteoblast-specific genes were markedly decreased after differentiation culture for 0, 7 or 14days. Fortunately, MGF E peptide inhibited HIF-1α expression and transferring into the nucleus. Cell proliferation and osteogenic differentiation of BMSCs could be recovered by MGF E peptide pretreatment. MEK-ERK1/2 and PI3K-Akt signaling pathway were confirmed to be involved in MGF E peptide regulating the abovementioned indexes of BMSCs. What's more, short-time treatment with MGF E peptide alone promoted the osteogenic differentiation of BMSCs as well. Our study provides new evidence for the role of MGF E peptide in regulating proliferation and osteogenic differentiation of BMSCs under severe hypoxia, which may potentially have therapeutic implication for bone defect repair. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Osteogenic differentiation of MC3T3-E1 cells on poly(L-lactide)/Fe{sub 3}O{sub 4} nanofibers with static magnetic field exposure

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Qing [State Key Laboratory of Organic–inorganic Composites, Beijing University of Chemical Technology, Beijing 100029 (China); Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing 100029 (China); Shi, Yuzhou; Shan, Dingying; Jia, Wenkai; Duan, Shun [Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing 100029 (China); Deng, Xuliang [Department of Geriatric Dentistry, Peking University School and Hospital of Stomatology, Beijing 100081 (China); Yang, Xiaoping, E-mail: yangxp@mail.buct.edu.cn [State Key Laboratory of Organic–inorganic Composites, Beijing University of Chemical Technology, Beijing 100029 (China); Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing 100029 (China)

    2015-10-01

    Proliferation and differentiation of bone-related cells are modulated by many factors such as scaffold design, growth factor, dynamic culture system, and physical simulation. Nanofibrous structure and moderate-intensity (1 mT–1 T) static magnetic field (SMF) have been identified as capable of stimulating proliferation and differentiation of osteoblasts. Herein, magnetic nanofibers were prepared by electrospinning mixture solutions of poly(L-lactide) (PLLA) and ferromagnetic Fe{sub 3}O{sub 4} nanoparticles (NPs). The PLLA/Fe{sub 3}O{sub 4} composite nanofibers demonstrated homogeneous dispersion of Fe{sub 3}O{sub 4} NPs, and their magnetism depended on the contents of Fe{sub 3}O{sub 4} NPs. SMF of 100 mT was applied in the culture of MC3T3-E1 osteoblasts on pure PLLA and PLLA/Fe{sub 3}O{sub 4} composite nanofibers for the purpose of studying the effect of SMF on osteogenic differentiation of osteoblastic cells on magnetic nanofibrous scaffolds. On non-magnetic PLLA nanofibers, the application of external SMF could enhance the proliferation and osteogenic differentiation of MC3T3-E1 cells. In comparison with pure PLLA nanofibers, the incorporation of Fe{sub 3}O{sub 4} NPs could also promote the proliferation and osteogenic differentiation of MC3T3-E1 cells in the absence or presence of external SMF. The marriage of magnetic nanofibers and external SMF was found most effective in accelerating every aspect of biological behaviors of MC3T3-E1 osteoblasts. The findings demonstrated that the magnetic feature of substrate and microenvironment were applicable ways in regulating osteogenesis in bone tissue engineering. - Highlights: • Magnetic nanofibers containing well-dispersed Fe{sub 3}O{sub 4} nanoparticles were produced. • Static magnetic field (SMF) was applied to perform the culture of osteoblasts. • Osteogenic differentiation was enhanced on magnetic substrate with exposure to SMF.

  17. MiR-27a is Essential for the Shift from Osteogenic Differentiation to Adipogenic Differentiation of Mesenchymal Stem Cells in Postmenopausal Osteoporosis

    Directory of Open Access Journals (Sweden)

    Li You

    2016-06-01

    enhancer factor 2 c (Mef2c, a transcription factor, was the direct target of miR-27a using a dual luciferase assay. An inverse relationship between miR-27a expression and Mef2c expression in osteoporotic patients was shown. Silencing of miR-27a decreased bone formation, confirming the role of miR-27a in bone formation in vivo. Conclusion: In summary, miR-27a was essential for the shift of MSCs from osteogenic differentiation to adipogenic differentiation in osteoporosis by targeting Mef2c.

  18. MiR-27a is Essential for the Shift from Osteogenic Differentiation to Adipogenic Differentiation of Mesenchymal Stem Cells in Postmenopausal Osteoporosis.

    Science.gov (United States)

    You, Li; Pan, Ling; Chen, Lin; Gu, Wensha; Chen, Jinyu

    2016-01-01

    , was the direct target of miR-27a using a dual luciferase assay. An inverse relationship between miR-27a expression and Mef2c expression in osteoporotic patients was shown. Silencing of miR-27a decreased bone formation, confirming the role of miR-27a in bone formation in vivo. In summary, miR-27a was essential for the shift of MSCs from osteogenic differentiation to adipogenic differentiation in osteoporosis by targeting Mef2c. © 2016 S. Karger AG, Basel.

  19. LncRNA MEG3 inhibited osteogenic differentiation of bone marrow mesenchymal stem cells from postmenopausal osteoporosis by targeting miR-133a-3p.

    Science.gov (United States)

    Wang, Qiujun; Li, Ying; Zhang, Yuanxia; Ma, Lan; Lin, Lin; Meng, Jia; Jiang, Lihong; Wang, Liping; Zhou, Ping; Zhang, Yina

    2017-05-01

    Long non-coding RNA (lncRNA) MEG3 has proven to be an important regulator involved in the pathogenesis and development of various human diseases. However, the functional involvement of MEG3 in postmenopausal osteoporosis (PMOP) and its mechanism is still unclear. Bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured from mouse pathologic models and patients with PMOP, respectively. The expression of MEG3 and miR-133a-3p in BMSCs was detected using qRT-PCR. The recombinant expression vector was constructed and transfected into BMSCs to regulate the endogenous expression of MEG3 and miR-133a-3p. The mineralized nodules formation, alkaline phosphatase (ALP) activity and Runx2, OCN, OPN expressions were used as specific markers for the differentiation of osteoblasts. The expressions of MEG3 and miR-133a-3p in BMSCs from PMOP were increased, and there was a positive correlation between MEG3 and miR-133a-3p expression in BMSCs. In the differentiation process from BMSCs to osteoblasts, the expressions of MEG3 and miR-133a-3p were markedly decreased, and MEG3 overexpression reversed the osteogenic induction-mediated downregulation of miR-133a-3p, which was accompanied by significant decline in SLC39A1 expression. Furthermore, miR-133a-3p silencing or upregulation eliminated the effects of MEG3 on the osteogenic differentiation of BMSCs through direct binding. The research indicated that MEG3 regulated the expression of miR-133a-3p, and inhibited the osteogenic differentiation of BMSCs induced PMOP. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  20. Porcine brain extract promotes osteogenic differentiation of bone marrow derived mesenchymal stem cells and bone consolidation in a rat distraction osteogenesis model.

    Directory of Open Access Journals (Sweden)

    Jia Xu

    Full Text Available Distraction osteogenesis (DO is the gold standard to treat large bone defects, but long consolidation period is a major limitation. Innovative efforts to promote osteogenesis are needed. Porcine brain extract (PBE was reported to enhance the proliferation and differentiation of multiple primary cells. In this study, we aimed to develop a method for collecting PBE and investigate its effects on osteogenic differentiation of rat bone marrow derived mesenchymal stem cells (rBMSCs and bone consolidation in a rat DO model. The PBE was collected from neonatal brain tissues of porcine fetus and was used to treat rBMSCs. Following PBE treatment (700 ng/ml, osteogenic differentiation was assessed. Further, we locally injected PBE (7 μg/ml, 100μl or PBS (100μl into the gap in a Sprague-Dawley (SD male rat DO model every three days till termination. X-rays, micro-computed tomography, mechanical testing, histology and immunohischemistry examinations were used to exam the quality of the regenerates. The alkaline phosphatase, calcium deposits, and steogenic markers in the PBE treated rBMSCs were significantly increased. In the rat model, new bone properties of bone volume/total tissue volume and mechanical strength were higher in the PBE treated group. Histological analysis also confirmed more mineralized bone after PBE treatment. The current study reports a standard protocol for PBE collection and demonstrated its positive effects on osteogenic differentiation and bone consolidation in DO. Since the PBE is readily available and very cost effective, PBE may be a potential new bio-source to promote bone formation in patients undergo DO treatment.

  1. Role of human amnion-derived mesenchymal stem cells in promoting osteogenic differentiation by influencing p38 MAPK signaling in lipopolysaccharide -induced human bone marrow mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yuli; Wu, Hongxia; Shen, Ming; Ding, Siyang; Miao, Jing; Chen, Ning, E-mail: 2927410849@qq.com

    2017-01-01

    Periodontitis is a chronic inflammatory disease induced by bacterial pathogens, which not only affect connective tissue attachments but also cause alveolar bone loss. In this study, we investigated the anti-inflammatory effects of Human amnion-derived mesenchymal stem cells (HAMSCs) on human bone marrow mesenchymal stem cells (HBMSCs) under lipopolysaccharide (LPS)-induced inflammatory conditions. Proliferation levels were measured by flow cytometry and immunofluorescence staining of 5-ethynyl-2′-deoxyuridine (EdU). Osteoblastic differentiation and mineralization were investigated using chromogenic alkaline phosphatase activity (ALP) activity substrate assays, Alizarin red S staining, and RT-PCR analysis of HBMSCs osteogenic marker expression. Oxidative stress induced by LPS was investigated by assaying reactive oxygen species (ROS) level and superoxide dismutase (SOD) activity. Here, we demonstrated that HAMSCs increased the proliferation, osteoblastic differentiation, and SOD activity of LPS-induced HBMSCs, and down-regulated the ROS level. Moreover, our results suggested that the activation of p38 MAPK signal transduction pathway is essential for reversing the LPS-induced bone-destructive processes. SB203580, a selective inhibitor of p38 MAPK signaling, significantly suppressed the anti-inflammatory effects in HAMSCs. In conclusion, HAMSCs show a strong potential in treating inflammation-induced bone loss by influencing p38 MAPK signaling. - Highlights: • LPS inhibites osteogenic differentiation in HBMSCs via suppression of p38 MAPK signaling pathway. • HAMSCs promote LPS-induced HBMSCs osteogenic differentiation through p38 MAPK signaling pathway. • HAMSCs reverse LPS-induced oxidative stress in LPS-induced HBMSCs through p38 MAPK signaling pathway.

  2. Molecular and cellular characterization of SEL-OB/SVEP1 in osteogenic cells in vivo and in vitro.

    Science.gov (United States)

    Shur, I; Socher, R; Hameiri, M; Fried, A; Benayahu, D

    2006-02-01

    We describe a novel human gene, named SEL-OB/SVEP1, expressed by skeletal tissues in vivo and by cultured osteogenic cells. The mRNA expression was analyzed on frozen tissues retrieved by laser-capture microscope dissection (LCM) and was detected in osteogenic tissues (periosteum and bone) but not in cartilage or skeletal muscle. The SEL-OB/SVEP1 cDNA of 11,139 bp was in silico translated into a 3574AA protein with expected molecular weight of 370 kDa. The protein is composed of multiple domains including complement control protein (CCP) modules with selectin superfamily signature; sushi and other domains, such as vWA, EGF, PTX, and HYR. Stromal osteogenic cells were analyzed for the protein expression using anti-SEL-OB/SVEP1 for immuno-precipitation and Western blot application confirm the presence of high molecular weight protein. Immuno-histochemistry and fluorescence-activated cell sorting (FACS) were applied to detect SEL-OB/SVEP1 on the surface of stromal cells. ELISA quantified the dependence of protein expression on cell density. Bioinformatic analysis of SEL-OB/SVEP1 revealed domains compositions recognized in cell surface molecules and suggested its role in cell adhesion. Analysis of mesechymal osteogenic cells' adhesion in presence of anti-SEL-OB/SVEP1 antibody demonstrated its interference with initial adhesion stages. In summary, present study describes novel SEL-OB/SVEP1 protein with a unique composition of functional domains, restricted pattern of expression in skeletal cells and demonstrated involvement in attachment of mesenchymal cells. The unusual composition of functional domains puts SEL-OB/SVEP1 in the discrete new group of membrane proteins involved in cell adhesion processes. All together makes SEL-OB/SVEP1 an attractive marker for studying the role of stromal osteogenic cells and their interactions within the bone marrow microenvironment creating a network that regulates the skeletal homeostasis. Copyright (c) 2005 Wiley-Liss, Inc.

  3. Effect of calcium phosphate particle shape and size on their antibacterial and osteogenic activity in the delivery of antibiotics in vitro.

    Science.gov (United States)

    Uskoković, Vuk; Batarni, Samir Shariff; Schweicher, Julien; King, Andrew; Desai, Tejal A

    2013-04-10

    Powders composed of four morphologically different calcium phosphate particles were prepared by precipitation from aqueous solutions: flaky, brick-like, elongated orthogonal, and spherical. The particles were then loaded with either clindamycin phosphate as the antibiotic of choice or fluorescein, a model molecule used to assess the drug release properties. A comparison was carried out of the effect of such antibiotic-releasing materials on: sustained drug release profiles; Staphylococcus aureus growth inhibition; and osteogenic propensities in vitro. Raman spectroscopic analysis indicated the presence of various calcium phosphate phases, including monetite (flaky and elongated orthogonal particles), octacalcium phosphate (brick-shaped particles), and hydroxyapatite (spherical particles). Testing the antibiotic-loaded calcium phosphate powders for bacterial growth inhibition demonstrated satisfying antibacterial properties both in broths and on agar plates. All four calcium-phosphate-fluorescein powders exhibited sustained drug release over 21 days. The calcium phosphate sample with the highest specific surface area and the smallest, spherical particle size was the most effective in both drug loading and release, consequently having the highest antibacterial efficiency. Moreover, the highest cell viability, the largest gene expression upregulation of three different osteogenic markers--osteocalcin, osteopontin, and Runx2--as well as the least disrupted cell cytoskeleton and cell morphologies were also noticed for the calcium phosphate powder composed of the smallest, spherical nanosized particles. Still, all four powders exerted a viable effect on osteoblastic MC3T3-E1 cells in vitro, as evidenced by both morphological assessments on fluorescently stained cells and measurements of their mitochondrial activity. The obtained results suggest that the nanoscale particle size and the corresponding coarseness of the surface of particle conglomerates as the cell

  4. Benefits of biphasic calcium phosphate hybrid scaffold-driven osteogenic differentiation of mesenchymal stem cells through upregulated leptin receptor expression.

    Science.gov (United States)

    Niu, Chi-Chien; Lin, Song-Shu; Chen, Wen-Jer; Liu, Shih-Jung; Chen, Lih-Huei; Yang, Chuen-Yung; Wang, Chao-Jan; Yuan, Li-Jen; Chen, Po-Han; Cheng, Hsiao-Yang

    2015-07-16

    The use of mesenchymal stem cells (MSCs) and coralline hydroxyapatite (HA) or biphasic calcium phosphate (BCP) as a bone substitute for posterolateral spinal fusion has been reported. However, the genes and molecular signals by which MSCs interact with their surrounding environment require further elucidation. MSCs were harvested from bone grafting patients and identified by flow cytometry. A composite scaffold was developed using poly(lactide-co-glycolide) (PLGA) copolymer, coralline HA, BCP, and collagen as a carrier matrix for MSCs. The gene expression profiles of MSCs cultured in the scaffolds were measured by microarrays. The alkaline phosphatase (ALP) activity of the MSCs was assessed, and the expression of osteogenic genes and proteins was determined by quantitative polymerase chain reaction (Q-PCR) and Western blotting. Furthermore, we cultured rabbit MSCs in BCP or coralline HA hybrid scaffolds and transplanted these mixtures into rabbits for spinal fusion. We investigated the differences between BCP and coralline HA hybrid scaffolds by dual-energy X-ray absorptiometry (DEXA) and computed tomography (CT). Tested in vitro, the cells were negative for hematopoietic cell markers and positive for MSC markers. There was higher expression of 80 genes and lower of 101 genes of MSCs cultured in BCP hybrid scaffolds. Some of these genes have been shown to play a role in osteogenesis of MSCs. In addition, MSCs cultured in BCP hybrid scaffolds produced more messenger RNA (mRNA) for osteopontin, osteocalcin, Runx2, and leptin receptor (leptin-R) than those cultured in coralline HA hybrid scaffolds. Western blotting showed more Runx2 and leptin-R protein expression in BCP hybrid scaffolds. For in vivo results, 3D reconstructed CT images showed continuous bone bridges and fusion mass incorporated with the transverse processes. Bone mineral content (BMC) values were higher in the BCP hybrid scaffold group than in the coralline HA hybrid scaffold group. The BCP hybrid

  5. Metformin Decreases Reactive Oxygen Species, Enhances Osteogenic Properties of Adipose-Derived Multipotent Mesenchymal Stem Cells In Vitro, and Increases Bone Density In Vivo

    Directory of Open Access Journals (Sweden)

    Krzysztof Marycz

    2016-01-01

    Full Text Available Due to its pleiotropic effects, the commonly used drug metformin has gained renewed interest among medical researchers. While metformin is mainly used for the treatment of diabetes, recent studies suggest that it may have further application in anticancer and antiaging therapies. In this study, we investigated the proliferative potential, accumulation of oxidative stress factors, and osteogenic and adipogenic differentiation potential of mouse adipose-derived stem cells (MuASCs isolated from mice treated with metformin for 8 weeks. Moreover, we investigated the influence of metformin supplementation on mice bone density and bone element composition. The ASCs isolated from mice who were treated with metformin for 8 weeks showed highest proliferative potential, generated a robust net of cytoskeletal projections, had reduced expression of markers associated with cellular senescence, and decreased amount of reactive oxygen species in comparison to control group. Furthermore, we demonstrated that these cells possessed greatest osteogenic differentiation potential, while their adipogenic differentiation ability was reduced. We also demonstrated that metformin supplementation increases bone density in vivo. Our result stands as a valuable source of data regarding the in vivo influence of metformin on ASCs and bone density and supports a role for metformin in regenerative medicine.

  6. Incorporation of Cerium Oxide into Hydroxyapatite Coating Protects Bone Marrow Stromal Cells Against H2O2-Induced Inhibition of Osteogenic Differentiation.

    Science.gov (United States)

    Li, Kai; Shen, Qingyi; Xie, Youtao; You, Mingyu; Huang, Liping; Zheng, Xuebin

    2018-03-01

    Oxidative stress exerts a key influence in osteoporosis in part by inhibiting osteogenic differentiation of bone marrow stromal cells (BMSCs). With their unique antioxidant properties and reported biocompatibility, cerium oxide (CeO 2 ) ceramics exhibit promising potential for the treatment of osteoporosis resulting from oxidative stress. In this study, protective effects of CeO 2 -incorporated hydroxyapatite coatings (HA-10Ce and HA-30Ce) on the viability and osteogenic differentiation of H 2 O 2 -treated BMSCs were examined. CeO 2 -incorporated HA coatings enhanced cell viability and attenuated cell apoptosis caused by H 2 O 2 . An increase in CeO 2 content in HA coatings better alleviated H 2 O 2 -induced inhibition of osteogenic differentiation by increasing alkaline phosphatase (ALP) activity, calcium deposition activity, and mRNA expression levels of osteogenesis markers runt-related transcription factor 2 (Runx2), ALP, and osteocalcin (OCN) in BMSCs. Furthermore, the H 2 O 2 -induced decrease of gene and protein expressions of β-catenin and cyclin D1 in the Wnt/β-catenin signaling pathway was successfully rescued by the CeO 2 incorporated HA coatings. Besides, the decreased expression of receptor activator of nuclear factor kappa-B ligand (RANKL) and the increased ratio of osteoprotegerin (OPG)/RANKL in BMSCs on the CeO 2 -modified coatings was observed, indicating the inhibition of osteoclastogenesis. The above results were mediated by the antioxidant properties of CeO 2 . The CeO 2 -incorporated HA coatings reversed the decreased superoxide dismutase (SOD) activity, reduced reactive oxygen species (ROS) generation, and suppressed the malondiadehyde (MDA) formation. The findings suggested that CeO 2 -modified HA coatings may be promising coating materials for osteoporotic bone regeneration.

  7. The Effect of the Nanoscale Structure of Nanobioceramics on Their In Vitro Bioactivity and Cell Differentiation Properties

    Directory of Open Access Journals (Sweden)

    Cristian Covarrubias

    2015-01-01

    Full Text Available The effect of the nanoscale structure of bioceramics on their in vitro bioactivity and capacity to osteogenically differentiate stem cell is studied. Nanoparticles of hydroxyapatite (nHA, bioactive glass (nBG, nanoporous bioactive glass (MBG, and nanoporous bioactive glass nanospheres (nMBG are investigated. The nanometric particle size of bioceramics seems to be more determining in controlling the ability to induce bone-like apatite as compared to the nanoporous structure. At short incubation time, nBG also produces a bioactive extracellular medium capable of upregulating key osteogenic markers involved in the development of a mineralizing phenotype in DPSCs. The bioactive properties of nBG are promissory for accelerating the bone regeneration process in tissue engineering applications.

  8. L-type calcium channels play a crucial role in the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Wen, Li [Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi' an 710032 (China); Wang, Yu [Department of Oncology, Xijing Hospital, Fourth Military Medical University, Xi' an 710032 (China); Wang, Huan [Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi' an 710032 (China); Kong, Lingmin [Department of Fundamental Medicine, Cell Engineering Research Centre, Fourth Military Medical University, Xi' an 710032 (China); Zhang, Liang [Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi' an 710032 (China); Chen, Xin [Department of General Dentistry, The 174th Hospital of Chinese PLA, Xiamen 361003 (China); Ding, Yin, E-mail: dingyin@fmmu.edu.cn [Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi' an 710032 (China)

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer We detect the functional Ca{sup 2+} currents and mRNA expression of VDCC{sub L} in rMSCs. Black-Right-Pointing-Pointer Blockage of VDCC{sub L} exert antiproliferative and apoptosis-inducing effects on rMSCs. Black-Right-Pointing-Pointer Inhibiting VDCC{sub L} can suppress the ability of rMSCs to differentiate into osteoblasts. Black-Right-Pointing-Pointer {alpha}1C of VDCC{sub L} may be a primary functional subunit in VDCC{sub L}-regulating rMSCs. -- Abstract: L-type voltage-dependent Ca{sup 2+} channels (VDCC{sub L}) play an important role in the maintenance of intracellular calcium homeostasis, and influence multiple cellular processes. They have been confirmed to contribute to the functional activities of osteoblasts. Recently, VDCC{sub L} expression was reported in mesenchymal stem cells (MSCs), but the role of VDCC{sub L} in MSCs is still undetermined. The aim of this study was to determine whether VDCC{sub L} may be regarded as a new regulator in the proliferation and osteogenic differentiation of rat MSC (rMSCs). In this study, we examined functional Ca{sup 2+} currents (I{sub Ca}) and mRNA expression of VDCC{sub L} in rMSCs, and then suppressed VDCC{sub L} using nifedipine (Nif), a VDCC{sub L} blocker, to investigate its role in rMSCs. The proliferation and osteogenic differentiation of MSCs were analyzed by MTT, flow cytometry, alkaline phosphatase (ALP), Alizarin Red S staining, RT-PCR, and real-time PCR assays. We found that Nif exerts antiproliferative and apoptosis-inducing effects on rMSCs. ALP activity and mineralized nodules were significantly decreased after Nif treatment. Moreover, the mRNA levels of the osteogenic markers, osteocalcin (OCN), bone sialoprotein (BSP), and runt-related transcription factor 2 (Runx2), were also down-regulated. In addition, we transfected {alpha}1C-siRNA into the cells to further confirm the role of VDCC{sub L} in rMSCs, and a similar effect on osteogenesis was found. These

  9. Enhanced growth and osteogenic differentiation of human osteoblast-like cells on boron-doped nanocrystalline diamond thin films.

    Directory of Open Access Journals (Sweden)

    Lubica Grausova

    Full Text Available 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 CH(4:H(2 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/cm(2, respectively, compared to 113,000 ± 10,000 cells/cm(2 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.

  10. Assessment of Effects of Si-Ca-P Biphasic Ceramic on the Osteogenic Differentiation of a Population of Multipotent Adult Human Stem Cells

    Directory of Open Access Journals (Sweden)

    Patricia Ros-Tárraga

    2016-11-01

    Full Text Available A new type of bioceramic with osteogenic properties, suitable for hard tissue regeneration, was synthesised. The ceramic was designed and obtained in the Nurse’s A-phase-silicocarnotite subsystem. The selected composition was that corresponding to the eutectoid 28.39 wt % Nurse’s A-phase-71.61 wt % silicocarnotite invariant point. We report the effect of Nurse’s A-phase-silicocarnotite ceramic on the capacity of multipotent adult human mesenchymal stem cells (ahMSCs cultured under experimental conditions, known to adhere, proliferate and differentiate into osteoblast lineage cells. The results at long-term culture (28 days on the material confirmed that the undifferentiated ahMSCs cultured and in contact with the material surface adhered, spread, proliferated, and produced a mineralised extracellular matrix on the studied ceramic, and finally acquired an osteoblastic phenotype. These findings indicate that it underwent an osteoblast differentiation process. All these findings were more significant than when cells were grown on plastic, in the presence and absence of this osteogenic supplement, and were more evident when this supplement was present in the growth medium (GM. The ceramic evaluated herein was bioactive, cytocompatible and capable of promoting the proliferation and differentiation of undifferentiated ahMSCs into osteoblasts, which may be important for bone integration into the clinical setting.

  11. Osteogenic differentiation of umbilical cord and adipose derived stem cells onto highly porous 45S5 Bioglass®-based scaffolds.

    Science.gov (United States)

    Detsch, Rainer; Alles, Sonja; Hum, Jasmin; Westenberger, Peter; Sieker, Frank; Heusinger, Dominik; Kasper, Cornelia; Boccaccini, Aldo R

    2015-03-01

    In the context of bone tissue engineering (BTE), combinations of bioactive scaffolds with living cells are investigated to optimally yield functional bone tissue for implantation purposes. Bioactive glasses are a class of highly bioactive, inorganic materials with broad application potential in BTE strategies. The aim of this study was to evaluate bioactive glass (45S5 Bioglass(®)) samples of composition: 45 SiO2, 24.5 CaO, 24.5 Na2O, and 6 P2O5 (in wt%) as scaffold materials for mesenchymal stem cells (MSC). Pore architecture of the scaffolds as well as cell behavior in the three-dimensional environment was evaluated by several methods. Investigations concerned the osteogenic cell attachment, growth and differentiation of adipose tissue derived MSC (adMSC) compared with MSC from human full term umbilical cord tissues (ucMSC) on porous Bioglass(®)-based scaffolds over a cultivation period of 5 weeks. Differences in lineage-specific osteogenic differentiation of adMSC and ucMSC on Bioglass(®) samples were demonstrated. The investigation led to positive results in terms of cell attachment, proliferation, and differentiation of MSC onto Bioglass(®)-based scaffolds confirming the relevance of these matrices for BTE applications. © 2014 Wiley Periodicals, Inc.

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

    Directory of Open Access Journals (Sweden)

    Yin-Hua Zhao

    2015-05-01

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

  13. Atlantic salmon (Salmo salar) muscle precursor cells differentiate into osteoblasts in vitro: polyunsaturated fatty acids and hyperthermia influence gene expression and differentiation.

    Science.gov (United States)

    Ytteborg, Elisabeth; Vegusdal, Anne; Witten, P Eckhard; Berge, Gerd Marit; Takle, Harald; Østbye, Tone-Kari; Ruyter, Bente

    2010-02-01

    The formation and mineralisation of bone are two critical processes in fast-growing Atlantic salmon (Salmo salar). The mechanisms of these processes, however, have not been described in detail. Thus, in vitro systems that allow the study of factors that influence bone formation in farmed Atlantic salmon are highly warranted. We describe here a method by which unspecialized primary cells from salmon white muscle can differentiate to osteoblasts in vitro. We have subsequently used the differentiated cells as a model system to study the effects of two factors that influence bone formation in Atlantic salmon under commercial farming conditions, namely polyunsaturated fatty acids, PUFAs, and temperature. Muscle precursor cells changed their morphology from triangular or spindle-shaped cells to polygonal or cubical cells after 3 weeks in osteogenic medium. In addition, gene expression studies showed that marker genes for osteoblastogenesis; alp, col1a1, osteocalcin, bmp2 and bmp4 increased after 3 weeks of incubation in osteogenic media showing that these cells have differentiated to osteoblasts at this stage. Adding CLA or DHA to the osteoblast media resulted in a reduced PGE(2) production and increased expression of osteocalcin. Further, temperature studies showed that differentiating osteoblasts are highly sensitive to increased incubation temperature at early stages of differentiation. Our studies show that unspecialized precursor cells isolated from salmon muscle tissue can be caused to differentiate to osteoblasts in vitro. Furthermore, this model system appears to be suitable for the study of osteoblast biology in vitro. 2009 Elsevier B.V. All rights reserved.

  14. Osteogenic activity and antibacterial effect of zinc oxide/carboxylated graphene oxide nanocomposites: Preparation and in vitro evaluation.

    Science.gov (United States)

    Chen, Junyu; Zhang, Xin; Cai, He; Chen, Zhiqiang; Wang, Tong; Jia, Lingling; Wang, Jian; Wan, Qianbing; Pei, Xibo

    2016-11-01

    The aim of this study was to prepare nanocomposites of carboxylated graphene oxide (GO-COOH) sheets decorated with zinc oxide (ZnO) nanoparticles (NPs) and investigate their advantages in the field of bone tissue engineering. First, ZnO/GO-COOH nanocomposites were synthesized by facile reactions, including the carboxylation of graphene oxide (GO) and the nucleation of ZnO on GO-COOH sheets. The synthesized ZnO/GO-COOH nanocomposites were then characterized by X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), Raman spectra, and transmission electron microscopy (TEM). The biocompatibility, osteogenic activity and antibacterial effect of ZnO/GO-COOH nanocomposites were further investigated. In the nanocomposites, ZnO nanoparticles with a size of approximately 12nm were uniformly decorated on GO-COOH sheets. Compared with GO-COOH and the control group, ZnO/GO-COOH nanocomposites significantly enhanced ALP activity, osteocalcin production and extracellular matrix mineralization as well as up-regulated osteogenic-related genes (ALP, OCN, and Runx2) in MG63 osteoblast-like cells. Moreover, ZnO/GO-COOH nanocomposites had an antibacterial effect against Streptococcus mutans. These results indicated that ZnO/GO-COOH nanocomposites exhibited both osteogenic activity and antibacterial effect and had great potential for designing new biomaterials in the field of bone tissue engineering. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Calcium Hydroxide-induced Proliferation, Migration, Osteogenic Differentiation, and Mineralization via the Mitogen-activated Protein Kinase Pathway in Human Dental Pulp Stem Cells.

    Science.gov (United States)

    Chen, Luoping; Zheng, Lisha; Jiang, Jingyi; Gui, Jinpeng; Zhang, Lingyu; Huang, Yan; Chen, Xiaofang; Ji, Jing; Fan, Yubo

    2016-09-01

    Calcium hydroxide has been extensively used as the gold standard for direct pulp capping in clinical dentistry. It induces proliferation, migration, and mineralization in dental pulp stem cells (DPSCs), but the underlying mechanisms are still unclear. The aim of this study was to investigate the role of the mitogen-activated protein (MAP) kinase pathway in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Human DPSCs between passages 3 and 6 were used. DPSCs were preincubated with inhibitors of MAP kinases and cultured with calcium hydroxide. The phosphorylated MAP kinases were detected by Western blot analysis. Cell viability was analyzed via the methylthiazol tetrazolium assay. Cell migration was estimated using the wound healing assay. Alkaline phosphatase (ALP) expression was analyzed using the ALP staining assay. Mineralization was studied by alizarin red staining analysis. Calcium hydroxide significantly promoted the phosphorylation of the c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase. The inhibition of JNK and p38 signaling abolished calcium hydroxide-induced proliferation of DPSCs. The inhibition of JNK, p38, and extracellular signal-regulated kinase signaling suppressed the migration, ALP expression, and mineralization of DPSCs. Our study showed that the MAP kinase pathway was involved in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  16. Adhesion, proliferation and osteogenic differentiation of human MSCs cultured under perfusion with a marine oxygen carrier on an allogenic bone substitute.

    Science.gov (United States)

    Le Pape, Fiona; Richard, Gaëlle; Porchet, Emmanuelle; Sourice, Sophie; Dubrana, Frédéric; Férec, Claude; Polard, Valérie; Pace, Richard; Weiss, Pierre; Zal, Franck; Delépine, Pascal; Leize, Elisabeth

    2017-08-22

    Tissue engineering strategies have been developed to optimize osseointegration in dental implant surgery. One of the major problems is the non-homogeneous spatial cell distribution in the scaffold, as well as subsequent matrix production. Insufficient nutrient and oxygen supplies inside the scaffold are factors in this phenomenon. To mediate this gradient formation, we have implemented a perfusion culture method to seed human bone marrow mesenchymal stem cells (MSCs) into three-dimensional (3-D)-allogenic bone scaffolds in combination with a marine haemoglobin, HEMOXCell(®), for oxygen delivery. Cell culture was performed under static and perfusion conditions, with standard and osteogenic media, with and without HEMOXCell(®). The cell seeding efficiency, as well as MSC/scaffold cytocompatibly were assessed using viability and proliferation assays. Scaffolds' cellularization and extracellular matrix (ECM) formation were analyzed using scanning electron microscopy and histological staining. Cell differentiation was investigated with osteogenic biomarkers gene expression analysis. The perfusion culture was observed to significantly promote MSC proliferation and differentiation throughout the scaffolds, especially when using the induction medium w/HEMOXCell(®). Our data suggest that perfusion culture of MSC into allogenic bone substitute with HEMOXCell(®) as a natural oxygen carrier is promising for tissue engineering applications to oxygenate hypoxic areas and to promote cellular proliferation.

  17. A diels-alder modulated approach to control and sustain the release of dexamethasone and induce osteogenic differentiation of human mesenchymal stem cells

    Science.gov (United States)

    Koehler, Kenneth C.; Alge, Daniel L.; Anseth, Kristi S.; Bowman, Christopher N.

    2013-01-01

    We report a new approach to controlled drug release based upon exploiting the dynamic equilibrium that exists between Diels-Alder reactants and products, demonstrating the release of a furan containing dexamethasone peptide (dex-KGPQG-furan) from a maleimide containing hydrogel. Using a reaction-diffusion model, the release kinetics were tuned to achieve sustained concentrations conducive to osteogenic differentiation of human mesenchymal stem cells (hMSCs). Efficacy was first demonstrated in a 2D culture model, in which dexamethasone release induced significant increases in alkaline phosphatase (ALP) activity and mineral deposition in hMSCs compared to a dexamethasone-free treatment. The results were similar to that observed with a soluble dexamethasone treatment. More dramatic differences were observed in 3D culture, where co-encapsulation of a dexamethasone releasing hydrogel depot within an hMSC-laden extracellular matrix mimetic poly(ethylene glycol) hydrogel resulted in a local and robust osteogenic differentiation. ALP activity reached levels that were up to six times higher than the dexamethasone free treatment. Interestingly, at 5 and 10 day time points, the ALP activity exceeded the dexamethasone positive control, suggesting a potential benefit of sustained release in 3D culture. After 21 days, substantial mineralization comparable to the positive control was also observed in the hydrogels. Collectively, these results demonstrate Diels-Alder modulated release as an effective and versatile new platform for controlled drug delivery that may prove especially beneficial for sustaining the release of low molecular weight molecules in hydrogel systems. PMID:23465826

  18. Irisin Enhances Osteoblast Differentiation In Vitro

    Directory of Open Access Journals (Sweden)

    Graziana Colaianni

    2014-01-01

    Full Text Available It has been recently demonstrated that exercise activity increases the expression of the myokine Irisin in skeletal muscle, which is able to drive the transition of white to brown adipocytes, likely following a phenomenon of transdifferentiation. This new evidence supports the idea that muscle can be considered an endocrine organ, given its ability to target adipose tissue by promoting energy expenditure. In accordance with these new findings, we hypothesized that Irisin is directly involved in bone metabolism, demonstrating its ability to increase the differentiation of bone marrow stromal cells into mature osteoblasts. Firstly, we confirmed that myoblasts from mice subjected to 3 weeks of free wheel running increased Irisin expression compared to nonexercised state. The conditioned media (CM collected from myoblasts of exercised mice induced osteoblast differentiation in vitro to a greater extent than those of mice housed in resting conditions. Furthermore, the differentiated osteoblasts increased alkaline phosphatase and collagen I expression by an Irisin-dependent mechanism. Our results show, for the first time, that Irisin directly targets osteoblasts, enhancing their differentiation. This finding advances notable perspectives in future studies which could satisfy the ongoing research of exercise-mimetic therapies with anabolic action on the skeleton.

  19. Effect of grafting RGD and BMP-2 protein-derived peptides to a hydrogel substrate on osteogenic differentiation of marrow stromal cells.

    Science.gov (United States)

    He, Xuezhong; Ma, Junyu; Jabbari, Esmaiel

    2008-11-04

    Osteogenic differentiation and mineralization of bone marrow stromal (BMS) cells depends on the cells' interactions with bioactive peptides associated with the matrix proteins. The RGD peptides of ECM proteins interact with BMS cells through integrin surface receptors to facilitate cell spreading and adhesion. The BMP peptide corresponding to residues 73-92 of bone morphogenetic protein-2 promotes differentiation and mineralization of BMS cells. The objective of this work was to investigate the effects of RGD and BMP peptides, grafted to a hydrogel substrate, on osteogenic differentiation and mineralization of BMS cells. RGD peptide was acrylamide-terminated by reacting acrylic acid with the N-terminal amine group of the peptide to produce the functionalized Ac-GRGD peptide. The PEGylated BMP peptide was reacted with 4-carboxybenzenesulfonazide to produce an azide functionalized Az-mPEG-BMP peptide. Poly (lactide-co-ethylene oxide- co-fumarate) (PLEOF) macromer was cross-linked with Ac-GRGD peptide and propargyl acrylate to produce an RGD conjugated hydrogel. Az-mPEG-BMP peptide was grafted to the hydrogel by "click chemistry". The RGD and BMP peptide density on the hydrogel surface was 1.62+/-0.37 and 5.2+/-0.6 pmol/cm2, respectively. BMS cells were seeded on the hydrogels and the effect of RGD and BMP peptides on osteogenesis was evaluated by measuring ALPase activity and calcium content with incubation time. BMS cells cultured on RGD conjugated, BMP peptide grafted, and RGD+BMP peptide modified hydrogels showed 3, 2.5, and 5-fold increase in ALPase activity after 14 days incubation. BMS cells seeded on RGD+BMP peptides modified hydrogel showed 4.9- and 11.8-fold increase in calcium content after 14 and 21 days, respectively, which was significantly higher than RGD conjugated or BMP grafted hydrogels. These results demonstrate that RGD and BMP peptides, grafted to a hydrogel substrate, act synergistically to enhance osteogenic differentiation and mineralization

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

    Science.gov (United States)

    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. © 2013 Wiley Periodicals, Inc.

  1. Indirect coating of RGD peptides using a poly-L-lysine spacer enhances jaw periosteal cell adhesion, proliferation, and differentiation into osteogenic tissue.

    Science.gov (United States)

    Ardjomandi, N; Klein, C; Kohler, K; Maurer, A; Kalbacher, H; Niederländer, J; Reinert, S; Alexander, D

    2012-08-01

    The aim of our study was to generate a biofunctionalized, three-dimensional (3D) biomaterial to enhance jaw periosteal cell (JPC) adhesion and differentiation into osteogenic tissue. Therefore, open-cell polylactic acid (OPLA) scaffolds were coated covalently with different RGD peptides (a conserved recognition sequence of the most ECM proteins--arginine-glycine-asparagine) and different coating variants. The linear and cyclic RGD peptides were either applied directly or indirectly via a poly-L-lysine (PLL) spacer. JPCs were analyzed on coated constructs in 2D and 3D cultures and showed enhanced rates for indirectly coated scaffolds using the PLL spacer. By gene expression, we detected significantly increased levels of osteogenic marker genes, such as alkaline phosphatase, RUNX2, and AMELY in JPCs seeded onto PLL/linear RGD constructs compared to the otherwise-coated constructs. An analysis of the JPC mineralization capacity revealed the highest amounts of calcium-phosphate precipitates in cells growing within the PLL/linear scaffolds. Additionally, the JPC adhesion behavior on OPLA scaffolds seems to be mediated by ITGB3, ITGB1, and ITGAV, as shown by blocking assays. We concluded that coating of OPLA constructs with linear RGD peptides via PLL represents a suitable approach for functionalizing the polymer surface and enhancing adhesion, proliferation, and mineralization of JPCs. Copyright © 2012 Wiley Periodicals, Inc.

  2. Osteogenic induction of human bone marrow-derived mesenchymal progenitor cells in novel synthetic polymer-hydrogel matrices.

    Science.gov (United States)

    Endres, M; Hutmacher, D W; Salgado, A J; Kaps, C; Ringe, J; Reis, R L; Sittinger, M; Brandwood, A; Schantz, J T

    2003-08-01

    The aim of this project was to investigate the in vitro osteogenic potential of human mesenchymal progenitor cells in novel matrix architectures built by means of a three-dimensional bioresorbable synthetic framework in combination with a hydrogel. Human mesenchymal progenitor cells (hMPCs) were isolated from a human bone marrow aspirate by gradient centrifugation. Before in vitro engineering of scaffold-hMPC constructs, the adipogenic and osteogenic differentiation potential was demonstrated by staining of neutral lipids and induction of bone-specific proteins, respectively. After expansion in monolayer cultures, the cells were enzymatically detached and then seeded in combination with a hydrogel into polycaprolactone (PCL) and polycaprolactone-hydroxyapatite (PCL-HA) frameworks. This scaffold design concept is characterized by novel matrix architecture, good mechanical properties, and slow degradation kinetics of the framework and a biomimetic milieu for cell delivery and proliferation. To induce osteogenic differentiation, the specimens were cultured in an osteogenic cell culture medium and were maintained in vitro for 6 weeks. Cellular distribution and viability within three-dimensional hMPC bone grafts were documented by scanning electron microscopy, cell metabolism assays, and confocal laser microscopy. Secretion of the osteogenic marker molecules type I procollagen and osteocalcin was analyzed by semiquantitative immunocytochemistry assays. Alkaline phosphatase activity was visualized by p-nitrophenyl phosphate substrate reaction. During osteogenic stimulation, hMPCs proliferated toward and onto the PCL and PCL-HA scaffold surfaces and metabolic activity increased, reaching a plateau by day 15. The temporal pattern of bone-related marker molecules produced by in vitro tissue-engineered scaffold-cell constructs revealed that hMPCs differentiated better within the biomimetic matrix architecture along the osteogenic lineage.

  3. Collagenous matrix supported by a 3D-printed scaffold for osteogenic differentiation of dental pulp cells.

    Science.gov (United States)

    Fahimipour, Farahnaz; Dashtimoghadam, Erfan; Rasoulianboroujeni, Morteza; Yazdimamaghani, Mostafa; Khoshroo, Kimia; Tahriri, Mohammadreza; Yadegari, Amir; Gonzalez, Jose A; Vashaee, Daryoosh; Lobner, Douglas C; Jafarzadeh Kashi, Tahereh S; Tayebi, Lobat

    2017-10-17

    A systematic characterization of hybrid scaffolds, fabricated based on combinatorial additive manufacturing technique and freeze-drying method, is presented as a new platform for osteoblastic differentiation of dental pulp cells (DPCs). The scaffolds were consisted of a collagenous matrix embedded in a 3D-printed beta-tricalcium phosphate (β-TCP) as the mineral phase. The developed construct design was intended to achieve mechanical robustness owing to 3D-printed β-TCP scaffold, and biologically active 3D cell culture matrix pertaining to the Collagen extracellular matrix. The β-TCP precursor formulations were investigated for their flow-ability at various temperatures, which optimized for fabrication of 3D printed scaffolds with interconnected porosity. The hybrid constructs were characterized by 3D laser scanning microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and compressive strength testing. The in vitro characterization of scaffolds revealed that the hybrid β-TCP/Collagen constructs offer superior DPCs proliferation and alkaline phosphatase (ALP) activity compared to the 3D-printed β-TCP scaffold over three weeks. Moreover, it was found that the incorporation of TCP into the Collagen matrix improves the ALP activity. The presented results converge to suggest the developed 3D-printed β-TCP/Collagen hybrid constructs as a new platform for osteoblastic differentiation of DPCs for craniomaxillofacial bone regeneration. Copyright © 2017. Published by Elsevier Ltd.

  4. Effects of BMP-2 and dexamethasone on osteogenic differentiation of rat dental follicle progenitor cells seeded on three-dimensional beta-TCP

    Energy Technology Data Exchange (ETDEWEB)

    Xu Lulu; Jin Zuolin; Duan Yinzhong [Department of Orthodontics, Stomatological College, Fourth Military Medical University, Xi' an 710032 (China); Liu Hongchen; Wang Dongsheng; E Lingling [Department of Stomatology, China PLA General Hospital, Beijing 100853 (China); Xu Lin, E-mail: jinzuolin88@yahoo.com.c, E-mail: duanyinzhong@yahoo.com.c [Department of Stomatology, the First Hospital of PLA, Lanzhou 730000 (China)

    2009-12-15

    The aim of this study was to investigate the effects of BMP-2 and dexamethasone (Dex) on osteogenic differentiation of rat dental follicle progenitor cells (RDFCs) seeded on three-dimensional beta-TCP. The alkaline phosphatase (ALP), the calcium and phosphonium, the osteocalcin in media of the third passage RDFCs on biomaterial beta-TCP after 1-3, 3-7, 7-14 days of culture were examined respectively. The growth of cells on the scaffolds was observed by scanning electron microscope (SEM) after 3, 7 days of culture and by implanting in the backs of severe combined immunodeficient (SCID) mice for bone regeneration. The third passage RDFCs could be seen adhered, extended and proliferated on the beta-TCP by scanning electron microscopy. The ALP activity, the calcium and phosphoniums and the osteocalcin content of dexamethasone (10{sup -8} M) or/and BMP-2 (100 ng ml{sup -1}) were significantly higher than their existence in the control group. They were the significantly highest among four groups after joint application of BMP-2 and dexamethasone. After 8 weeks of implantation, the percentage of the new bones formed area in the RDFCs+beta-TCP+BMP-2+Dex group was significantly higher than that in the RDFCs+beta-TCP+BMP-2 group. In contrast, beta-TCP, RDFCs+beta-TCP+Dex and control constructs lacked new bone formation by histological staining and histomorphometric analysis. The BMP-2+Dex could significantly promote osteogenic differentiation of RDFCs on beta-TCP. beta-TCP supported fast cellular adhesion, proliferation and differentiation of RDFCs. The feasibility of its application in periodontal tissue engineering was also proved.

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

    Directory of Open Access Journals (Sweden)

    Sardar M Z Uddin

    Full Text Available 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.

  6. Femtosecond laser microstructured Alumina toughened Zirconia: A new strategy to improve osteogenic differentiation of hMSCs

    Science.gov (United States)

    Carvalho, Angela; Cangueiro, Liliana; Oliveira, Vítor; Vilar, Rui; Fernandes, Maria H.; Monteiro, Fernando J.

    2018-03-01

    The use of topographic patterns has been a continuously growing area of research for tissue engineering and it is widely accepted that the surface topography of biomaterials can influence and modulate the initial biological response. Ultrafast lasers are extremely powerful tools to machine and pattern the surface of a wide range of biomaterials, however, only few work has been performed on ceramics with the intent of biomedical applications, and the biological characterization of these structured materials is scarce. In this work, relevance is given to the biological performance of such materials. A femtosecond laser ablation technique was used to modify Alumina toughened Zirconia (ATZ) surface topography, developing surfaces structured at the micro and nanoscale levels (μATZ), in a controlled and reproducible manner. Materials characterization was performed before and after laser treatment, and both materials were compared in terms of osteogenic response of human bone marrow derived mesenchymal stem cells cultured under basal conditions, expecting that the micro/nanofeatures will improve the biological response of cells. Cells metabolic activity and proliferation increased with the culture time and surface microtopography modulated cells alignment and guided proliferation. The modified surface, displayed significantly higher expression of osteogenic transcription factors and genes and, additionally, the formation of a mineralized extracellular matrix, when compared to the control surface, i.e. unmodified ATZ.

  7. Human serum promotes osteogenic differentiation of human dental pulp stem cells in vitro and in vivo

    National Research Council Canada - National Science Library

    Pisciotta, Alessandra; Riccio, Massimo; Carnevale, Gianluca; Beretti, Francesca; Gibellini, Lara; Maraldi, Tullia; Cavallini, Gian Maria; Ferrari, Adriano; Bruzzesi, Giacomo; De Pol, Anto

    2012-01-01

    Human dental pulp is a promising alternative source of stem cells for cell-based tissue engineering in regenerative medicine, for the easily recruitment with low invasivity for the patient and for the...

  8. Stimulation of osteogenic differentiation in human osteoprogenitor cells by pulsed electromagnetic fields: An in vitro study

    NARCIS (Netherlands)

    J.H.W. Jansen (Justus); O.P. van der Jagt (Olav); B.J. Punt (Bas); J.A.N. Verhaar (Jan); J.P.T.M. van Leeuwen (Hans); H.H. Weinans (Harrie); H. Jahr (Holger)

    2010-01-01

    textabstractBackground: Although pulsed electromagnetic field (PEMF) stimulation may be clinically beneficial during fracture healing and for a wide range of bone disorders, there is still debate on its working mechanism. Mesenchymal stem cells are likely mediators facilitating the observed clinical

  9. Sustained Release of BMP-2 in Bioprinted Alginate for Osteogenicity in Mice and Rats

    Science.gov (United States)

    Poldervaart, Michelle T.; Wang, Huanan; van der Stok, Johan; Weinans, Harrie; Leeuwenburgh, Sander C. G.; Öner, F. Cumhur; Dhert, Wouter J. A.; Alblas, Jacqueline

    2013-01-01

    The design of bioactive three-dimensional (3D) scaffolds is a major focus in bone tissue engineering. Incorporation of growth factors into bioprinted scaffolds offers many new possibilities regarding both biological and architectural properties of the scaffolds. This study investigates whether the sustained release of bone morphogenetic protein 2 (BMP-2) influences osteogenicity of tissue engineered bioprinted constructs. BMP-2 loaded on gelatin microparticles (GMPs) was used as a sustained release system, which was dispersed in hydrogel-based constructs and compared to direct inclusion of BMP-2 in alginate or control GMPs. The constructs were supplemented with goat multipotent stromal cells (gMSCs) and biphasic calcium phosphate to study osteogenic differentiation and bone formation respectively. BMP-2 release kinetics and bioactivity showed continuous release for three weeks coinciding with osteogenicity. Osteogenic differentiation and bone formation of bioprinted GMP containing constructs were investigated after subcutaneous implantation in mice or rats. BMP-2 significantly increased bone formation, which was not influenced by the release timing. We showed that 3D printing of controlled release particles is feasible and that the released BMP-2 directs osteogenic differentiation in vitro and in vivo. PMID:23977328

  10. Sustained release of BMP-2 in bioprinted alginate for osteogenicity in mice and rats.

    Directory of Open Access Journals (Sweden)

    Michelle T Poldervaart

    Full Text Available The design of bioactive three-dimensional (3D scaffolds is a major focus in bone tissue engineering. Incorporation of growth factors into bioprinted scaffolds offers many new possibilities regarding both biological and architectural properties of the scaffolds. This study investigates whether the sustained release of bone morphogenetic protein 2 (BMP-2 influences osteogenicity of tissue engineered bioprinted constructs. BMP-2 loaded on gelatin microparticles (GMPs was used as a sustained release system, which was dispersed in hydrogel-based constructs and compared to direct inclusion of BMP-2 in alginate or control GMPs. The constructs were supplemented with goat multipotent stromal cells (gMSCs and biphasic calcium phosphate to study osteogenic differentiation and bone formation respectively. BMP-2 release kinetics and bioactivity showed continuous release for three weeks coinciding with osteogenicity. Osteogenic differentiation and bone formation of bioprinted GMP containing constructs were investigated after subcutaneous implantation in mice or rats. BMP-2 significantly increased bone formation, which was not influenced by the release timing. We showed that 3D printing of controlled release particles is feasible and that the released BMP-2 directs osteogenic differentiation in vitro and in vivo.

  11. Osteogenic potency of human bone marrow mesenchymal stem cells from femoral atrophic non-union fracture site

    Directory of Open Access Journals (Sweden)

    Ismail Hadisoebroto Dilogo

    2014-06-01

    Full Text Available Objective: Mesenchymal stem cells (MSCs exist in the site of atrophic non-union fracture. The aim of this study was to evaluate the osteogenic potency of MSCs in order to have a better understanding of the unclear pathophysiology of atrophic non-union fracture Methods: This is an in vitro experimental study. Sample was obtained from the non-union site of a patient with a 6-years-history of atrophic non-union fracture of right femur. The MSCs was isolated from the fracture site and was cultured in the growth medium. Confirmation of the MSCs was performed and then osteogenic differentiation was performed in mono-layered MSC grown in both home-made and commercial osteogenic media. To evaluate the osteogenic differentiation, we performed Alizarin red staining and colorimetric assay for alkaline phosphatase (ALP. Results: From Alizarin red staining, most cells in the osteoblast medium were stained red by the staining. The result of colorimetric assessment of ALP shows that peak concentration was reached after 4 minutes in osteogenic group and control group. Conclusion: The presence of ALP activity and positive Alizarin red staining in our study showed that MSCs stem cells obtained from site of atrophic non-union is capable to be differentiated into osteogenic cells. . J Clin Exp Invest 2014; 5 (2: 159-163

  12. The effects of platelet-rich plasma derived from human umbilical cord blood on the osteogenic differentiation of human dental stem cells.

    Science.gov (United States)

    Lee, Jung-Yeon; Nam, Hyun; Park, Yoon-Jeong; Lee, Seung-Jin; Chung, Chong-Pyoung; Han, Soo-Boo; Lee, Gene

    2011-02-01

    Platelet-rich plasma (PRP) is an emerging therapeutic application because PRP contains various growth factors that have beneficial effects on tissue regeneration and engineering. Mesenchymal stem cells and PRP derived from peripheral blood have been well studied. In this study, we investigated the effects of PRP derived from human umbilical cord blood (UCB-PRP) on proliferation, alkaline phosphatase (ALP) activity, and osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHEDs), dental pulp stem cells (DPSCs), and periodontal ligament stem cells (PDLSCs). Three types of dental stem cells were primarily isolated and characterized by flow cytometric analysis. Dental stem cells were exposed to various concentrations of UCB-PRP, which resulted in the proliferation of dental stem cells. Treatment with 2% UCB-PRP resulted in the highest level of proliferation. The ALP activity of DPSCs and PDLSCs increased following treatment with UCB-PRP in a dose-dependent manner up to a concentration of 2%. ALP activity decreased with higher concentration of UCB-PRP. The effects of UCB-PRP on calcium deposition were similar to those on proliferation and ALP activity. Treatment with 2% UCB-PRP resulted in the highest calcium depositions in DPSCs and PDLSCs; however, treatment with 1% UCB-PRP resulted in the highest calcium deposition in SHEDs. The concentrations of platelet-derived growth factor-AB and transforming growth factor-β1 in UCB-PRP were investigated and found to be comparable to the amounts in peripheral blood. Overall, UCB-PRP had beneficial effects on the proliferation and osteogenic differentiation of dental stem cells. Determination of the optimal concentration of UCB-PRP requires further investigation for clinical applications.

  13. Strontium-containing mesoporous bioactive glass scaffolds with improved osteogenic/cementogenic differentiation of periodontal ligament cells for periodontal tissue engineering.

    Science.gov (United States)

    Wu, Chengtie; Zhou, Yinghong; Lin, Chucheng; Chang, Jiang; Xiao, Yin

    2012-10-01

    To achieve the ultimate goal of periodontal tissue engineering, it is of great importance to develop bioactive scaffolds which can stimulate the osteogenic/cementogenic differentiation of periodontal ligament cells (PDLCs) for the favorable regeneration of alveolar bone, root cementum and periodontal ligament. Strontium (Sr) and Sr-containing biomaterials have been found to induce osteoblast activity. However, there has been no systematic report about the interaction between Sr or Sr-containing biomaterials and PDLCs for periodontal tissue engineering. The aims of this study were to prepare Sr-containing mesoporous bioactive glass (Sr-MBG) scaffolds and investigate whether the addition of Sr could stimulate osteogenic/cementogenic differentiation of PDLCs in a tissue-engineering scaffold system. The composition, microstructure and mesopore properties (specific surface area, nanopore volume and nanopore distribution) of Sr-MBG scaffolds were characterized. The proliferation, alkaline phosphatase (ALP) activity and osteogenesis/cementogenesis-related gene expression (ALP, Runx2, Col I, OPN and CEMP1) of PDLCs on different kinds of Sr-MBG scaffolds were systematically investigated. The results show that Sr plays an important role in influencing the mesoporous structure of MBG scaffolds in which high contents of Sr decreased the well-ordered mesopores as well as their surface area/pore volume. Sr(2+) ions could be released from Sr-MBG scaffolds in a controlled way. The incorporation of Sr into MBG scaffolds has significantly stimulated ALP activity and osteogenesis/cementogenesis-related gene expression of PDLCs. Furthermore, Sr-MBG scaffolds in a simulated body fluid environment still maintained excellent apatite-mineralization ability. The study suggests that the incorporation of Sr into MBG scaffolds is a viable way to stimulate the biological response of PDLCs. Sr-MBG scaffolds are a promising bioactive material for periodontal tissue-engineering applications

  14. Bifunctional coating based on carboxymethyl chitosan with stable conjugated alkaline phosphatase for inhibiting bacterial adhesion and promoting osteogenic differentiation on titanium

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Dong; Neoh, Koon Gee, E-mail: chenkg@nus.edu.sg; Kang, En-Tang

    2016-01-01

    Graphical abstract: - Highlights: • Alkaline phosphatase was immobilized on carboxymethyl chitosan coating on Ti. • The coating is bifunctional; resists bacterial adhesion and enhances cell functions. • Osteogenic differentiation of osteoblasts and stem cells is enhanced on the coating. • The coating remains stable and functional after ethanol treatment and autoclaving. - Abstract: 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/cm{sup 2} 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.

  15. Induction of osteogenic differentiation of stem cells via a lyophilized microRNA reverse transfection formulation on a tissue culture plate

    Science.gov (United States)

    Wu, Kaimin; Xu, Jie; Liu, Mengyuan; Song, Wen; Yan, Jun; Gao, Shan; Zhao, Lingzhou; Zhang, Yumei

    2013-01-01

    MicroRNA (miRNA) regulation is a novel approach to manipulating the fate of mesenchymal stem cells, but an easy, safe, and highly efficient method of transfection is required. In this study, we developed an miRNA reverse transfection formulation by lyophilizing Lipofectamine 2000-miRNA lipoplexes on a tissue culture plate. The lipoplexes can be immobilized on a tissue culture plate with an intact pseudospherical structure and lyophilization without any lyoprotectant. In this study, reverse transfection resulted in highly efficient cellular uptake of miRNA and enabled significant manipulation of the intracellular target miRNA level. Reverse transfection formulations containing Lipofectamine 2000 1 μL per well generated much higher transfection efficiency without obvious cytotoxicity compared with conventional and other transfection methods. Further, the transfection efficiency of the reverse transfection formulations did not deteriorate during 90 days of storage at 4°C and −20°C. We then assessed the efficiency of the miRNA reverse transfection formulation in promoting osteogenic differentiation of mesenchymal stem cells. We found that transfection with anti-miR-138 and miR-148b was efficient for enhancing osteogenic differentiation, as indicated by enhanced osteogenesis-related gene expression, amount of alkaline phosphatase present, production of collagen, and matrix mineralization. Overall, the miRNA reverse transfection formulation developed in this study is a promising approach for miRNA transfection which can control stem cell fate and is suitable for loading miRNAs onto various biomaterials. PMID:23662054

  16. Effect of biomimetic zinc-containing tricalcium phosphate (Zn-TCP) on the growth and osteogenic differentiation of mesenchymal stem cells.

    Science.gov (United States)

    Chou, Joshua; Hao, Jia; Hatoyama, Hirokazu; Ben-Nissan, Besim; Milthorpe, Bruce; Otsuka, Makoto

    2015-07-01

    Several studies have shown the effectiveness of zinc-tricalcium phosphate (Zn-TCP) for bone tissue engineering. In this study, marine calcareous foraminifera possessing uniform pore size distribution were hydrothermally converted to Zn-TCP. The ability of a scaffold to combine effectively with mesenchymal stem cells (MSCs) is a key tissue-engineering aim. In order to demonstrate the osteogenic ability of MSCs with Zn-TCP, the scaffolds were cultured in an osteogenic induction medium to elicit an osteoblastic response. The physicochemical properties of Zn-TCP were characterized by XRD, FT-IR and ICP-MS. MSCs were aspirated from rat femurs and cultured for 3 days before indirectly placing four samples into each respective well. After culture for 7, 10 and 14 days, osteoblastic differentiation was evaluated using alizarin red S stain, measurement of alkaline phosphatase (ALP) levels, cell numbers and cell viability. XRD and FT-IR patterns both showed the replacement of CO(3)(2-) with PO(4)(3-). Chemical analysis showed zinc incorporation of 5 mol%. Significant increases in cell numbers were observed at 10 and 14 days in the Zn-TCP group, while maintaining high levels of cell viability (> 90%). ALP activity in the Zn-TCP group was statistically higher at 10 days. Alizarin red S staining also showed significantly higher levels of calcium mineralization in Zn-TCP compared with the control groups. This study showed that MSCs in the presence of biomimetically derived Zn-TCP can accelerate their differentiation to osteoblasts and could potentially be useful as a scaffold for bone tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.

  17. Induction of osteogenic differentiation of stem cells via a lyophilized microRNA reverse transfection formulation on a tissue culture plate.

    Science.gov (United States)

    Wu, Kaimin; Xu, Jie; Liu, Mengyuan; Song, Wen; Yan, Jun; Gao, Shan; Zhao, Lingzhou; Zhang, Yumei

    2013-01-01

    MicroRNA (miRNA) regulation is a novel approach to manipulating the fate of mesenchymal stem cells, but an easy, safe, and highly efficient method of transfection is required. In this study, we developed an miRNA reverse transfection formulation by lyophilizing Lipofectamine 2000-miRNA lipoplexes on a tissue culture plate. The lipoplexes can be immobilized on a tissue culture plate with an intact pseudospherical structure and lyophilization without any lyoprotectant. In this study, reverse transfection resulted in highly efficient cellular uptake of miRNA and enabled significant manipulation of the intracellular target miRNA level. Reverse transfection formulations containing Lipofectamine 2000 1 μL per well generated much higher transfection efficiency without obvious cytotoxicity compared with conventional and other transfection methods. Further, the transfection efficiency of the reverse transfection formulations did not deteriorate during 90 days of storage at 4°C and -20°C. We then assessed the efficiency of the miRNA reverse transfection formulation in promoting osteogenic differentiation of mesenchymal stem cells. We found that transfection with anti-miR-138 and miR-148b was efficient for enhancing osteogenic differentiation, as indicated by enhanced osteogenesis-related gene expression, amount of alkaline phosphatase present, production of collagen, and matrix mineralization. Overall, the miRNA reverse transfection formulation developed in this study is a promising approach for miRNA transfection which can control stem cell fate and is suitable for loading miRNAs onto various biomaterials.

  18. Intrinsic differentiation potential of adolescent human tendon tissue: an in-vitro cell differentiation study

    Directory of Open Access Journals (Sweden)

    Weinans Harrie

    2007-02-01

    Full Text Available Abstract Background Tendinosis lesions show an increase of glycosaminoglycan amount, calcifications, and lipid accumulation. Therefore, altered cellular differentiation might play a role in the etiology of tendinosis. This study investigates whether adolescent human tendon tissue contains a population of cells with intrinsic differentiation potential. Methods Cells derived from adolescent non-degenerative hamstring tendons were characterized by immunohistochemistry and FACS-analysis. Cells were cultured for 21 days in osteogenic, adipogenic, and chondrogenic medium and phenotypical evaluation was carried out by immunohistochemical and qPCR analysis. The results were compared with the results of similar experiments on adult bone marrow-derived stromal cells (BMSCs. Results Tendon-derived cells stained D7-FIB (fibroblast-marker positive, but α-SMA (marker for smooth muscle cells and pericytes negative. Tendon-derived cells were 99% negative for CD34 (endothelial cell marker, and 73% positive for CD105 (mesenchymal progenitor-cell marker. In adipogenic medium, intracellular lipid vacuoles were visible and tendon-derived fibroblasts showed upregulation of adipogenic markers FABP4 (fatty-acid binding protein 4 and PPARG (peroxisome proliferative activated receptor γ. In chondrogenic medium, some cells stained positive for collagen 2 and tendon-derived fibroblasts showed upregulation of collagen 2 and collagen 10. In osteogenic medium Von Kossa staining showed calcium deposition although osteogenic markers remained unaltered. Tendon-derived cells and BMCSs behaved largely comparable, although some distinct differences were present between the two cell populations. Conclusion This study suggests that our population of explanted human tendon cells has an intrinsic differentiation potential. These results support the hypothesis that there might be a role for altered tendon-cell differentiation in the pathophysiology of tendinosis.

  19. Characterization and in vitro biological evaluation of mineral/osteogenic growth peptide nanocomposites synthesized biomimetically on titanium

    Science.gov (United States)

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

    2015-04-01

    Nanocomposite layers of mineral/osteogenic growth peptide (OGP) were synthesized on calcium phosphate coated titanium substrates by immersing in calcium-phosphate buffer solution containing OGP. Peptide incorporated mineral was characterized by determining quantity loaded, effects on mineral morphology and structure. Also, the biological activity was investigated by cell adhesion, proliferation assay, and measurement of alkaline phosphatase (ALP) activity. X-ray photoelectron spectroscopy (XPS) and micro-bicinchoninic acid (BCA) assay revealed that OGP was successfully incorporated with mineral and the amount was increased with immersion time. Incorporated OGP changed the mineral morphology from sharp plate-like shape to more rounded one, and the octacalcium phosphate structure of the mineral was gradually transformed into apatite. With confocal microscopy to examine the incorporation of fluorescently labeled peptide, OGP was evenly distributed throughout mineral layers. Mineral/OGP nanocomposites promoted cell adhesion and proliferation, and also increased ALP activity of mesenchymal stem cells (MSCs). Results presented here indicated that the mineral/OGP nanocomposites formed on titanium substrates had the potential for applications in dental implants.

  20. Characterization and in vitro biological evaluation of mineral/osteogenic growth peptide nanocomposites synthesized biomimetically on titanium

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Cen; Kong, Xiangdong [Bio-X Center, College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018 (China); Zhang, Sheng-Min [Advanced Biomaterials and Tissue Engineering Center, Huazhong University of Science and Technology, Wuhan 430074 (China); Lee, In-Seop, E-mail: inseop@yonsei.ac.kr [Bio-X Center, College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018 (China); Institute of Natural Sciences, Yonsei University, Seoul 120-749 (Korea, Republic of)

    2015-04-15

    Graphical abstract: - Highlights: • Mineral/OGP nanocomposite layers were synthesized biomimetically on Ti substrates. • Incorporated OGP affected the morphology and ultimate structure of mineral. • Incorporated OGP improved the MSCs adhesion, proliferation, and ALP activity. - Abstract: Nanocomposite layers of mineral/osteogenic growth peptide (OGP) were synthesized on calcium phosphate coated titanium substrates by immersing in calcium-phosphate buffer solution containing OGP. Peptide incorporated mineral was characterized by determining quantity loaded, effects on mineral morphology and structure. Also, the biological activity was investigated by cell adhesion, proliferation assay, and measurement of alkaline phosphatase (ALP) activity. X-ray photoelectron spectroscopy (XPS) and micro-bicinchoninic acid (BCA) assay revealed that OGP was successfully incorporated with mineral and the amount was increased with immersion time. Incorporated OGP changed the mineral morphology from sharp plate-like shape to more rounded one, and the octacalcium phosphate structure of the mineral was gradually transformed into apatite. With confocal microscopy to examine the incorporation of fluorescently labeled peptide, OGP was evenly distributed throughout mineral layers. Mineral/OGP nanocomposites promoted cell adhesion and proliferation, and also increased ALP activity of mesenchymal stem cells (MSCs). Results presented here indicated that the mineral/OGP nanocomposites formed on titanium substrates had the potential for applications in dental implants.

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

    Science.gov (United States)

    de Peppo, Giuseppe Maria; Marolt, Darja

    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.

  2. Effect of the fiber diameter and porosity of non-woven PET fabrics on the osteogenic differentiation of mesenchymal stem cells.

    Science.gov (United States)

    Takahashi, Yoshitake; Tabata, Yasuhiko

    2004-01-01

    The proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) was investigated in three-dimensional non-woven fabrics prepared from polyethylene terephthalate (PET) fiber with different diameters. When seeded into the fabrics of cell scaffold, more MSC attached in the fabric of thicker PET fibers than that of thinner ones, irrespective of the fabric porosity. The morphology of cells attached became more spreaded with an increase in the fiber diameter of fabrics. The rate of MSC proliferation depended on the PET fiber diameter and porosity of fabrics: the bigger the fiber diameter of fabrics with higher porosity, the higher their proliferation rate. When the alkaline phosphatase (ALP) activity and osteocalcin content of MSC cultured in different types of fabrics was measured to evaluate the ostegenic differentiation, they became maximum for the non-woven fabrics with a fiber diameter of 9.0 microm, although the values of low-porous fabrics were significantly high compared with those of high porous fabrics. We concluded that the attachment, proliferation and bone differentiation of MSC was influenced by the fiber diameter and porosity of non-woven fabrics as the scaffold.

  3. Influence of Poly(L-Lactic Acid Nanofibers and BMP-2–Containing Poly(L-Lactic Acid Nanofibers on Growth and Osteogenic Differentiation of Human Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Markus D. Schofer

    2008-01-01

    Full Text Available The aim of this study was to characterize synthetic poly-(L-lactic acid (PLLA nanofibers concerning their ability to promote growth and osteogenic differentiation of stem cells in vitro, as well as to test their suitability as a carrier system for growth factors. Fiber matrices composed of PLLA or BMP-2–incorporated PLLA were seeded with human mesenchymal stem cells and cultivated over a period of 22 days under growth and osteoinductive conditions, and analyzed during the course of culture, with respect to gene expression of alkaline phosphatase (ALP, osteocalcin (OC, and collagen I (COL-I. Furthermore, COL-I and OC deposition, as well as cell densities and proliferation, were analyzed using fluorescence microscopy. Although the presence of nanofibers diminished the dexamethasone-induced proliferation, there were no differences in cell densities or deposition of either COL-I or OC after 22 days of culture. The gene expression of ALP, OC, and COL-I decreased in the initial phase of cell cultivation on PLLA nanofibers as compared to cover slip control, but normalized during the course of cultivation. The initial down-regulation was not observed when BMP-2 was directly incorporated into PLLA nanofibers by electrospinning, indicating that growth factors like BMP-2 might survive the spinning process in a bioactive form.

  4. Enhancement of osteogenic differentiation of rat adipose tissue-derived mesenchymal stem cells by zinc sulphate under electromagnetic field via the PKA, ERK1/2 and Wnt/β-catenin signaling pathways.

    Directory of Open Access Journals (Sweden)

    Ezzatollah Fathi

    Full Text Available Zinc ion as an essential trace element and electromagnetic fields (EMFs has been reported to be involved in the regulation of bone metabolism. The aim of this study was to elucidate the effects of zinc sulphate (ZnSO4 on the osteogenic differentiation of adipose tissue-derived mesenchymal stem cells (ADSCs in the presence of EMF as a strategy in osteoporosis therapy. Alkaline phophatase (ALP activity measurement, calcium assay and expression of several osteoblastic marker genes were examined to assess the effect of ZnSO4 on the osteogenic differentiation of ADSCs under EMF. The expression of cAMP and PKA was evaluated by ELISA. The expression of β-catenin, Wnt1, Wnt3a, low-density lipoprotein receptor-related protein 5 (LRP5 and reduced dickkopf1 (DKK1 genes were used to detect the Wnt/β-catenin pathway. It was found that ZnSO4, in the presence of EMF, resulted in an increase in the expression of osteogenic genes, ALP activity and calcium levels. EMF, in the presence of ZnSO4, increased the cAMP level and protein kinase A (PKA activity. Treatment of ADSCs with (MAPK/ERK kinase 1/2 inhibitor, or PKA inhibitor, significantly inhibited the promotion of osteogenic markers, indicating that the induction of osteogenesis was dependent on the ERK and PKA signaling pathways. Real-time PCR analysis showed that ZnSO4, in the presence of EMF, increased the mRNA expressions of β-catenin, Wnt1, Wnt3a, LRP5 and DKK1. In this study, it was shown that 0.432 μg/ml ZnSO4, in the presence of 50 Hz, 20 mT EMF, induced the osteogenic differentiation of ADSCs via PKA, ERK1/2 and Wnt/β-catenin signaling pathways.

  5. Independent effects of the chemical and microstructural surface properties of polymer/ceramic composites on proliferation and osteogenic differentiation of human MSCs.

    Science.gov (United States)

    Sun, Lanying; Danoux, Charlène B; Wang, Qibao; Pereira, Daniel; Barata, David; Zhang, Jingwei; LaPointe, Vanessa; Truckenmüller, Roman; Bao, Chongyun; Xu, Xin; Habibovic, Pamela

    2016-09-15

    Within the general aim of finding affordable and sustainable regenerative solutions for damaged and diseased tissues and organs, significant efforts have been invested in developing synthetic alternatives to natural bone grafts, such as autografts. Calcium phosphate (CaP) ceramics are among widely used synthetic bone graft substitutes, but their mechanical properties and bone regenerative capacity are still outperformed by their natural counterparts. In order to improve the existing synthetic bone graft substitutes, it is imperative to understand the effects of their individual properties on a biological response, and to find a way to combine the desired properties into new, improved functional biomaterials. To this end, we studied the independent effects of the chemical composition and surface microstructure of a poly(lactic acid)/hydroxyapatite (PLA/HA) composite material on the proliferation and osteogenic differentiation of clinically relevant bone marrow-derived human mesenchymal stromal cells (hMSCs). While the molecular weight of the polymer and presence/absence of the ceramic phase were used as the chemical variables, a soft embossing technique was used to pattern the surfaces of all materials with either pits or pillars with identical microscale dimensions. The results indicated that, while cell morphology was affected by both the presence and availability of HA and by the surface microstructure, the effect of the latter parameter on cell proliferation was negligible. The osteogenic differentiation of hMSCs, and in particular the expression of bone morphogenetic protein 2 (BMP-2) and osteopontin (OP) were significantly enhanced when cells were cultured on the composite based on low-molecular-weight PLA, as compared to the high-molecular-weight PLA-based composite and the two pure polymers. The OP expression on the low-molecular-weight PLA-based composite was further enhanced when the surface was patterned with pits. Taken together, within this experimental

  6. Effect of ternary phosphate-based glass compositions on osteoblast and osteoblast-like proliferation, differentiation and death in vitro.

    Science.gov (United States)

    Skelton, K L; Glenn, J V; Clarke, S A; Georgiou, G; Valappil, S P; Knowles, J C; Nazhat, S N; Jordan, G R

    2007-07-01

    There is currently a need to expand the range of graft materials available to orthopaedic surgeons. This study investigated the effect of ternary phosphate-based glass (PBG) compositions on the behaviour of osteoblast and osteoblast-like cells. PBGs of the formula (in mol.%) P(2)O(5)(50)-CaO(50-X)-Na(2)O(X), where X is either 2, 4, 6, 8 or 10, were produced and their influence on the proliferation, differentiation and death in vitro of adult human bone marrow stromal cells (hBMSCs) and human fetal osteoblast 1.19 (HFOB 1.19) cells were assessed. Tissue culture plastic (TCP) and hydroxyapatite (HA) were used as controls. Exposure to PBGs in culture inhibited cell adhesion and proliferation and increased cell death in both cell types studied. There was no significant difference in percentage cell death between the PBGs, which was significantly greater than the controls. However, compared with other PBGs, a greater number of cells were found on the 48mol.% CaO which may have been due to either increased adherence or proliferation, or both. This composition was capable of supporting osteogenic proliferation and early differentiation, and supports the notion that chemical modification of the glass could lead to a more biologically compatible substrate with the potential to support osteogenic grafting. Realisation of this potential should lead to the development of novel grafting strategies for the treatment of problematic bone defects.

  7. Osteogenic tumors of bone; Osteogene Tumoren

    Energy Technology Data Exchange (ETDEWEB)

    Jobke, B. [Deutsches Krebsforschungszentrum (DKFZ), Abtl. Radiologie, Heidelberg (Germany); Werner, M. [MVZ des HELIOS Klinikum Emil von Behring, Orthopaedische Pathologie - Referenzzentrum, Institut fuer Gewebediagnostik Berlin, Berlin (Germany)

    2016-06-15

    Osteogenic tumors include malignant and benign tumors that produce tumor osteoid and/or bone tissue. Osteosarcoma is the most common malignant bone tumor, especially in children and young adults. The entities with their characteristic morphological features are described to enable the reader to come to a diagnosis and differential diagnosis on the basis of patient age, history and predominant location of the tumor. For this review we selectively used mainly large published patient cohorts. Our own and externally published data on widely accepted tumor criteria were also compared. Detection is the initial diagnostic step for an osseous lesion, and is determined by the sensitivity of the method applied. Plain X-ray films in two planes and CT are the basics in the radiological toolkit for osteogenic tumors. For evaluation of local tumor extension and biopsy planning MRI or scintigraphy should be combined. MRI as a stand-alone diagnostic tool is insufficient. For malignant bone tumors staging should be performed, applying a variable combination of thoracic CT, MRI, scintigraphy, and positron emission tomography (PET). Osteosarcoma, along with Ewing sarcoma and chondrosarcoma, are the most common malignant bone tumors; all sub-entities are significantly rarer. Among benign bone tumors, osteoid osteomas have the highest incidence, presenting with typical pain, location, and age predilection. Diagnostics and treatment of malignant bone tumors should preferably be performed in specialized centers because of significant therapeutic implications for patients. In uncertain cases, a second opinion should always be obtained. (orig.) [German] Osteogene Tumoren umfassen maligne und benigne Tumoren, die eine tumoreigene Produktion von Osteoid und/oder Knochengewebe aufweisen. Das Osteosarkom ist der haeufigste maligne Knochentumor v. a. bei Kindern und jungen Erwachsenen. Es werden die Entitaeten mit ihren morphologischen Charakteristika beschrieben, um anhand wichtiger

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

    Science.gov (United States)

    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. Copyright © 2015 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  9. Studies on culture and osteogenic induction of human mesenchymal stem cells under CO2-independent conditions.

    Science.gov (United States)

    Chen, Jian; Zhang, Cui; Feng, Yiding; Zong, Chen; Chen, Jiarong; Tang, Zihua; Jia, Bingbing; Tong, Xiangming; Zheng, Qiang; Wang, Jinfu

    2013-04-01

    Human mesenchymal stem cells (hMSCs) are one of the important factors that regulate bone anabolism. Osteoporosis resulting from microgravity during spaceflight may possibly be due to a decrease in osteogenesis mediated by hMSCs. This speculation should be verified through culture and osteogenic induction of hMSCs in a microgravity environment during spaceflight. Control of CO2 is a key component in current experimental protocols for growth, survival, and proliferation of in vitro cultured cells. However, carrying CO2 tanks on a spaceflight and devoting space/mass allowances for classical CO2 control protocols make experimentation on culture and osteogenesis difficult during most missions. Therefore, an experimental culture and osteogenic medium was developed through modifying the components of buffer salts in conventional culture medium. This experimental medium was used to culture and induce hMSCs under CO2-independent conditions. The results showed that culture and induction of hMSCs with conventional culture medium and conventional osteogenic medium under CO2-independent conditions resulted in an increase of pH in medium. The proliferation of hMSCs was also inhibited. hMSCs cultured with experimental culture medium under CO2-independent conditions showed a proliferation potential that was the same as those cultured with conventional culture medium under CO2-dependent conditions. The experimental osteogenic medium could promote hMSCs to differentiate into osteoblast-like cells under CO2-independent conditions. Cells induced by this induction system showed high alkaline phosphatase activity. The expression levels of osteogenic genes in cells induced with experimental osteogenic medium under CO2-independent conditions were not significantly different from those cells induced with conventional osteogenic medium under CO2-dependent conditions. These results suggest that the experimental culture and induction system could be used to culture hMSCs and induce the

  10. Bifunctional coating based on carboxymethyl chitosan with stable conjugated alkaline phosphatase for inhibiting bacterial adhesion and promoting osteogenic differentiation on titanium

    Science.gov (United States)

    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.

  11. Influence of poly(3-hydroxybutyrate-co-4-hydroxybutyrate-co-3-hydroxyhexanoate) on growth and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Wei, Xing; Hu, Ya-Jun; Xie, Wen-Peng; Lin, Ruo-Ling; Chen, Guo-Qiang

    2009-09-01

    As a new member of polyhydroxyalkanoate (PHA) family, the novel polyester poly(3-hydroxybutyrate-co-4-hydroxybutyrate-co-3-hydroxyhexanoate) (P(3HB-4HB-3HHx)) was produced by recombinant Aeromonas hydrophila 4AK4 and used for the first time to test its biocompatibility. It was shown that P(3HB-4HB-3HHx) had higher hydrophobicity, surface energy, and rougher surface than the well-studied polymers poly(L-lactic acid) (PLA) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). Human mesenchymal stem cells (MSCs) attached better on P(3HB-4HB-3HHx) film than on tissue culture plates (TCPs), PLA film, and PHBHHx film. MSC proliferation on P(3HB-4HB-3HHx) film was 126% higher than that on TCPs, 84% higher than that on PHBHHx film, and 312% higher than that on PLA film (p < 0.01). P(3HB-4HB-3HHx) also supported osteogenic differentiation of MSCs. Previous studies found that all PHA materials tested were either less than or equal to TCPs for supporting cell growth. Among all PHA materials tested, P(3HB-4HB-3HHx) was the only PHA material to significantly promote cell proliferation compared with TCPs. P(3HB-4HB-3HHx) could be exploited for applications in bone tissue engineering.

  12. In vitro differentiation of mouse embryonic stem cells into functional ...

    African Journals Online (AJOL)

    In vitro differentiation of mouse embryonic stem cells into functional hepatocytes by sodium butyrate, hepatocyte growth factor and dexamethasone under ... under chemically defined conditions, which might be useful as an in vitro system for hepatocyte transplantation therapy and toxicity screening in drug discovery.

  13. Nonpulsed sinusoidal electromagnetic fields as a noninvasive strategy in bone repair: the effect on human mesenchymal stem cell osteogenic differentiation.

    Science.gov (United States)

    Ledda, Mario; D'Emilia, Enrico; Giuliani, Livio; Marchese, Rodolfo; Foletti, Alberto; Grimaldi, Settimio; Lisi, Antonella

    2015-02-01

    In vivo control of osteoblast differentiation is an important process needed to maintain the continuous supply of mature osteoblast cells for growth, repair, and remodeling of bones. The regulation of this process has also an important and significant impact on the clinical strategies and future applications of cell therapy. In this article, we studied the effect of nonpulsed sinusoidal electromagnetic field radiation tuned at calcium-ion cyclotron frequency of 50 Hz exposure treatment for bone differentiation of human mesenchymal stem cells (hMSCs) alone or in synergy with dexamethasone, their canonical chemical differentiation agent. Five days of continuous exposure to calcium-ion cyclotron resonance affect hMSC proliferation, morphology, and cytoskeletal actin reorganization. By quantitative real-time polymerase chain reaction, we also observed an increase of osteoblast differentiation marker expression such as Runx2, alkaline phosphatase (ALP), osteocalcin (OC), and osteopontin (OPN) together with the osteoprotegerin mRNA modulation. Moreover, in these cells, the increase of the protein expression of OPN and ALP was also demonstrated. These results demonstrate bone commitment of hMSCs through a noninvasive and biocompatible differentiating physical agent treatment and highlight possible applications in new regenerative medicine protocols.

  14. The effect of newer anti-rheumatic drugs on osteogenic cell proliferation: an in-vitro study

    Directory of Open Access Journals (Sweden)

    Laing Patrick

    2009-05-01

    Full Text Available Abstract Background Disease modifying anti-rheumatic drugs (DMARDs may interfere with bone healing. Previous studies give conflicting advice regarding discontinuation of these drugs in the peri-operative setting. No consensus exists in current practice especially with the newer DMARDs such as Leflunomide, Etanercept, and Infliximab. The aim of this study was to assess the in-vitro effect of these drugs alone and in relevant clinical combinations on Osteoblast activity. Methods Osteoblasts were cultured from femoral heads obtained from five young otherwise healthy patients undergoing total hip replacement. The cells were cultured using techniques that have been previously described. A full factorial design was used to set up the experiment on samples obtained from the five donors. Normal therapeutic concentrations of the various DMARDs were added alone and in combination to the media. The cell proliferation was estimated after two weeks using spectrophotometric technique using Roche Cell proliferation Kit. Multilevel regression analysis was used to estimate which drugs or combination of drugs significantly affected cell proliferation. Results Infliximab and Leflunomide had an overall significant inhibitory effect (p Conclusion Our study indicates that in-vitro osteoblast proliferation can be inhibited by the presence of certain DMARDs. Combinations of drugs had an influence and could negate the action of a drug on osteoblast proliferation. The response to drugs may be donor-dependent.

  15. Effect of dynamic 3-D culture on proliferation, distribution, and osteogenic differentiation of human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Stiehler, Maik; Bünger, Cody; Baatrup, Anette

    2009-01-01

    Ex vivo engineering of autologous bone tissue as an alternative to bone grafting is a major clinical need. In the present study, we evaluated the effect of 3-D dynamic spinner flask culture on the proliferation, distribution, and differentiation of human mesenchymal stem cells (MSCs). Immortalize...

  16. Inhibition of Viability, Proliferation, Cytokines Secretion, Surface Antigen Expression, and Adipogenic and Osteogenic Differentiation of Adipose-Derived Stem Cells by Seven-Day Exposure to 0.5 T Static Magnetic Fields

    Directory of Open Access Journals (Sweden)

    Jian Wang

    2016-01-01

    Full Text Available After seven-day exposure to 0.5-Tesla Static Magnetic Field (SMF, Adipose-derived Stem Cells (ASCs and those labeled by superparamagnetic iron oxide (SPIO nanoparticles were examined for viability by methyl thiazol tetrazolium (MTT assay, proliferation by cell counting and bromodeoxyuridine (BrdU incorporation, DNA integrity by single cell gel electrophoresis, surface antigen by flow cytometry analysis, and the expression of cytokines and genetic markers by reverse transcription-PCR and underwent adipogenic and osteogenic differentiation assessed by quantifying related specific genes expression. The SMF slightly reduced cell viability and proliferation and inhibited the expression of CD49d, CD54, and CD73 but did not damage DNA integrity. The SMF slightly downregulated the expression of cytokines including Vascular Endothelial Growth Factor (VEGF, Insulin-like Growth Factor-1 (IGF-1, Transforming Growth Factor Beta 1 (TGF-β1, genetic markers comprising Stem Cell Antigen-1 (Sca1, Octamer-4 (Oct-4, ATP-binding Cassette Subfamily B Member 1 (ABCB1, adipogenic marker genes containing Lipoprotein Lipase (LPL, Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ, and osteogenic marker genes including Secreted Phosphor-protein 1 (SPP1 and Osterix (OSX. Exposure to 0.5 T SMF for seven days inhibited viability, proliferation, surface antigen expression, cytokine secretion, stem cell genetic marker expression, and adipogenic and osteogenic differentiation but did not affect the DNA integrity in ASCs with or without SPIO labeling.

  17. Ibandronate promotes osteogenic differentiation of periodontal ligament stem cells by regulating the expression of microRNAs

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Qiang [Department of General Dentistry and Emergency, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Zhao, Zhi-Ning [Clinical Laboratory, 451 Hospital of Chinese PLA, Xi' an 710054 (China); Cheng, Jing-Tao [Department of Special Dentistry, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Zhang, Bin [Department of Orthodontics, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Xu, Jie [Department of Periodontology, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Huang, Fei; Zhao, Rui-Ni [Department of General Dentistry and Emergency, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Chen, Yong-Jin, E-mail: cyj1229@fmmu.edu.cn [Department of General Dentistry and Emergency, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China)

    2011-01-07

    Research highlights: {yields} Ibandronate significantly promote the proliferation of PDLSC cells. {yields} Ibandronate enhanced the expression of ALP, COL-1, OPG, OCN, Runx2. {yields} The expression of a class of miRNAs, e.g., miR-18a, miR-133a, miR-141 and miR-19a, was significantly modified in PDLSC cells cultured with ibandronate. {yields} Ibandronate regulates the expression of diverse bone formation-related genes via miRNAs in PDLSCs. {yields} Ibandronate can suppress the activity of osteoclast while promoting the proliferation of osteoblast by regulating the expression of microRNAs. -- Abstract: Bisphosphonates (BPs) have a profound effect on bone resorption and are widely used to treat osteoclast-mediated bone diseases. They suppress bone resorption by inhibiting the activity of mature osteoclasts and/or the formation of new osteoclasts. Osteoblasts may be an alternative target for BPs. Periodontal ligament stem cells (PDLSCs) exhibit osteoblast-like features and are capable of differentiating into osteoblasts or cementoblasts. This study aimed to determine the effects of ibandronate, a nitrogen-containing BP, on the proliferation and the differentiation of PDLSCs and to identify the microRNAs (miRNAs) that mediate these effects. The PDLSCs were treated with ibandronate, and cell proliferation was measured using the MTT (3-dimethylthiazol-2,5-diphenyltetrazolium bromide) assay. The expression of genes and miRNAs involved in osteoblastic differentiation was assayed using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). Ibandronate promoted the proliferation of PDLSCs and enhanced the expression of alkaline phosphatase (ALP), type I collagen (COL-1), osteoprotegerin (OPG), osteocalcin (OCN), and Runx2. The expression of miRNAs, including miR-18a, miR-133a, miR-141 and miR-19a, was significantly altered in the PDLSCs cultured with ibandronate. In PDLSCs, ibandronate regulates the expression of diverse bone formation

  18. Enhanced cell proliferation and osteogenic differentiation in electrospun PLGA/hydroxyapatite nanofibre scaffolds incorporated with graphene oxide.

    Directory of Open Access Journals (Sweden)

    Chuan Fu

    Full Text Available One of the goals of bone tissue engineering is to mimic native ECM in architecture and function, creating scaffolds with excellent biocompatibility, osteoinductive ability and mechanical properties. The aim of this study was to fabricate nanofibrous matrices by electrospinning a blend of poly (L-lactic-co-glycolic acid (PLGA, hydroxyapatite (HA, and grapheme oxide (GO as a favourable platform for bone tissue engineering. The morphology, biocompatibility, mechanical properties, and biological activity of all nanofibrous matrices were compared. The data indicate that the hydrophilicity and protein adsorption rate of the fabricated matrices were significantly increased by blending with a small amount of HA and GO. Furthermore, GO significantly boosted the tensile strength of the nanofibrous matrices, and the PLGA/GO/HA nanofibrous matrices can serve as mechanically stable scaffolds for cell growth. For further test in vitro, MC3T3-E1 cells were cultured on the PLGA/HA/GO nanofbrous matrices to observe various cellular activities and cell mineralization. The results indicated that the PLGA/GO/HA nanofibrous matrices significantly enhanced adhesion, and proliferation in MCET3-E1 cells and functionally promoted alkaline phosphatase (ALP activity, the osteogenesis-related gene expression and mineral deposition. Therefore, the PLGA/HA/GO composite nanofibres are excellent and versatile scaffolds for applications in bone tissue regeneration.

  19. Synergistic acceleration in the osteogenic and angiogenic differentiation of human mesenchymal stem cells by calcium silicate-graphene composites.

    Science.gov (United States)

    Shie, Ming-You; Chiang, Wei-Hung; Chen, I-Wen Peter; Liu, Wen-Yi; Chen, Yi-Wen

    2017-04-01

    Recent exciting findings of the biological interactions of graphene materials have shed light on potential biomedical applications of graphene-containing composites. Owing to the superior mechanical properties and low coefficient of thermal expansion, graphene has been widely used in the reinforcement of biocomposites. In the present study, various ratios of graphene (0.25wt%, 0.5wt% and 1.0wt%) were reinforced into calcium silicate (CS) for bone graft application. Results show that the graphene was embedded in the composites homogeneously. Adding 1wt% graphene into CS increased the young's modulus by ~47.1%. The formation of bone-like apatite on a range of composites with graphene weight percentages ranging from 0 to 1 has been investigated in simulated body fluid. The presence of a bone-like apatite layer on the composites surface after immersion in simulated body fluid was considered by scanning electron microscopy. In vitro cytocompatibility of the graphene-contained CS composites was evaluated using human marrow stem cells (hMSCs). The proliferation and alkaline phosphatase, osteopontin and osteocalcin osteogenesis-related protein expression of the hMSCs on the 1wt% graphene-contained specimens showed better results than on the pure CS. In addition, the angiogenesis-related protein (vWF and ang-1) secretion of cells was significantly stimulated when the graphene concentration in the composites was increased. These results suggest that graphene-contained CS bone graft are promising materials for bone tissue engineering applications. Copyright © 2016. Published by Elsevier B.V.

  20. Effect of internal structure of collagen/hydroxyapatite scaffold on the osteogenic differentiation of mesenchymal stem cells.

    Science.gov (United States)

    Chen, Guobao; Lv, Yonggang; Dong, Chanjuan; Yang, Li

    2015-01-01

    Consisting of seed cells and scaffold, regenerative medicine provides a new way for the repair and regeneration of tissue and organ. Collagen/hydroxyapatite (HA) biocomposite scaffold is highlighted due to its advantageous features of two major components of bone matrix: collagen and HA. The aim of this study is to investigate the effect of internal structure of collagen/HA scaffold on the fate of rat mesenchymal stem cells (MSCs). The internal structure of collagen/HA scaffold was characterized by micro-CT. It is found that the porosity decreased while average compressive modulus increased with the increase of collagen proportion. Within the collagen proportion of 0.35%, 0.5% and 0.7%, the porosities were 89.08%, 78.37% and 75.36%, the pore sizes were 140.6±75.5 μm, 133.9±48.4 μm and 160.7±119.6 μm, and the average compressive moduli were 6.74±1.16 kPa, 8.82±2.12 kPa and 23.61±8.06 kPa, respectively. Among these three kinds of scaffolds, MSCs on the Col 0.35/HA 22 scaffold have the highest viability and the best cell proliferation. On the contrary, the Col 0.7/HA 22 scaffold has the best ability to stimulate MSCs to differentiate into osteoblasts in a relatively short period of time. In vivo research also demonstrated that the internal structure of collagen/HA scaffold has significant effect on the cell infiltration. Therefore, precise control of the internal structure of collagen/HA scaffold can provide a more efficient carrier to the repair of bone defects.

  1. Human Adipose-Derived Mesenchymal Stem Cells Are Resistant to HBV Infection during Differentiation into Hepatocytes in Vitro

    Directory of Open Access Journals (Sweden)

    Ying Wang

    2014-04-01

    Full Text Available The therapeutic methods for chronic hepatitis B are limited. The shortage of organ donors and hepatitis B virus (HBV reinfection obstruct the clinical application of orthotopic liver transplantation (OLT. In the present study, adipose-derived mesenchymal stem cells (AD-MSCs and bone marrow-derived mesenchymal stem cells (BM-MSCs were isolated from chronic hepatitis B patients and characterized for morphology, growth potency, surface phenotype and the differentiation potential. The results showed that both MSCs had adipogenic, osteogenic and neuron differentiation potential, and nearly all MSCs expressed CD105, CD44 and CD29. Compared with AD-MSCs, BM-MSCs of chronic hepatitis B patients proliferated defectively. In addition, the ability of AD-MSCs to differentiate into hepatocyte was evaluated and the susceptibility to HBV infection were assessed. AD-MSCs could differentiate into functional hepatocyte-like cells. These cells express the hepatic-specific markers and have glycogen production and albumin secretion function. AD-MSCs and hepatic differentiation AD-MSCs were not susceptible to infection by HBV in vitro. Compared with BM-MSCs, AD-MSCs may be alternative stem cells for chronic hepatitis B patients.

  2. In vitro differentiation of mouse embryonic stem cells into functional ...

    African Journals Online (AJOL)

    Jane

    2011-08-22

    Aug 22, 2011 ... Key words: Embryonic stem cells, hepatic-like cells, in vitro differentiation, sodium butyrate, hepatocyte growth factor, dexamethason. INTRODUCTION. The liver is the major organ that provides multiple metabolic functions critical for the maintenance of homeostasis. One of the major causes of morbidity and.

  3. Osteogenic ability of Cu-bearing stainless steel.

    Science.gov (United States)

    Ren, Ling; Wong, Hoi Man; Yan, Chun Hoi; Yeung, Kelvin W K; Yang, Ke

    2015-10-01

    A newly developed copper-bearing stainless steel (Cu-SS) by directly immobilizing proper amount of Cu into a medical stainless steel (317L SS) during the metallurgical process could enable continuous release of trace amount of Cu(2+) ions, which play the key role to offer the multi-biofunctions of the stainless steel, including the osteogenic ability in the present study. The results of in vitro experiments clearly demonstrated that Cu(2+) ions from Cu-SS could promote the osteogenic differentiation by stimulating the Alkaline phosphatase enzyme activity and the osteogenic gene expressions (Col1a1, Opn, and Runx2), and enhancing the adhesion and proliferation of osteoblasts cultured on its surface. The in vivo test further proved that more new bone tissue formed around the Cu-SS implant with more stable bone-to-implant contact in comparison with the 317L SS. In addition, Cu-SS showed satisfied biocompatibility according to the results of in vitro cytotoxicity and in vivo histocompatibility, and its daily released amount of Cu(2+) ions in physiological saline solution was at trace level of ppb order (1.4 ppb/cm(2) ), which is rather safe to human health. Apart from these results, it was also found that Cu-SS could inhibit the happening of inflammation with lower TNF-α expression in the bone tissue post implantation compared with 317L SS. In addition to good biocompatibility, the overall findings demonstrated that the Cu-SS possessed obvious ability of promoting osteogenesis, indicating a unique application advantage in orthopedics. © 2014 Wiley Periodicals, Inc.

  4. The Osteogenic Potential of Human Non-differentiated and Pre-differentiated Mesenchymal Stem Cells Combined with an Osteoconductive Scaffold – Early Stage Healing

    Directory of Open Access Journals (Sweden)

    Luboš Tuček

    2017-06-01

    Full Text Available Despite the huge research into stem cells and their regenerative properties for bone healing, there are still unanswered questions including the recipient’s respond to the presence of the stem cells, the fate of stem cells inside the bone defect and the possible advantage in utilizing pre-differentiated cells. To address these problems, we used human multipotent mesenchymal stromal/stem cells (MSCs, GMP Grade, in a rat model of bone formation. In a “bioreactor concept” approach seven Wistar rats were implanted with 0.2 g of synthetic bone scaffold seeded with 2 × 106 MSCs, seven Wistar rats were implanted with 0.2 g of synthetic bone scaffold seeded with 1 × 106 predifferentiated osteoblasts and 1 × 106 pre-differentiated endothelial cells and 14 Wistar rats were implanted with 0.2 g of synthetic bone scaffold without seeded cells into an intramuscular pocket on the left side of their back. The right side of each rat was used as a control, and 0.2 g of synthetic bone scaffold was implanted into the intramuscular pocket alone. To see the early stage healing the samples were harvested 14 days after the implantation, MSCs were detected by positive DAPI and MTCO2 staining in 43% of all the samples implanted with MSCs, and no inflammation signs were present in any implanted animal. New vessels could be found in both groups implanted with MSCs, but not in the control group of animals. However, hematoxylin-eosin staining could not detect newly created bone within the implant in any of the groups. These results were in line with COLL1 staining, where we could detect positive staining only in three cases, all of which were implanted with un-differentiated MSCs. According to our findings, there were no benefits of using the pre-differentiated of MSC.

  5. Oxy133, a novel osteogenic agent, promotes bone regeneration in an intramembranous bone-healing model.

    Science.gov (United States)

    Li, Andrew; Hokugo, Akishige; Segovia, Luis Andres; Yalom, Anisa; Rezzadeh, Kameron; Zhou, Situo; Zhang, Zheyu; Parhami, Farhad; Stappenbeck, Frank; Jarrahy, Reza

    2017-05-01

    Current reconstructive techniques for complex craniofacial osseous defects are challenging and are associated with significant morbidity. Oxysterols are naturally occurring cholesterol oxidation products with osteogenic potential. In this study, we investigated the effects of a novel semi-synthetic oxysterol, Oxy133, on in vitro osteogenesis and an in vivo intramembranous bone-healing model. Rabbit bone marrow stromal cells (BMSCs) were treated with either Oxy133 or BMP-2. Alkaline phosphatase (ALP) activity, expression of osteogenic gene markers and in vitro mineralization were all examined. Next, collagen sponges carrying either Oxy133 or BMP-2 were used to reconstruct critical-sized cranial defects in mature rabbits and bone regeneration was assessed. To determine the mechanism of action of Oxy133 both in vitro and in vivo, rabbit BMSCs cultures and collagen sponge/Oxy133 implants were treated with the Hedgehog signalling pathway inhibitor, cyclopamine, and similar outcomes were measured. ALP activity in rabbit BMSCs treated with 1 μm Oxy133 was induced and was significantly higher than in control cells. These results were mitigated in cultures treated with cyclopamine. Expression of osteogenic gene markers and mineralization in BMSCs treated with 1 μm Oxy133 was significantly higher than in control groups. Complete bone regeneration was noted in vivo when cranial defects were treated with Oxy133; healing was incomplete, however, when cyclopamine was added. Collectively, these results demonstrate that Oxy133 has the ability to induce osteogenic differentiation in vitro in rabbit BMSCs and to promote robust bone regeneration in vivo in an animal model of intramembranous bone healing. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  6. In vitro germ cell differentiation during sex differentiation in a teleost fish.

    Science.gov (United States)

    Kobayashi, Tohru

    2010-01-01

    To clarify the sexually dimorphic mechanisms of gonadal sex differentiation, we established an in vitro culture system for gonadal sex differentiation using the teleost fish Oreochromis niloticus. In vivo, the entry of germ cells into meiosis occurs around 35 days after hatching (dah) in XX gonads, whereas in XY gonads, meiotic cells became differentiated around 85 dah. In our in vitro culture system using gonads from young fry at 23 dah, the meiotic cells in the XX gonads appeared after 21 days of culture. In contrast, in the XY gonads, no meiotic cells were detected after 21 days. These results indicate that germ cell differentiation in this culture system progresses in a manner similar to that in vivo. To identify the gene products that are involved in the entry of germ cells into meiosis or in the arrest of germ cells at the gonial stage of gonadal sex differentiation, we performed subtractive hybridization screening with this in vitro culture system. From the screening process, we identified the female-related gene, FR-3, which is a homolog of zebrafish nanos-related gene (nos). The nos gene was expressed after gonadal formation around 35 dah in XX gonads, but not in XY gonads. In situ hybridization indicated that nos is expressed in oogenic meiotic cells, but not in spermatogenic meiotic cells. Further examination revealed that nos was expressed in oogenic meiotic cells after gonadal formation, specifically in teleost fish. Together, nos may be also involved in oogenic meiosis, with the exception of primordial germ cell migration.

  7. Biocompatibility and osteogenic properties of porous tantalum

    OpenAIRE

    Wang, Qian; Zhang, Hui; LI, QIJIA; Ye, Lei; Gan, Hongquan; Liu, Yingjie; Wang, Hui; Wang, Zhiqiang

    2015-01-01

    Porous tantalum has been reported to be a promising material for use in bone tissue engineering. In the present study, the biocompatibility and osteogenic properties of porous tantalum were studied in vitro and in vivo. The morphology of porous tantalum was observed using scanning electron microscopy (SEM). Osteoblasts were cultured with porous tantalum, and cell morphology, adhesion and proliferation were investigated using optical microscopy and SEM. In addition, porous tantalum rods were i...

  8. In Vitro Studies on the Degradability, Bioactivity, and Cell Differentiation of PRP/AZ31B Mg Alloys Composite Scaffold

    Directory of Open Access Journals (Sweden)

    Jian Zou

    2017-01-01

    Full Text Available In recent years, more and more methods have been developed to improve the bioactivity of the biodegradable materials in bone tissue regeneration. In present study, we used rat mesenchymal stem cells (rMSCs to evaluate the outcomes of Mg alloys (AZ31B, Magnesium, and Aluminum and Platelet-rich plasma (PRP/Mg alloys on rMSCs biocompatibility and osteogenic differentiation. Water absorption experiments indicated that both bare AZ31B and PRP/AZ31B were capable of absorbing large amounts of water. But the water absorption ratio for PRP/AZ31B was significantly higher than that for bare AZ31B. The degradability experiments implied that both samples degraded at same speed. rMSCs on the surface of AZ31B distributed more and better than those on the AZ31B scaffold. In ALP activity experiment, the activity of rMSCs on the PRP/AZ31B was markedly higher than that on the AZ31B scaffolds on the 7th day and 14th day. qRT-PCR also showed that OPN and OCN were expressed in both samples. OPN and OCN expression in PRP/AZ31B sample were higher than those in bare AZ31B samples. In summary, the in vitro study implied that AZ31B combined with PRP could remarkably improve cell seeding, attachment, proliferation, and differentiation.

  9. Treatment with a growth factor-protein mixture inhibits formation of mineralized nodules in osteogenic cell cultures grown on titanium.

    Science.gov (United States)

    de Oliva, Marcos Andrade; Maximiano, William Marcatti Amarú; de Castro, Larissa Moreira Spínola; da Silva, Paulo Eliandro; Fernandes, Roger Rodrigo; Ciancaglini, Pietro; Beloti, Márcio Mateus; Nanci, Antonio; Rosa, Adalberto Luiz; de Oliveira, Paulo Tambasco

    2009-03-01

    Despite wide clinical application, the efficacy of platelet-rich plasma (PRP) for repairing bone defects and enhancing osseointegration of metal implants is still subject of debate. This study aimed to evaluate the effects of a well-defined PRP-like mixture containing platelet-derived growth factor-BB, transforming growth factor (TGF)-beta1, TGF-beta2, albumin, fibronectin, and thrombospondin [growth factors (GFs) + proteins] on the development of the osteogenic phenotype on titanium (Ti) in vitro. Human alveolar bone-derived osteoblastic cells were subcultured on Ti discs and exposed during the first 7 days to osteogenic medium supplemented with GFs + proteins and to osteogenic medium alone thereafter up to 14 days. Control cultures were exposed to only osteogenic medium. Dose-response experiments were carried out using rat primary calvarial cells exposed to GFs + proteins and 1:10 or 1:100 dilutions of the mixture. Treated human-derived cell cultures exhibited a significantly higher number of cycling cells at days 1 and 4 and of total cells at days 4 and 7, significantly reduced alkaline phosphatase (ALP) activity at days 4, 7, and 10, and no Alizarin red-stained areas (calcium deposits) at day 14, indicating an impairment in osteoblast differentiation. Although the 1:10 and 1:100 dilutions of the mixture restored the proliferative activity of rat-derived osteogenic cells to control levels and promoted a significant increase in ALP activity at day 10 compared with GFs + proteins, mineralized nodule formation was only observed with the 1:100 dilution ( approximately 50% of the control). These results showed that a PRP-like protein mixture inhibits development of the osteogenic phenotype in both human and rat osteoblastic cell cultures grown on Ti.

  10. Evaluation of osteogenic cell differentiation in response to bone morphogenetic protein or demineralized bone matrix in a critical sized defect model using GFP reporter mice.

    Science.gov (United States)

    Alaee, Farhang; Hong, Seung-Hyun; Dukas, Alex G; Pensak, Michael J; Rowe, David W; Lieberman, Jay R

    2014-09-01

    We evaluated the osteoprogenitor response to rhBMP-2 and DBM in a transgenic mouse critical sized defect. The mice expressed Col3.6GFPtopaz (a pre-osteoblastic marker), Col2.3GFPemerald (an osteoblastic marker) and α-smooth muscle actin (α-SMA-Cherry, a pericyte/myofibroblast marker). We assessed defect healing at various time points using radiographs, frozen, and conventional histologic analyses. GFP signal in regions of interest corresponding to the areas of new bone formation was quantified using a novel computer assisted algorithm. All defects treated with rhBMP-2 healed. In contrast, the majority of the defects in the DBM (27/30) and control (28/30) groups did not heal. Quantitation of pre-osteoblasts demonstrated a maximal response (% GFP + cells/TV) in the Col3.6GFPtopaz mice at day 7 (7.2% ± 6.0, p < 0.05 compared to days 14, 21, 28, and 56). The maximal response of the Col2.3GFP cells was seen at days 14 (8.04% ± 5.0) and 21 (8.31% ± 4.32), p < 0.05. In contrast, DBM and control groups showed a limited osteogenic response at all time points. In conclusion, we demonstrated that the BMP and DBM induce vastly different osteogenic responses which should influence their clinical application as bone graft substitutes. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  11. Preparation of well-distributed titania nanopillar arrays on Ti6Al4V surface by induction heating for enhancing osteogenic differentiation of stem cells

    Science.gov (United States)

    Li, Ning-Bo; Sun, Sheng-Jun; Bai, Han-Ying; Xiao, Gui-Yong; Xu, Wen-Hua; Zhao, Jun-Han; Chen, Xin; Lu, Yu-Peng; Zhang, Yi-Lin

    2018-01-01

    Great effort has recently been devoted to the preparation of nanoscale surfaces on titanium-based implants to achieve clinically fast osteoinduction and osseointegration, which relies on the unique characteristics of the nanostructure. In this work, we used induction heating treatment (IHT) as a rapid oxidation method to fabricate a porous nanoscale oxide layer on the Ti6Al4V surface for better medical application. Well-distributed vertical nanopillars were yielded by IHT for 20–35 s on the alloy surface. The composition of the oxides contained rutile/anatase TiO2 and a small amount of Al2O3 between the TiO2 grain boundaries (GBs). This technology resulted in a reduction and subsequent increase of surface roughness of 26–32 nm when upregulating the heating time, followed by the successive enhancement of the thickness, wettability and adhesion strength of the oxidation layer to the matrix. The surface hardness also distinctly rose to 554 HV in the IHT-35 s group compared with the 350 HV of bare Ti6Al4V. The massive small-angle GBs in the bare alloy promoted the formation of nanosized oxide crystallites. The grain refinement and deformation texture reduction further improved the mechanical properties of the matrix after IHT. Moreover, in vitro experiments on a mesenchymal stem cell (BMSC) culture derived from human bone marrow for 1–7 days indicated that the nanoscale layers did not cause cytotoxicity, and facilitated cell differentiation in osteoblasts by enhancing the gene and osteogenesis-related protein expressions after 1–3 weeks of culturing. The increase of the IHT time slightly advanced the BMSC proliferation and differentiation, especially during long-term culture. Our findings provide strong evidence that IHT oxidation technology is a novel nanosurface modification technology, which is potentially promising for further clinical development.

  12. RGD and BMP-2 mimetic peptide crosstalk enhances osteogenic commitment of human bone marrow stem cells.

    Science.gov (United States)

    Bilem, I; Chevallier, P; Plawinski, L; Sone, E D; Durrieu, M C; Laroche, G

    2016-05-01

    Human bone marrow mesenchymal stem cells (hBMSCs) commitment and differentiation are dictated by bioactive molecules sequestered within their Extra Cellular Matrix (ECM). One common approach to mimic the physiological environment is to functionalize biomaterial surfaces with ECM-derived peptides able to recruit stem cells and trigger their linage-specific differentiation. The objective of this work was to investigate the effect of RGD and BMP-2 ligands crosstalk and density on the extent of hBMSCs osteogenic commitment, without recourse to differentiation medium. RGD peptide promotes cell adhesion via cell transmembrane integrin receptors, while BMP-2 peptide, corresponding to residues 73-92 of Bone Morphogenetic Protein-2, was shown to induce hBMSCs osteoblast differentiation. The immobilization of peptides on aminated glass was ascertained by X-ray Photoelectron Spectroscopy (XPS), the density of grafted peptides was quantified by fluorescence microscopy and the surface roughness was evaluated using Atomic Force Microscopy (AFM). The osteogenic commitment of hBMSCs cultured on RGD and/or BMP-2 surfaces was characterized by immunohistochemistry using STRO-1 as specific stem cells marker and Runx-2 as an earlier osteogenic marker. Biological results showed that the osteogenic commitment of hBMSCs was enhanced on bifunctionalized surfaces as compared to surfaces containing BMP-2, while on RGD surfaces cells mainly preserved their stemness character. These results demonstrated that RGD and BMP-2 mimetic peptides act synergistically to enhance hBMSCs osteogenesis without supplementing the media with osteogenic factors. These findings contribute to the development of biomimetic materials, allowing a deeper understanding of signaling pathways that govern the transition of stem cells towards the osteoblastic lineage. For a long time, scientists thought that the differentiation of Mesenchymal Stem Cells (MSCs) into bone cells was dictated by growth factors. This

  13. Semaphorin 3A Shifts Adipose Mesenchymal Stem Cells towards Osteogenic Phenotype and Promotes Bone Regeneration In Vivo

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    Xiangwei Liu

    2016-01-01

    Full Text Available Adipose mesenchymal stem cells (ASCs are considered as the promising seed cells for bone regeneration. However, the lower osteogenic differentiation capacity limits its therapeutic efficacy. Identification of the key molecules governing the differences between ASCs and BMSCs would shed light on manipulation of ASCs towards osteogenic phenotype. In this study, we screened semaphorin family members in ASCs and BMSCs and identified Sema3A as an osteogenic semaphorin that was significantly and predominantly expressed in BMSCs. The analyses in vitro showed that the overexpression of Sema3A in ASCs significantly enhanced the expression of bone-related genes and extracellular matrix calcium deposition, while decreasing the expression of adipose-related genes and thus lipid droplet formation, resembling a BMSCs phenotype. Furthermore, Sema3A modified ASCs were then engrafted into poly(lactic-co-glycolic acid (PLGA scaffolds to repair the critical-sized calvarial defects in rat model. As expected, Sema3A modified ASCs encapsulation significantly promoted new bone formation with higher bone volume fraction and bone mineral density. Additionally, Sema3A was found to simultaneously increase multiple Wnt related genes and thus activating Wnt pathway. Taken together, our study here identifies Sema3A as a critical gene for osteogenic phenotype and reveals that Sema3A-modified ASCs would serve as a promising candidate for bettering bone defect repair.

  14. MBG-Modified β-TCP Scaffold Promotes Mesenchymal Stem Cells Adhesion and Osteogenic Differentiation via a FAK/MAPK Signaling Pathway.

    Science.gov (United States)

    Liu, Yutong; Ma, Yifan; Zhang, Jing; Xie, Qing; Wang, Zi; Yu, Shuang; Yuan, Yuan; Liu, Changsheng

    2017-09-13

    The β-TCP scaffold has been widely used as a bone graft substitute, but the traditional PMMA molding method-induced undesirable mechanical strength and poor interconnectivity still have not been addressed until now. In this study, a MBG-based PU foam templating method was developed to fabricate β-TCP scaffolds with desirable microtopography. The MBG gel, as both binder and modifier, prepared by a modified sol-gel method with controlled viscosity is incorporated with β-TCP powder and thereafter is impregnated into PU foam. The resultant hybrid scaffolds exhibited interconnected macropores (200-500 μm) and distinctive micropores (0.2-1.5 μm), especially for the TCP/25MBG (with 25 wt % content MBG). As expected, the compression strength of β-TCP/MBG composite scaffolds was enhanced with increasing MBG content, and TCP/50MBG (with 50 wt % content MBG) exhibited almost 100-fold enhancement compared to the pure β-TCP. Intriguingly, the cell affinity and osteogenic capacity of rBMSCs were also dramatically improved the best on TCP/25MBG. Further investigation found that the subtle, grainy-like microtopography, not the chemical composition, of the TCP/25MBG favored the adsorption of Fn and expression of integrin α5β1 and further facilitated FA formation and the expression of p-FAK, following activation of the MAPK/ERK signaling pathway and ultimately upregulated expression of osteogenic genes. Further in vivo experiments confirmed the promoted osteogenesis of TCP/25MBG in vivo. The results suggest that such a novel MBG-based PU foam templating method offers new guidance to construct hierarchically porous scaffolds, and the prepared MBG-modified β-TCP scaffold will have great potential for future use in bone tissue regeneration.

  15. * Harnessing the Osteogenicity of In Vitro Stem Cell-Derived Mineralized Extracellular Matrix as 3D Biotemplate to Guide Bone Regeneration.

    Science.gov (United States)

    Chai, Yoke Chin; Bolander, Johanna; Papantoniou, Ioannis; Patterson, Jennifer; Vleugels, Jef; Schrooten, Jan; Luyten, Frank P

    2017-09-01

    Advanced biomaterials that are capable of guiding robust bone regeneration are highly demanded for translational therapy of bone defects or bone augmentation in clinics. One of the strategic approaches is to produce tissue engineering (TE) constructs that mediate bone regeneration by recapitulating the natural bone formation or healing process. In this study, we aimed at producing devitalized mineralized carriers with augmented bone forming capacity via a modified culture protocol (i.e., culture conditions with high calcium and/or phosphate concentrations) that first promotes cell growth and, subsequently, mineralized extracellular matrix (ECM) deposition by human periosteum-derived osteoprogenitor cells (hPDCs) on additive manufactured three-dimensional (3D) porous titanium (Ti)-based scaffolds. Qualitative and quantitative analysis was performed to characterize the physicochemical properties of the produced devitalized mineralized carriers, as well as their effects as carriers on in vitro cell growth and osteochondrogenic differentiation of hPDCs under a perfusion bioreactor culture set-up. The results showed that the modified culture protocol was useful to produce devitalized mineralized carriers with different amount, distribution, composition, and morphology of mineralized matrix that resembled hydroxyapatite, and exhibited different Ca2+ release kinetics, distinct human bone morphogenetic protein (hBMP)-2, human vascular endothelial growth factor (hVEGF) proteins, and collagen contents. The produced devitalized mineralized carriers supported 3D growth of hPDCs, with minor osteochondrogenic differentiation effects under the perfusion bioreactor culture condition. Subcutaneous implantation of hPDC-seeded devitalized mineralized carriers in athymic nude rats showed nearly five-fold augmentation in the ectopic bone-forming capacity, with no bone induction obtained for unseeded, devitalized mineralized carriers and plain Ti scaffolds. Implantation of devitalized

  16. Disulfiram attenuates osteoclast differentiation in vitro: a potential antiresorptive agent.

    Directory of Open Access Journals (Sweden)

    Hua Ying

    Full Text Available Disulfiram (DSF, a cysteine modifying compound, has long been clinically employed for the treatment of alcohol addiction. Mechanistically, DSF acts as a modulator of MAPK and NF-κB pathways signaling pathways. While these pathways are crucial for osteoclast (OC differentiation, the potential influence of DSF on OC formation and function has not been directly assessed. Here, we explore the pharmacological effects of DSF on OC differentiation, activity and the modulation of osteoclastogenic signaling cascades. We first analyzed cytotoxicity of DSF on bone marrow monocytes isolated from C57BL/6J mice. Upon the establishment of optimal dosage, we conducted osteoclastogenesis and bone resorption assays in the presence or absence of DSF treatment. Luciferase assays in RAW264.7 cells were used to examine the effects of DSF on major transcription factors activation. Western blot, reverse transcription polymerase chain reaction, intracellular acidification and proton influx assays were employed to further dissect the underlying mechanism. DSF treatment dose-dependently inhibited both mouse and human osteoclastogenesis, especially at early stages of differentiation. This inhibition correlated with a decrease in the expression of key osteoclastic marker genes including CtsK, TRAP, DC-STAMP and Atp6v0d2 as well as a reduction in bone resorption in vitro. Suppression of OC differentiation was found to be due, at least in part, to the blockade of several key receptor activators of nuclear factor kappa-B ligand (RANKL-signaling pathways including ERK, NF-κB and NFATc1. On the other hand, DSF failed to suppress intracellular acidification and proton influx in mouse and human osteoclasts using acridine orange quenching and microsome-based proton transport assays. Our findings indicate that DSF attenuates OC differentiation via the collective suppression of several key RANKL-mediated signaling cascades, thus making it an attractive agent for the treatment of OC

  17. IL-4 Induces Cholinergic Differentiation of Retinal Cells In Vitro.

    Science.gov (United States)

    Granja, Marcelo Gomes; Braga, Luis Eduardo Gomes; Carpi-Santos, Raul; de Araujo-Martins, Leandro; Nunes-Tavares, Nilson; Calaza, Karin C; Dos Santos, Aline Araujo; Giestal-de-Araujo, Elizabeth

    2015-07-01

    Interleukin-4 (IL-4) is a pleiotropic cytokine that regulates several phenomena, among them survival and differentiation of neuronal and glial cells. The aim of this work was to investigate the effect of IL-4 on the cholinergic differentiation of neonatal rat retinal cells in vitro, evaluating its effect on the levels of cholinergic markers (CHT1-high-affinity choline transporter; VAChT-vesicular acetylcholine transporter, ChAT-choline acetyltransferase, AChE-acetylcholinesterase), muscarinic receptors, and on the signaling pathways involved. Lister Hooded rat pups were used in postnatal days 0-2 (P0-P2). Our results show that IL-4 treatment (50 U/mL) for 48 h increases the levels of the cholinergic transporters VAChT and CHT1, the acetylcholinesterase activity, and the number of ChAT-positive cells. It also induces changes in muscarinic receptor levels, leading to a small decrease in M1 levels and a significant increase in M3 and M5 levels after 48 h of treatment. We also showed that IL-4 effect on M3 receptors is dependent on type I IL-4 receptor and on an increase in NFκB phosphorylation. These results indicate that IL-4 stimulates cholinergic differentiation of retinal cells.

  18. Lin28a enhances in vitro osteoblastic differentiation of human periosteum-derived cells.

    Science.gov (United States)

    Park, Jin-Ho; Park, Bong-Wook; Kang, Young-Hoon; Byun, Sung-Hoon; Hwang, Sun-Chul; Kim, Deok Ryong; Woo, Dong Kyun; Byun, June-Ho

    2017-12-01

    Despite a capacity for proliferation and an ability to differentiate into multiple cell types, in long-term culture and with ageing, stem cells show a reduction in growth, display a decrease in differentiation potential, and enter senescence without evidence of transformation. The Lin28a gene encodes an RNA-binding protein that plays a role in regulating stem cell activity, including self-renewal and differentiation propensity. However, the effect of the Lin28a gene on cultured human osteoprecursor cells is poorly understood. In the present study, alkaline phosphatase activity, alizarin red-positive mineralization, and calcium content, positive indicators of osteogenic differentiation, were significantly higher in cultured human periosteum-derived cells (hPDCs) with Lin28a overexpression compared with cells without Lin28a overexpression. Lin28a overexpression by hPDCs also increased mitochondrial activity, which is essential for cellular proliferation, as suggested by a reduced presence of reactive oxygen species and significantly enhanced lactate levels and ATP production. Our results suggest that, in hPDCs, the Lin28a gene enhances osteoblastic differentiation and increases mitochondrial activity. Although Lin28a is known as a marker of undifferentiated human embryogenic stem cell, there is limited evidence regarding the influence of Lin28a on osteoblastic differentiation of cultured osteoprecursor cells. This study was to examine the impact of Lin28a on osteogenic phenotypes of human periosteum-derived cells. Their phenotypes can be similar to those of mesenchymal stem cells. Our results suggest that the Lin28a gene enhances the osteoblastic differentiation of human periosteum-derived cells. In addition, the Lin28a gene increases mitochondrial activity in human periosteum-derived cells. Copyright © 2017 John Wiley & Sons, Ltd.

  19. Indomethacin induces differential effects on in vitro endochondral ossification depending on the chondrocyte's differentiation stage.

    Science.gov (United States)

    Caron, Marjolein M J; Emans, Pieter J; Cremers, Andy; Surtel, Don A M; van Rhijn, Lodewijk W; Welting, Tim J M

    2017-04-01

    Heterotopic ossification (HO) is the abnormal formation of bone in soft tissues and is a frequent complication of hip replacement surgery. Heterotopic ossifications are described to develop via endochondral ossification and standard treatment is administration of indomethacin. It is currently unknown how indomethacin influences heterotopic ossification on a molecular level; therefore, we aimed to determine whether indomethacin might influence heterotopic ossification via impairing the chondrogenic phase of endochondral ossification. Progenitor cell models differentiating in the chondrogenic lineage (ATDC5, primary human bone marrow stem cells and ex vivo periosteal agarose cultures) were treated with increasing concentrations of indomethacin and a decrease in gene- and protein expression of chondrogenic and hypertrophic markers (measured by RT-qPCR and immunoblotting) as well as decreased glycosamino-glycan content (by alcian blue histochemistry) was observed. Even when hypertrophic differentiation was provoked, the addition of indomethacin resulted in decreased hypertrophic marker expression. Interestingly, when mature chondrocytes were treated with indomethacin, a clear increase in collagen type 2 expression was observed. Similarly, when ATDC5 cells and bone marrow stem cells were pre-differentiated to obtain a chondrocyte phenotype and indomethacin was added from this time point onward, low concentrations of indomethacin also resulted in increased chondrogenic differentiation. Indomethacin induces differential effects on in vitro endochondral ossification, depending on the chondrocyte's differentiation stage, with complete inhibition of chondrogenic differentiation as the most pronounced action. This observation may provide a rational behind the elusive mode of action of indomethacin in the treatment of heterotopic ossifications. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:847-857, 2017. © 2016 Orthopaedic Research

  20. Calcium-mediated differentiation of ameloblast lineage cells in vitro.

    Science.gov (United States)

    Chen, James; Zhang, Yan; Mendoza, Joseph; Denbesten, Pamela

    2009-07-15

    Calcium is a key component of the mineralized enamel matrix, but may also have a role in ameloblast cell differentiation. In this study we used human ameloblast lineage cells to determine the effect of calcium on cell function. Primary human ameloblast lineage cells were isolated from human fetal tooth buds. Cells were treated with calcium ranging from 0.05 to -1.8 mM. Cell morphology was imaged by phase contrast microscopy, and amelogenin was immunolocalized. Proliferation of cells treated with calcium was measured by BrdU immunoassay. The effect of calcium on mRNA expression of amelogenin, Type 1 collagen, DSPP, amelotin, and KLK-4 was compared by PCR analysis. Von Kossa staining was used to detect mineral formation after cells were pretreated with calcium. Calcium induced cell organization and clustering at 0.1 and 0.3 mM concentrations. Increasing concentrations of calcium significantly reduced ameloblast lineage cell proliferation. The addition of 0.1 mM calcium to the cultures upregulated expression of amelogenin, Type I collagen, and amelotin. After pretreatment with 0.3 mM calcium, the cells could form a mineralized matrix. These studies, which utilized human ameloblast lineage cells grown in vitro, showed that the addition of calcium at 0.1 and 0.3 mM, induced cell differentiation and upregulation of amelogenin Type I collagen and amelotin. (c) 2009 Wiley-Liss, Inc.

  1. Amyloid Beta Peptides Differentially Affect Hippocampal Theta Rhythms In Vitro

    Directory of Open Access Journals (Sweden)

    Armando I. Gutiérrez-Lerma

    2013-01-01

    Full Text Available Soluble amyloid beta peptide (Aβ is responsible for the early cognitive dysfunction observed in Alzheimer's disease. Both cholinergically and glutamatergically induced hippocampal theta rhythms are related to learning and memory, spatial navigation, and spatial memory. However, these two types of theta rhythms are not identical; they are associated with different behaviors and can be differentially modulated by diverse experimental conditions. Therefore, in this study, we aimed to investigate whether or not application of soluble Aβ alters the two types of theta frequency oscillatory network activity generated in rat hippocampal slices by application of the cholinergic and glutamatergic agonists carbachol or DHPG, respectively. Due to previous evidence that oscillatory activity can be differentially affected by different Aβ peptides, we also compared Aβ25−35 and Aβ1−42 for their effects on theta rhythms in vitro at similar concentrations (0.5 to 1.0 μM. We found that Aβ25−35 reduces, with less potency than Aβ1−42, carbachol-induced population theta oscillatory activity. In contrast, DHPG-induced oscillatory activity was not affected by a high concentration of Aβ25−35 but was reduced by Aβ1−42. Our results support the idea that different amyloid peptides might alter specific cellular mechanisms related to the generation of specific neuronal network activities, instead of exerting a generalized inhibitory effect on neuronal network function.

  2. An axial distribution of seeding, proliferation, and osteogenic differentiation of MC3T3-E1 cells and rat bone marrow-derived mesenchymal stem cells across a 3D Thai silk fibroin/gelatin/hydroxyapatite scaffold in a perfusion bioreactor

    Energy Technology Data Exchange (ETDEWEB)

    Sinlapabodin, Salita; Amornsudthiwat, Phakdee; Damrongsakkul, Siriporn; Kanokpanont, Sorada, E-mail: sorada.k@chula.ac.th

    2016-01-01

    In cell culture, a perfusion bioreactor provides effective transportation of nutrients, oxygen, and waste removal to and from the core of the scaffold. In addition, it provides mechanical stimuli for enhancing osteogenic differentiation. In this study, we used an axial distribution of cell numbers, alkaline phosphatase (ALP) enzyme activity, and calcium content across 4 cross-sections of 10 mm thick scaffold, made of Thai silk fibroin (SF)/gelatin (G)/hydroxyapatite (HA), as a tool to evaluate the suitable perfusion flow rate. These evaluations cover all cellular developmental phases starting from seeding, to proliferation, and later osteogenic differentiation. Mouse pre-osteoblastic MC3T3-E1 cell lines were used as a cell model during seeding and proliferation. The bioreactor seeded scaffold provided more uniform cell distribution across the scaffold compared to centrifugal and agitation seeding, while the overall number of adhered cells from bioreactor seeding was slightly lower than agitation seeding. The dynamic culture using 1 ml/min perfusion flow rate (initial shear stress of 0.1 dyn/cm{sup 2}) enabled statistically higher MC3T3-E1 proliferation, ALP activity, and calcium deposition than those observed in the static-culturing condition. However, the perfusion flow rate of 1 ml/min seemed not to be enough for enhancing ALP expression across all sections of the scaffold. Rat bone marrow derived stromal cells (rMSC) were used in the detachment test and osteogenic differentiation. It was found that perfusion flow rate of 5 ml/min caused statistically higher cell detachment than that of 1 and 3 ml/min. The perfusion flow rate of 3 ml/min gave the highest rMSC osteogenic differentiation on a SF/G/HA scaffold than other flow rates, as observed from the significantly highest number of ALP enzyme activity and the calcium content without any significant cell growth. In addition, all of these parameters were evenly distributed across all scaffold sections. - Highlights

  3. Cell proliferation, viability, and in vitro differentiation of equine mesenchymal stem cells seeded on bacterial cellulose hydrogel scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Favi, Pelagie M.; Benson, Roberto S. [Department of Materials Science and Engineering, College of Engineering, University of Tennessee, Knoxville, TN 37996 (United States); Neilsen, Nancy R. [Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996 (United States); Hammonds, Ryan L. [Department of Materials Science and Engineering, College of Engineering, University of Tennessee, Knoxville, TN 37996 (United States); Bates, Cassandra C. [Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996 (United States); Stephens, Christopher P. [Department of Surgery, Graduate School of Medicine, University of Tennessee, Knoxville, TN 37996 (United States); Center for Materials Processing, University of Tennessee, Knoxville, TN 37996 (United States); Dhar, Madhu S., E-mail: mdhar@utk.edu [Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996 (United States)

    2013-05-01

    The culture of multipotent mesenchymal stem cells on natural biopolymers holds great promise for treatments of connective tissue disorders such as osteoarthritis. The safety and performance of such therapies relies on the systematic in vitro evaluation of the developed stem cell-biomaterial constructs prior to in vivo implantation. This study evaluates bacterial cellulose (BC), a biocompatible natural polymer, as a scaffold for equine-derived bone marrow mesenchymal stem cells (EqMSCs) for application in bone and cartilage tissue engineering. An equine model was chosen due to similarities in size, load and types of joint injuries suffered by horses and humans. Lyophilized and critical point dried BC hydrogel scaffolds were characterized using scanning electron microscopy (SEM) to confirm nanostructure morphology which demonstrated that critical point drying induces fibre bundling unlike lyophilisation. EqMSCs positively expressed the undifferentiated pluripotent mesenchymal stem cell surface markers CD44 and CD90. The BC scaffolds were shown to be cytocompatible, supporting cellular adhesion and proliferation, and allowed for osteogenic and chondrogenic differentiation of EqMSCs. The cells seeded on the BC hydrogel were shown to be viable and metabolically active. These findings demonstrate that the combination of a BC hydrogel and EqMSCs are promising constructs for musculoskeletal tissue engineering applications. - Highlights: ► Critical point drying induces fibre bundling unlike lyophilisation. ► Cells positively expressed undifferentiated pluripotent stem cell markers. ► BCs were cytocompatible, supported cell adhesion, proliferation and differentiation ► Cells seeded on BC scaffolds were viable and metabolically active. ► Findings demonstrate that BC and EqMSCs are promising tissue engineered constructs.

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

    Science.gov (United States)

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

    2015-09-01

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

  5. Osteogenic potential of in situ TiO{sub 2} nanowire surfaces formed by thermal oxidation of titanium alloy substrate

    Energy Technology Data Exchange (ETDEWEB)

    Tan, A.W. [Department of Biomedical Engineering, University of Malaya, 50603 Kuala Lumpur (Malaysia); Ismail, R.; Chua, K.H. [Department of Physiology, Faculty of Medicine, National University of Malaysia, 50300 Kuala Lumpur (Malaysia); Ahmad, R. [Department of Mechanical Engineering, University of Malaya, 50603 Kuala Lumpur (Malaysia); Akbar, S.A. [Department of Materials Science and Engineering, The Ohio State University, Columbus, OH 43210 (United States); Pingguan-Murphy, B., E-mail: bpingguan@um.edu.my [Department of Biomedical Engineering, University of Malaya, 50603 Kuala Lumpur (Malaysia)

    2014-11-30

    Highlights: • In situ titanium dioxide (TiO{sub 2}) nanowire surface structures were fabricated on Ti-6Al-4V substrate using thermal oxidation. • Initial cell adhesion, cell proliferation, cell differentiation, cell mineralization, and osteogenic related gene expression of primary human osteoblasts were examined on the TiO{sub 2} nanowire surfaces. • TiO{sub 2} nanowire surfaces showed enhanced osteogenic potential as compared to the planar surface. - Abstract: Titanium dioxide (TiO{sub 2}) nanowire surface structures were fabricated in situ by a thermal oxidation process, and their ability to enhance the osteogenic potential of primary osteoblasts was investigated. Human osteoblasts were isolated from nasal bone and cultured on a TiO{sub 2} nanowires coated substrate to assess its in vitro cellular interaction. Bare featureless Ti-6Al-4V substrate was used as a control surface. Initial cell adhesion, cell proliferation, cell differentiation, cell mineralization, and osteogenic related gene expression were examined on the TiO{sub 2} nanowire surfaces as compared to the control surfaces after 2 weeks of culturing. Cell adhesion and cell proliferation were assayed by field emission scanning electron microscope (FESEM) and Alamar Blue reduction assay, respectively. The nanowire surfaces promoted better cell adhesion and spreading than the control surface, as well as leading to higher cell proliferation. Our results showed that osteoblasts grown onto the TiO{sub 2} nanowire surfaces displayed significantly higher production levels of alkaline phosphatase (ALP), extracellular (ECM) mineralization and genes expression of runt-related transcription factor (Runx2), bone sialoprotein (BSP), ostoepontin (OPN) and osteocalcin (OCN) compared to the control surfaces. This suggests the potential use of such surface modification on Ti-6Al-4V substrates as a promising means to improve the osteointegration of titanium based implants.

  6. Osteogenic potential of mesenchymal cells derived from canine umbilical cord matrix co-cultured with platelet-rich plasma and demineralized bone matrix.

    Science.gov (United States)

    Souza, Talita F B; Sakamoto, Silmara S; Ferreira, Gabriel T N M; Gameiro, Roberto; Marinho, Marcia; de Andrade, Alexandre L; Cardoso, Tereza C

    2015-01-01

    Canine mesenchymal cells (MSCs) derived from Wharton's jelly were co-cultured, then supplemented or not supplemented with platelet rich plasma (PRP) and demineralized bone matrix (DBM) to verify osteogenic differentiation. Osteoblastic differentiation followed by mineralized bone matrix production was found to be significantly higher (p < 0.05) when MSCs were associated with PRP/DBM in culture after 14-21-days of induction. Osteopontin and osteocalcin gene expression were significantly superior (p < 0.05) under the same culture conditions after 21 days of observation. In conclusion, addition of PRP to DBM co-cultured with MSCs successfully induced osteogenesis in vitro.

  7. Estrogen modulates osteogenic activity and estrogen receptor mRNA in mesenchymal stem cells of women.

    Science.gov (United States)

    Chen, F-P; Hu, C-H; Wang, K-C

    2013-02-01

    To determine whether estrogen regulates mesenchymal stem cell (MSC) activity in bone marrow from osteoporotic postmenopausal women. MSCs were collected from bone marrows which were aspirated simultaneously during iliac bone graft procedures in spine fusion surgery in osteoporotic postmenopausal women. We investigated proliferation, differentiation, osteogenic activity, and estrogen receptor (ER) α and β mRNA expression of primary culture MSCs isolated from four osteoporotic postmenopausal women, treated in vitro with or without 17β-estradiol. The expression of alkaline phosphatase (ALP), osteocalcin, interleukin-6, ERα and ERβ mRNA was evaluated. The expression of ALP and osteocalcin mRNA was detected during the cultures of MSCs and was observed to increase up to day 20. As compared with MSCs not treated with estradiol, a significant increase in DNA content, ERα mRNA, and ALP mRNA expression was observed in cultures with estradiol. The mRNA expression of osteocalcin and interleukin-6 was significantly lower in MSCs treated with estradiol than those without estradiol. There was no significant difference in the mRNA expression of ERβ between MSCs cultured with and without estradiol. In the proper environment, MSCs from osteoporotic women can differentiate into osteoblasts and estrogen enhances the osteogenic activity possibly via ERα activity.

  8. The Osteogenic Potential of Human Nondifferentiated and Pre-differentiated Mesenchymal Stem Cells Combined with an Osteoconductive Scaffold - Early Stage Healing.

    Science.gov (United States)

    Tuček, Luboš; Kočí, Zuzana; Kárová, Kristýna; Doležalová, Helena; Suchánek, Jakub

    Despite the huge research into stem cells and their regenerative properties for bone healing, there are still unanswered questions including the recipient's respond to the presence of the stem cells, the fate of stem cells inside the bone defect and the possible advantage in utilizing pre-differentiated cells. To address these problems, we used human multipotent mesenchymal stromal/stem cells (MSCs), GMP Grade, in a rat model of bone formation. In a "bioreactor concept" approach seven Wistar rats were implanted with 0.2 g of synthetic bone scaffold seeded with 2 × 106 MSCs, seven Wistar rats were implanted with 0.2 g of synthetic bone scaffold seeded with 1 × 106 predifferentiated osteoblasts and 1 × 106 pre-differentiated endothelial cells and 14 Wistar rats were implanted with 0.2 g of synthetic bone scaffold without seeded cells into an intramuscular pocket on the left side of their back. The right side of each rat was used as a control, and 0.2 g of synthetic bone scaffold was implanted into the intramuscular pocket alone. To see the early stage healing the samples were harvested 14 days after the implantation, MSCs were detected by positive DAPI and MTCO2 staining in 43% of all the samples implanted with MSCs, and no inflammation signs were present in any implanted animal. New vessels could be found in both groups implanted with MSCs, but not in the control group of animals. However, hematoxylin-eosin staining could not detect newly created bone within the implant in any of the groups. These results were in line with COLL1 staining, where we could detect positive staining only in three cases, all of which were implanted with un-differentiated MSCs. According to our findings, there were no benefits of using the pre-differentiated of MSC.

  9. Effects of high glucose on mesenchymal stem cell proliferation and differentiation

    DEFF Research Database (Denmark)

    Li, Yu-Ming; Schilling, Tatjana; Benisch, Peggy

    2007-01-01

    High glucose (HG) concentrations impair cellular functions and induce apoptosis. Exposition of mesenchymal stem cells (MSC) to HG was reported to reduce colony forming activity and induce premature senescence. We characterized the effects of HG on human MSC in vitro using telomerase...... was not influenced by HG in both cell types. MSC treatment with HG favored osteogenic differentiation. MSC are resistant to HG toxicity, depending on the stemness of MSC. Proliferation and osteogenic differentiation are stimulated by HG. Effects of HG on the transient amplifying compartment of MSC may differ from...... those in mature cells. Further research is needed to unravel the molecular mechanisms of HG resistance of MSC...

  10. DIFFERENTIAL MODULATION OF CATECHOLAMINES BY CHLOROTRIAZINE HERBICIDES IN PHEOCHROMOCYTOMA (PC12) CELLS IN VITRO

    Science.gov (United States)

    Differential modulation of catecholamines by chlorotriazine herbicides in pheochromocytoma (PC12) cells in vitro.Das PC, McElroy WK, Cooper RL.Curriculum in Toxicology, University of North Carolina, Chapel Hill 27599, USA.Epidemiological, wildlife, and lab...

  11. Can magnetic resonance spectroscopy differentiate malignant and benign causes of lymphadenopathy? An in-vitro approach

    National Research Council Canada - National Science Library

    Lionel Buré; Louis-Martin Boucher; Miriam Blumenkrantz; Stefan Schob; Pierre Lafaye de Micheaux; Caroline Reinhold; Benoit Gallix

    2017-01-01

    .... The goal of our research was to assess whether magnetic resonance (MR) spectroscopy contains the necessary information to allow differentiation of benign from malignant lymph nodes in an in-vitro approach using a modern pattern recognition method...

  12. Hesperetin alleviates the inhibitory effects of high glucose on the osteoblastic differentiation of periodontal ligament stem cells.

    Directory of Open Access Journals (Sweden)

    So Yeon Kim

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

  13. In vitro differentiation of mouse embryonic stem cells into functional ...

    African Journals Online (AJOL)

    Jane

    2011-08-22

    Aug 22, 2011 ... Studies have shown that embryonic stem (ES) cells can be successfully differentiated into liver cells, which offer the potential unlimited cell source for a variety of end-stage liver disease. In our study, in order to induce mouse ES cells to differentiate into hepatocyte-like cells under chemically defined.

  14. In vitro differentiation of human umbilical cord blood mesenchymal ...

    African Journals Online (AJOL)

    Mesenchymal stem cells (MSCs) were isolated by gradient density centrifugation from umbilical cord blood. Spindle-shaped adherent cells were permitted to grow to 70% confluence in primary culture media which was reached by day 12. Induction of differentiation started by culturing cells with differentiation medium ...

  15. Poly(acrylic acid)-regulated Synthesis of Rod-Like Calcium Carbonate Nanoparticles for Inducing the Osteogenic Differentiation of MC3T3-E1 Cells.

    Science.gov (United States)

    Yang, Wei; Yao, Chenxue; Cui, Zhengyang; Luo, Dandan; Lee, In-Seop; Yao, Juming; Chen, Cen; Kong, Xiangdong

    2016-05-06

    Calcium carbonate, especially with nanostructure, has been considered as a good candidate material for bone regeneration due to its excellent biodegradability and osteoconductivity. In this study, rod-like calcium carbonate nanoparticles (Rod-CC NPs) with desired water dispersibility were achieved with the regulation of poly (acrylic acid). Characterization results revealed that the Rod-CC NPs had an average length of 240 nm, a width of 90 nm with an average aspect ratio of 2.60 and a negative ζ-potential of -22.25 ± 0.35 mV. The degradation study illustrated the nanoparticles degraded 23% at pH 7.4 and 45% at pH 5.6 in phosphate-buffered saline (PBS) solution within three months. When cultured with MC3T3-E1 cells, the Rod-CC NPs exhibited a positive effect on the proliferation of osteoblast cells. Alkaline phosphatase (ALP) activity assays together with the osteocalcin (OCN) and bone sialoprotein (BSP) expression observations demonstrated the nanoparticles could induce the differentiation of MC3T3-E1 cells. Our study developed well-dispersed rod-like calcium carbonate nanoparticles which have great potential to be used in bone regeneration.

  16. Poly(acrylic acid-regulated Synthesis of Rod-Like Calcium Carbonate Nanoparticles for Inducing the Osteogenic Differentiation of MC3T3-E1 Cells

    Directory of Open Access Journals (Sweden)

    Wei Yang

    2016-05-01

    Full Text Available Calcium carbonate, especially with nanostructure, has been considered as a good candidate material for bone regeneration due to its excellent biodegradability and osteoconductivity. In this study, rod-like calcium carbonate nanoparticles (Rod-CC NPs with desired water dispersibility were achieved with the regulation of poly (acrylic acid. Characterization results revealed that the Rod-CC NPs had an average length of 240 nm, a width of 90 nm with an average aspect ratio of 2.60 and a negative ζ-potential of −22.25 ± 0.35 mV. The degradation study illustrated the nanoparticles degraded 23% at pH 7.4 and 45% at pH 5.6 in phosphate-buffered saline (PBS solution within three months. When cultured with MC3T3-E1 cells, the Rod-CC NPs exhibited a positive effect on the proliferation of osteoblast cells. Alkaline phosphatase (ALP activity assays together with the osteocalcin (OCN and bone sialoprotein (BSP expression observations demonstrated the nanoparticles could induce the differentiation of MC3T3-E1 cells. Our study developed well-dispersed rod-like calcium carbonate nanoparticles which have great potential to be used in bone regeneration.

  17. Aberrant gene expression profiles, during in vitro osteoblast differentiation, of telomerase deficient mouse bone marrow stromal stem cells (mBMSCs)

    DEFF Research Database (Denmark)

    Saeed, H.; Iqtedar, M.

    2015-01-01

    . Osteogenic super array at day 10 of osteoblast differentiation revealed that telomerase deficiency strongly affected the osteoblast commitment by down-regulating Runx2, Twist and Vdr - known transcription regulators of osteogenesis. Similarly, in Terc(-/-) BMSCs a marked reduction in other genes engaged...

  18. Mussel-inspired bioceramics with self-assembled Ca-P/polydopamine composite nanolayer: preparation, formation mechanism, improved cellular bioactivity and osteogenic differentiation of bone marrow stromal cells.

    Science.gov (United States)

    Wu, Chengtie; Han, Pingping; Liu, Xiaoguo; Xu, Mengchi; Tian, Tian; Chang, Jiang; Xiao, Yin

    2014-01-01

    The nanostructured surface of biomaterials plays an important role in improving their in vitro cellular bioactivity as well as stimulating in vivo tissue regeneration. Inspired by the mussel's adhesive versatility, which is thought to be due to the plaque-substrate interface being rich in 3,4-dihydroxy-l-phenylalamine (DOPA) and lysine amino acids, in this study we developed a self-assembly method to prepare a uniform calcium phosphate (Ca-P)/polydopamine composite nanolayer on the surface of β-tricalcium phosphate (β-TCP) bioceramics by soaking β-TCP bioceramics in Tris-dopamine solution. It was found that the addition of dopamine, reaction temperature and reaction time are three key factors inducing the formation of a uniform Ca-P/polydopamine composite nanolayer. The formation mechanism of a Ca-P/polydopamine composite nanolayer involved two important steps: (i) the addition of dopamine to Tris-HCl solution decreases the pH value and accelerates Ca and P ionic dissolution from the crystal boundaries of β-TCP ceramics; (ii) dopamine is polymerized to form self-assembled polydopamine film and, at the same time, nanosized Ca-P particles are mineralized with the assistance of polydopamine, in which the formation of polydopamine occurs simultaneously with Ca-P mineralization (formation of nanosized microparticles composed of calcium phosphate-based materials), and finally a self-assembled Ca-P/polydopamine composite nanolayer forms on the surface of the β-TCP ceramics. Furthermore, the formed self-assembled Ca-P/polydopamine composite nanolayer significantly enhances the surface roughness and hydrophilicity of β-TCP ceramics, and stimulates the attachment, proliferation, alkaline phosphate (ALP) activity and bone-related gene expression (ALP, OCN, COL1 and Runx2) of human bone marrow stromal cells. Our results suggest that the preparation of self-assembled Ca-P/polydopamine composite nanolayers is a viable method to modify the surface of biomaterials by

  19. Potential osteogenic activity of ethanolic extract and oxoflavidin isolated from Pholidota articulata Lindley.

    Science.gov (United States)

    Sharma, Chetan; Dixit, Manisha; Singh, Rohit; Agrawal, Manali; Mansoori, Mohd Nizam; Kureel, Jyoti; Singh, Divya; Narender, Tadigoppula; Arya, Kamal Ram

    2015-07-21

    Pholidota articulata Lindley (PA) locally known as Hadjojen (bone jointer) belongs to family Orchidaceae is used for healing fractures in folklore tradition of Kumaon region of Uttarakhand, Himalaya, India. Bone is a dynamic organ and is constantly being remodeled in order to facilitate growth and repair. This process requires the involvement of bone forming osteoblast and bone resorbing osteoclast cells, which function in generating and mineralizing bone, giving strength and rigidity to the skeletal system. Present study was aimed to determine the therapeutic potential of ethanolic extract of PA and its isolated compound oxoflavidin, by characterizing their fracture healing properties. Ovariectomized (Ovx) estrogen deficient adult female Balb/c mice were used for in vivo evaluation of osteogenic or bone healing potential of ethanolic extract of PA. Further, its isolated compounds were tested for their osteogenic efficacy using alkaline phosphatase assay and mineralization assay in vitro in mice calvarial osteoblasts. The ethanolic extract of PA exhibited significant restoration of trabecular micro-architecture in both femoral and tibial bones. Additionally, treatment with PA extract led to better bone quality and devoid of any uterine estrogenicity in ovariectomized estrogen deficient mice. One of the isolated compound, oxoflavidin enhanced ALP activity (a marker of osteoblast differentiation), mineral nodule formation and mRNA levels of osteogenic markers like BMP-2, Type 1 Collagen, RUNX-2 and osteocalcin. These results warrant that ethanolic extract of PA and it's pure compound oxoflavidin have fracture healing properties. The extract and oxoflavidin exhibit a strong threapeutical potential for the treatment and management of postmenopausal osteoporosis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  20. Magnesium inhibits Wnt/β-catenin activity and reverses the osteogenic transformation of vascular smooth muscle cells.

    Science.gov (United States)

    Montes de Oca, Addy; Guerrero, Fatima; Martinez-Moreno, Julio M; Madueño, Juan A; Herencia, Carmen; Peralta, Alan; Almaden, Yolanda; Lopez, Ignacio; Aguilera-Tejero, Escolastico; Gundlach, Kristina; Büchel, Janine; Peter, Mirjam E; Passlick-Deetjen, Jutta; Rodriguez, Mariano; Muñoz-Castañeda, Juan R

    2014-01-01

    Magnesium reduces vascular smooth muscle cell (VSMC) calcification in vitro but the mechanism has not been revealed so far. This work used only slightly increased magnesium levels and aimed at determining: a) whether inhibition of magnesium transport into the cell influences VSMC calcification, b) whether Wnt/β-catenin signaling, a key mediator of osteogenic differentiation, is modified by magnesium and c) whether magnesium can influence already established vascular calcification. Human VSMC incubated with high phosphate (3.3 mM) and moderately elevated magnesium (1.4 mM) significantly reduced VSMC calcification and expression of the osteogenic transcription factors Cbfa-1 and osterix, and up-regulated expression of the natural calcification inhibitors matrix Gla protein (MGP) and osteoprotegerin (OPG). The protective effects of magnesium on calcification and expression of osteogenic markers were no longer observed in VSMC cultured with an inhibitor of cellular magnesium transport (2-aminoethoxy-diphenylborate [2-APB]). High phosphate induced activation of Wnt/β-catenin pathway as demonstrated by the translocation of β-catenin into the nucleus, increased expression of the frizzled-3 gene, and downregulation of Dkk-1 gene, a specific antagonist of the Wnt/β-catenin signaling pathway. The addition of magnesium however inhibited phosphate-induced activation of Wnt/β-catenin signaling pathway. Furthermore, TRPM7 silencing using siRNA resulted in activation of Wnt/β-catenin signaling pathway. Additional experiments were performed to test the ability of magnesium to halt the progression of already established VSMC calcification in vitro. The delayed addition of magnesium decreased calcium content, down-regulated Cbfa-1 and osterix and up-regulated MGP and OPG, when compared with a control group. This effect was not observed when 2-APB was added. In conclusion, magnesium transport through the cell membrane is important to inhibit VSMC calcification in vitro

  1. Osteogenic signaling on silk-based matrices.

    Science.gov (United States)

    Midha, Swati; Murab, Sumit; Ghosh, Sourabh

    2016-08-01

    Bone tissue engineering has mainly focused on generating 3D grafts to repair bone defects. However, the underlying signaling mechanisms responsible for development of such 3D bone equivalents have largely been ignored. Here we describe the crucial aspects of embryonic osteogenesis and bone development including cell sources and general signaling cascades that guide mesenchymal progenitors towards osteogenic lineage. Drawing from the knowledge of developmental biology, we then review how silk biomaterial can regulate osteogenic signaling by focusing on the expression of cell surface markers, functional genomic information (mRNA) of stem cells cultured on silk matrices. In an attempt to recapitulate exact in vivo microenvironment of osteogenesis, role of scaffold architecture and material chemistry in regulating cellular differentiation is elaborated. The generated knowledge will not only improve our understanding of cell-material interactions but reveal newer strategies beyond a conventional tissue engineering paradigm and open new prospects for developing silk-based therapies against clinically relevant bone disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. In vitro phenotypic differentiation towards commensal and pathogenic oral biofilms

    NARCIS (Netherlands)

    Janus, M.M.; Keijser, B.J.F.; Bikker, F.J.; Exterkate, R.A.M.; Crielaard, W.; Krom, B.P.

    2015-01-01

    Commensal oral biofilms, defined by the absence of pathology-related phenotypes, are ubiquitously present. In contrast to pathological biofilms commensal biofilms are rarely studied. Here, the effect of the initial inoculum and subsequent growth conditions on in vitro oral biofilms was studied.

  3. In vitro germ cell differentiation from cynomolgus monkey embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Kaori Yamauchi

    Full Text Available BACKGROUND: Mouse embryonic stem (ES cells can differentiate into female and male germ cells in vitro. Primate ES cells can also differentiate into immature germ cells in vitro. However, little is known about the differentiation markers and culture conditions for in vitro germ cell differentiation from ES cells in primates. Monkey ES cells are thus considered to be a useful model to study primate gametogenesis in vitro. Therefore, in order to obtain further information on germ cell differentiation from primate ES cells, this study examined the ability of cynomolgus monkey ES cells to differentiate into germ cells in vitro. METHODS AND FINDINGS: To explore the differentiation markers for detecting germ cells differentiated from ES cells, the expression of various germ cell marker genes was examined in tissues and ES cells of the cynomolgus monkey (Macaca fascicularis. VASA is a valuable gene for the detection of germ cells differentiated from ES cells. An increase of VASA expression was observed when differentiation was induced in ES cells via embryoid body (EB formation. In addition, the expression of other germ cell markers, such as NANOS and PIWIL1 genes, was also up-regulated as the EB differentiation progressed. Immunocytochemistry identified the cells expressing stage-specific embryonic antigen (SSEA 1, OCT-4, and VASA proteins in the EBs. These cells were detected in the peripheral region of the EBs as specific cell populations, such as SSEA1-positive, OCT-4-positive cells, OCT-4-positive, VASA-positive cells, and OCT-4-negative, VASA-positive cells. Thereafter, the effect of mouse gonadal cell-conditioned medium and growth factors on germ cell differentiation from monkey ES cells was examined, and this revealed that the addition of BMP4 to differentiating ES cells increased the expression of SCP1, a meiotic marker gene. CONCLUSION: VASA is a valuable gene for the detection of germ cells differentiated from ES cells in monkeys, and the

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

    Directory of Open Access Journals (Sweden)

    Yuning Zhou

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

  5. Multi-composite bioactive osteogenic sponges featuring mesenchymal stem cells, platelet-rich plasma, nanoporous silicon enclosures, and Peptide amphiphiles for rapid bone regeneration.

    Science.gov (United States)

    Murphy, Matthew B; Blashki, Daniel; Buchanan, Rachel M; Fan, Dongmei; De Rosa, Enrica; Shah, Ramille N; Stupp, Samuel I; Weiner, Bradley K; Simmons, Paul J; Ferrari, Mauro; Tasciotti, Ennio

    2011-06-21

    A novel bioactive sponge was created with a composite of type I collagen sponges or porous poly(e-caprolactone) (PCL) scaffolds, platelet-rich plasma (PRP), BMP2-loaded nanoporous silicon enclosure (NSE) microparticles, mineralizing peptide amphiphiles (PA), and mesenchymal stem cells (MSC). Primary MSC from cortical bone (CB)  tissue proved to form more and larger colony units, as well as produce more mineral matrix under osteogenic differentiation, than MSC from bone marrow (BM). Coating pre-treatments were optimized for maximum cell adhesion and mineralization, while a PRP-based gel carrier was created to efficiently deliver and retain MSC and  microparticles within a porous scaffold while simultaneously promoting cell recruitment, proliferation, and angiogenesis. Components and composite sponges were evaluated for osteogenic differentiation in vitro. Osteogenic sponges were loaded with MSC, PRP, PA, and NSE and implanted subcutaneously in rats to evaluate the formation of bone tissue and angiogenesis in vivo. It was found that the combination of a collagen sponge with CB MSC, PRP, PA, and the BMP2-releasing NSE formed the most bone and was most vascularized by four weeks compared to analogous composites featuring BM MSC or PCL or lacking PRP, PA, and NSE. This study indicates that CB MSC should be considered as an alternative to marrow as a source of stem cells, while the PRP-PA cell and microparticle delivery system may be utilized for diverse tissue engineering applications.

  6. In vitro differentiation of human umbilical cord blood mesenchymal ...

    African Journals Online (AJOL)

    May H. Hasan

    2016-08-05

    Aug 5, 2016 ... Abstract Mesenchymal stem cells (MSCs) were isolated by gradient density centrifugation from umbilical cord blood. Spindle-shaped adherent cells were permitted to grow to 70% confluence in primary culture media which was reached by day 12. Induction of differentiation started by cul- turing cells with ...

  7. Differentiation ability of rat postnatal dental pulp cells in vitro.

    NARCIS (Netherlands)

    Zhang, W.; Walboomers, X.F.; Wolke, J.G.C.; Bian, Z.; Fan, M.W.; Jansen, J.A.

    2005-01-01

    The current rapid progression in stem cell research has enhanced our knowledge of dental tissue regeneration. In this study, rat dental pulp cells were isolated and their differentiation ability was evaluated. First, dental pulp cells were obtained from maxillary incisors of male Wistar rats.

  8. In vitro evaluation of cardiomyogenic differentiation of bone marrow derived mesenchymal stem cells (MSCs)

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Min Hwan; Lee, Yong Jin; Kang, Joo Hyun [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2010-10-15

    Bone marrow derived mesenchymal stem cells (MSCs) are excellent candidate as therapeutic agent for cell therapy. MSCs can be expanded in vitro rapidly (more than 3-5 fold in a weeks), and maintained their stem cell properties for a long culture period. Recently, many investigators have suggested that MSCs have ability to differentiate into cardiomyocytes by given appropriate condition in vitro or in vivo. Although, MSCs may be useful cell therapeutic agents in heart disease, there are still exist major barriers to track their capacity to differentiate into functional cardiomyocytes. In our previous study, the transgenic mouse model expressing sodium iodide symporter (NIS) driven by {alpha}-myosin heavy chain ({alpha}-MHC) promoter was developed to image cardiomyocyte with {gamma}-camera and microPET in vivo. In this study, we investigate the monitoring availability of {alpha}-MHC driven NIS gene of MSCs from the transgenic mouse during cardiomyogenic differentiation in vitro

  9. Strontium Promotes Cementoblasts Differentiation through Inhibiting Sclerostin Expression In Vitro

    Directory of Open Access Journals (Sweden)

    Xingfu Bao

    2014-01-01

    Full Text Available Cementogenesis, performed by cementoblasts, is important for the repair of root resorption caused by orthodontic treatment. Based on recent studies, strontium has been applied for osteoporosis treatment due to its positive effect on osteoblasts. Although promising, the effect of strontium on cementoblasts is still unclear. So the aim of this research was to clarify and investigate the effect of strontium on cementogenesis via employing cementoblasts as model. A series of experiments including MTT, alkaline phosphatase activity, gene analysis, alizarin red staining, and western blot were carried out to evaluate the proliferation and differentiation of cementoblasts. In addition, expression of sclerostin was checked to analyze the possible mechanism. Our results show that strontium inhibits the proliferation of cementoblasts with a dose dependent manner; however, it can promote the differentiation of cementoblasts via downregulating sclerostin expression. Taking together, strontium may facilitate cementogenesis and benefit the treatment of root resorption at a low dose.

  10. Dietary relevant mixtures of phytoestrogens inhibit adipocyte differentiation in vitro

    DEFF Research Database (Denmark)

    Taxvig, Camilla; Specht, Ina Olmer; Boberg, Julie

    2013-01-01

    Phytoestrogens (PEs) are naturally occurring plant components, with the ability to induce biological responses in vertebrates by mimicking or modulating the action of endogenous hormones.Single isoflavones have been shown to affect adipocyte differentiation, but knowledge on the effect of dietary...... relevant mixtures of PEs, including for instance lignans, is lacking. In the current study dietary relevant mixtures of isoflavones and their metabolites, lignans and their metabolites, coumestrol, and a mixture containing all of them, were examined for effects on adipogenesis in 3T3-L1 adipocytes, as well...... as tested for their PPARγ activating abilities. The results showed that mixtures of isoflavonoid parent compounds and metabolites, respectively, a mixture of lignan metabolites, as well as coumestrol concentration-dependently inhibited adipocyte differentiation. Furthermore, a mixture of isoflavonoid parent...

  11. Strontium Promotes Cementoblasts Differentiation through Inhibiting Sclerostin Expression In Vitro

    Science.gov (United States)

    Bao, Xingfu; Liu, Xianjun; Zhang, Yi; Cui, Yue; Yao, Jindan

    2014-01-01

    Cementogenesis, performed by cementoblasts, is important for the repair of root resorption caused by orthodontic treatment. Based on recent studies, strontium has been applied for osteoporosis treatment due to its positive effect on osteoblasts. Although promising, the effect of strontium on cementoblasts is still unclear. So the aim of this research was to clarify and investigate the effect of strontium on cementogenesis via employing cementoblasts as model. A series of experiments including MTT, alkaline phosphatase activity, gene analysis, alizarin red staining, and western blot were carried out to evaluate the proliferation and differentiation of cementoblasts. In addition, expression of sclerostin was checked to analyze the possible mechanism. Our results show that strontium inhibits the proliferation of cementoblasts with a dose dependent manner; however, it can promote the differentiation of cementoblasts via downregulating sclerostin expression. Taking together, strontium may facilitate cementogenesis and benefit the treatment of root resorption at a low dose. PMID:25003114

  12. Osteogenic Potency of Nacre on Human Mesenchymal Stem Cells

    Science.gov (United States)

    Green, David W.; Kwon, Hyuk-Jae; Jung, Han-Sung

    2015-01-01

    Nacre seashell is a natural osteoinductive biomaterial with strong effects on osteoprogenitors, osteoblasts, and osteoclasts during bone tissue formation and morphogenesis. Although nacre has shown, in one study, to induce bridging of new bone across large non-union bone defects in 8 individual human patients, there have been no succeeding human surgical studies to confirm this outstanding potency. But the molecular mechanisms associated with nacre osteoinduction and the influence on bone marrow-derived mesenchymal stem cells (BMSC’s), skeletal stem cells or bone marrow stromal cells remain elusive. In this study we highlight the phenotypic and biochemical effects of Pinctada maxima nacre chips and the global nacre soluble protein matrix (SPM) on primary human bone marrow-derived stromal cells (hBMSCs) in vitro. In static co-culture with nacre chips, the hBMSCs secreted Alkaline phosphatase (ALP) at levels that exceeded bone morphogenetic protein (rhBMP-2) treatment. Concentrated preparation of SPM applied to Stro-1 selected hBMSC’s led to rapid ALP secretions, at concentrations exceeding the untreated controls even in osteogenic conditions. Within 21 days the same population of Stro-1 selected hBMSCs proliferated and secreted collagens I–IV, indicating the premature onset of an osteoblast phenotype. The same SPM was found to promote unselected hBMSC differentiation with osteocalcin detected at 7 days, and proliferation increased at 7 days in a dose-dependent manner. In conclusion, nacre particles and nacre SPM induced the early stages of human bone cell differentiation, indicating that they may be promising soluble factors with osteoinductive capacity in primary human bone cell progenitors such as, hBMSC’s. PMID:25666352

  13. Identification of suitable reference genes for quantitative gene expression analysis in rat adipose stromal cells induced to trilineage differentiation.

    Science.gov (United States)

    Santos, Bruno Paiva Dos; da Costa Diesel, Luciana Fraga; da Silva Meirelles, Lindolfo; Nardi, Nance Beyer; Camassola, Melissa

    2016-12-15

    This study was designed to (i) identify stable reference genes for the analysis of gene expression during in vitro differentiation of rat adipose stromal cells (rASCs), (ii) recommend stable genes for individual treatment conditions, and (iii) validate these genes by comparison with normalization results from stable and unstable reference genes. On the basis of a literature review, eight genes were selected: Actb, B2m, Hprt1, Ppia, Rplp0, Rpl13a, Rpl5, and Ywhaz. Genes were ranked according to their stability under different culture conditions as assessed using GenNorm, NormFinder, and RefFinder algorithms. Although the employed algorithms returned different rankings, the most frequently top-ranked genes were: B2m and/or Ppia for all 28day treatments (ALL28); Ppia and Hprt1 (adipogenic differentiation; A28), B2m (chondrogenic differentiation; C28), Rpl5 (controls maintained in complete culture medium; CCM), Rplp0 (osteogenic differentiation for 3days; O3), Rpl13a and Actb (osteogenic differentiation for 7days; O7), Rplp0 and Ppia (osteogenic differentiation for 14days; O14), Hprt1 and Ppia (osteogenic differentiation for 28days; O28), as well as Actb (all osteogenesis time points combined; ALLOSTEO). The obtained results indicate that the performance of reference genes depends on the differentiation protocol and on the analysis time, thus providing valuable information for the design of RT-PCR experiments. Copyright © 2016. Published by Elsevier B.V.

  14. Inhibition of in vitro myogenic differentiation by cellular transcription factor E2F1

    DEFF Research Database (Denmark)

    Wang, J; Helin, K; Jin, P

    1995-01-01

    Terminal differentiation of cultured myocytes requires withdrawal of the cells from the cell cycle. Constitutive overexpression of several oncogenes in myoblasts can inhibit in vitro myogenesis. Here we studied the role of the cellular transcription factor E2F1 on myogenic differentiation. E2F1...... expression is irreversibly down-regulated during differentiation of C2C12 myocytes. Furthermore, deregulated E2F1 expression in C2C12 cells prevented myogenic differentiation. This inhibition of myogenesis was associated with the repression of myogenin expression and an elevated cyclin D1 expression....... Moreover, E2F1-overexpressing myocytes failed to exit the