• Development and Reproduction
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• v.15 no.4
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• pp.339-347
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• 2011

Human eyelid adipose-derived stem cells (hEAs) and amniotic mesenchymal stem cells (hAMs) are very valuable sources for the cell therapeutics. Both types of cells have a great proliferating ability in vitro and a multipotency to differentiate into adipocytes, osteoblasts and chondrocytes. In the present study, we evaluated their stem cell characteristics after long-time cryopreservation for 6, 12 and 24 months. When frozen-thawed cells were cultivated in vitro, their cumulative cell number and doubling time were similar to freshly prepared cells. Also they expressed stem cell-related genes of SCF, NANOG, OCT4, and TERT, ectoderm-related genes of NCAM and FGF5, mesoderm/endoderm-related genes of CK18 and VIM, and immune-related genes of HLA-ABC and ${\beta}$2M. Following differentiation culture in appropriate culture media for 2-3 weeks, both types of cells exhibited well differentiation into adipocyte, osteoblast, and chondrocyte, as revealed by adipogenic, osteogenic or chondrogenic-specific staining and related genes, respectively. In conclusion, even after long-term storage hEAs and hAMs could maintain their stem cell characteristics, suggesting that they might be suitable for clinical application based on stem cell therapy.

• International Journal of Oral Biology
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• v.36 no.4
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• pp.173-178
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• 2011

Tumor necrosis factor alpha ($TNF{\alpha}$) is a multifunctional cytokine that is elevated in inflammatory diseases such as atherosclerosis, diabetes and rheumatoid arthritis. Recent evidence has suggested that ${\beta}2$ adrenergic receptor (${\beta}2AR$) activation in osteoblasts suppresses osteogenic activity. In the present study, we explored whether $TNF{\alpha}$ modulates ${\beta}AR$ expression in osteoblastic cells and whether this regulation is associated with the inhibition of osteoblast differentiation by $TNF{\alpha}$. In the experiments, we used C2C12 cells, MC3T3-E1 cells and primary cultured mouse bone marrow stromal cells. Among the three subtypes of ${\beta}AR$, ${\beta}2$ and ${\beta}3AR$ were found in our analysis to be upregulated by $TNF{\alpha}$. Moreover, isoproterenol-induced cAMP production was observed to be significantly enhanced in $TNF{\alpha}$-primed C2C12 cells, indicating that $TNF{\alpha}$ enhances ${\beta}2AR$ signaling in osteoblasts. $TNF{\alpha}$ was further found in C2C12 cells to suppress bone morphogenetic protein 2-induced alkaline phosphatase (ALP) activity and the expression of osteogenic marker genes including Runx2, ALP and osteocalcin. Propranolol, a ${\beta}2AR$ antagonist, attenuated this $TNF{\alpha}$ suppression of osteogenic differentiation. $TNF{\alpha}$ increased the expression of receptor activator of NF-${\kappa}B$ ligand (RANKL), an essential osteoclastogenic factor, in C2C12 cells which was again blocked by propranolol. In summary, our data show that $TNF{\alpha}$ increases ${\beta}2AR$ expression in osteoblasts and that a blockade of ${\beta}2AR$ attenuates the suppression of osteogenic differentiation and stimulation of RANKL expression by $TNF{\alpha}$. These findings imply that a crosstalk between $TNF{\alpha}$ and ${\beta}2AR$ signaling pathways might occur in osteoblasts to modulate their function.

• Journal of Periodontal and Implant Science
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• v.42 no.3
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• pp.73-80
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• 2012

Purpose: The purpose of this study was to investigate the effects of the immunosuppressants FK506 and cyclosporin A (CsA) on the osteogenic differentiation of rat mesenchymal stem cells (MSCs). Methods: The effect of FK506 and CsA on rat MSCs was assessed in vitro. The MTT assay was used to determine the deleterious effect of immunosuppressants on stem cell proliferation at 1, 3, and 7 days. Alkaline phosphatase (ALP) activity was analyzed on days 3, 7, and 14. Alizarin red S staining was done on day 21 to check mineralization nodule formation. Real-time polymerase chain reaction (RT-PCR) was also performed to detect the expressions of bone tissue-specific genes on days 1 and 7. Results: Cell proliferation was promoted more in the FK506 groups than the control or CsA groups on days 3 and 7. The FK506 groups showed increased ALP activity compared to the other groups during the experimental period. The ALP activity of the CsA groups did not differ from the control group in any of the assessments. Mineralization nodule formation was most prominent in the FK506 groups at 21 days. RT-PCR results of the FK506 groups showed that several bone-related genes-osteopontin, osteonectin, and type I collagen (Col-I)-were expressed more than the control in the beginning, but the intensity of expression decreased over time. Runx2 and Dlx5 gene expression were up-regulated on day 7. The effects of 50 nM CsA on osteonectin and Col-I were similar to those of the FK506 groups, but in the 500 nM CsA group, most of the genes were less expressed compared to the control. Conclusions: These results suggest that FK506 enhances the osteoblastic differentiation of rat MSCs. Therefore, FK506 might have a beneficial effect on bone regeneration when immunosuppressants are needed in xenogenic or allogenic stem cell transplantation to treat bone defects.

• Journal of Life Science
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• v.26 no.3
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• pp.331-337
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• 2016

Although fibroblast growth factor 23 (FGF23) is exclusively produced in osteoblasts and osteocytes, its main target is the kidney, where it decreases phosphate reabsorption by suppressing Na-phosphate cotransporters. Independently of its action on phosphate homeostasis, FGF23 also inhibits bone formation in vivo. In a calvarial osteoblastic cell model, FGF23 was shown to negatively affect extracellular matrix mineralization. This study investigated whether FGF23 had similar effects on osteoblast maturation, including differentiation and mineralization of bone marrow-derived mesenchymal stem cells (MSCs). D1 MSCs were cultured in an osteogenic medium containing β-glycerophosphate, ascorbic acid, and dexamethazone. Osteoblastic differentiation was evaluated by alkaline phosphatase (Alp) staining, and matrix mineralization was evaluated by alizarin red staining and calcium deposition. The expression of differentiation-stimulating genes Runx2, Alp, and osteocalcin and mineralization-inhibiting genes Enpp1 and Ank was analyzed using semiquantitative RT-PCR. Supraphysiological doses of FGF23 did not stimulate proliferation or osteoblastic differentiation of MSCs. Matrix mineralization 1, 2, and 3 weeks after the FGF23 treatment did not vary between control and FGF23 groups, although time-dependent enhancement of mineralization was obvious. Calcium deposition was also unchanged after the FGF23 treatment. mRNA expression levels of differentiation- and mineralization-related genes were also similar between the groups. Despite these negative findings, FGF23 signaling through FGF receptors seemed to function normally, with phosphorylation of the Erk protein more evident in the FGF23 group than in controls. These findings suggest that unlike calvarial osteoblasts, FGF23 is not likely to affect osteoblastic differentiation and mineralization of MSCs.

• The Journal of Korean Academy of Prosthodontics
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• v.43 no.3
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• pp.393-408
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• 2005

Statement of problem. In the process of bone formation, titanium (Ti) surface roughness is an important factor modulating osteoblastic function. Purpose. This study was carried out to determine the effect of different Ti surface on biologic responses of a human osteoblast-like cell line (MG63). Materials and methods. MG63 cells were cultured on S (smooth), SLA (sandblasted largegrit & acid etching), HA (hydroxyapatite) Ti. The morphology and attachment of the cells were examined by SEM. The cDNAs prepared from total RNAs of MG63 were hybridized to a human cDNA microarray (1,152 elements). Results. The appearances of the surfaces observed with SEM were different in the three types of dental substrates. The surface of SLA and HA were shown to be rougher than S. MG63 cells cultured on SLA and HA were cell-matrix interaction. In the expression of genes involved in osseointegration, upregulated genes were bone morphogenetic protein, Villin, Integrin, Insulin-like growth factors in different surfaces. Downregulated genes were fibroblast growth factor receptor 4, Bcl 2-related protein, collagen, CD4 in different surfaces. Conclusion. The attachment and expression of key osteogenic regulatory genes were enhanced by surface roughness of the dental materials.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.33 no.4
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• pp.308-313
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• 2011

Purpose: Beta-tricalcium phosphate (${\beta}$-TCP) is a synthetic calcium phosphate ceramic that has widely been used as a bone material to repair bone defects. Despite many clinical studies, the molecular mechanism whereby this biomaterial alters the gene expression in osteoblasts to promote bone formation is poorly understood. Thus, we attempted to address this question by using microarray techniques to identify the genes that are differentially regulated in osteoblasts exposed to ${\beta}$-TCP. Methods: By using DNA microarrays, we identified several genes whose expression levels were significantly up- or down-regulated in osteoblast-likeMG-63cells cultured with ${\beta}$-TCP at a concentration of 100 mg/10 ml for 24 hours. Results: The differentially expressed genes covered a broad range of functional activities: signal transduction, transcription, cell cycle regulation, vesicular transport, apoptosis, immunity, cytoskeletal elements and cell proliferation and differentiation. Conclusion: The gene expression changes related to cell proliferation and differentiation, vesicle transport, immunity and defense could affect the osteogenic activities of osteoblasts for bone regeneration. However, further studies will be required to verify the relative importance of these genes in bone formation, their temporal and spatial expression patterns and their interactions with each other.

• The Journal of the Korean dental association
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• v.54 no.4
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• pp.284-295
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• 2016

The aim of this study was to evaluate the effect of Epigallocatechin-3-Gallate (EGCG) on the alveolar bone metabolism in a collagen-induced arthritis (CIA) model in mice to enhance the understanding of rheumatoid arthritis (RA)-associated alveolar bone loss. Following the induction of CIA in animals (mice, n=16), mandibles were retrieved for micro-computed tomography (micro-CT) and isolation of alveolar bone cells (ABCs). In vitro osteogenic potentials of ABCs were evaluated and the mRNA expression of downstream effector genes was assessed. CIA was successfully induced in all animals, and micro-CT data showed that alveolar bone loss was significantly increased in the CIA group while the treatment of EGCG prevented the alveolar bone resorption. Osteogenesis by ABCs was significantly increased in the CIA+EGCG group in vitro. The analysis of mRNA expressions showed that osteoclastogenesis-associated genes were increased in CIA group while bone protecting genes were upregulated in EGCG treated group. The results demonstrate that EGCG downregulated the alveolar bone resorption in a CIA model in mice, and upregulation of bone protecting genes appear to be involved. Further studies are warranted.

• BMB Reports
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• v.47 no.8
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• pp.463-468
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• 2014

Osteoblasts are specialized mesenchymal cells that are responsible for bone formation. In this study, we examine the role of GATA4 in osteoblast differentiation. GATA4 was abundantly expressed in preosteoblast cells and gradually down-regulated during osteoblast differentiation. Overexpression of GATA4 in osteoblastic cells inhibited alkaline phosphatase activity and nodule formation in osteogenic conditioned cell culture system. In addition, overexpression of GATA4 attenuated expression of osteogenic marker genes, including Runx2, alkaline phosphatase, bone sialoprotein, and osteocalcin, all of which are important for osteoblast differentiation and function. Overexpression of GATA4 attenuated Runx2 promoter activity, whereas silencing of GATA4 increased Runx2 induction. We found that GATA4 interacted with Dlx5 and subsequently decreased Dlx5 binding activity to Runx2 promoter region. Our data suggest that GATA4 acts as a negative regulator in osteoblast differentiation by downregulation of Runx2.

• BMB Reports
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• v.44 no.10
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• pp.659-664
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• 2011

As part of the search for biologically active anti-osteoporotic agents that enhance differentiation and mineralization of osteoblastic MC3T3-E1 cells, we identified the ginsenoside Rh2(S), which is an active component in ginseng. Rh2(S) stimulates osteoblastic differentiation and mineralization, as manifested by the up-regulation of differentiation markers (alkaline phosphatase and osteogenic genes) and Alizarin Red staining, respectively. Rh2(S) activates p38 mitogen-activated protein kinase (MAPK) in time- and concentration-dependent manners, and Rh2(S)-induced differentiation and mineralization of osteoblastic cells were totally inhibited in the presence of the p38 MAPK inhibitor, SB203580. In addition, pretreatment with Go6976, a protein kinase D (PKD) inhibitor, significantly reversed the Rh2(S)-induced p38 MAPK activation, indicating that PKD might be an upstream kinase for p38 MAPK in MC3T3-E1 cells. Taken together, these results suggest that Rh2(S) induces the differentiation and mineralization of MC3T3-E1 cells through activation of PKD/p38 MAPK signaling pathways, and these findings provide a molecular basis for the osteogenic effect of Rh2(S).

• International Journal of Industrial Entomology and Biomaterials
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• v.27 no.1
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• pp.159-165
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• 2013

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor (TGF-${\beta}$) superfamily and are involved in osteoblastic differentiation. The largest TGF-${\beta}$ superfamily subgroup shares genetic homology with human BMPs (hBMPs) and silkworm decapentaplegic (dpp). In addition, hBMPs are functionally interchangeable with Drosophila dpp. Bombyx mori dpp may induce bone formation in mammalian cells. To test this hypothesis, we synthesized the 1,285-base pairs cDNA of full-length B. mori dpp using total RNAs obtained from the fat body of 3-day-old of the $5^{th}$ instar larvae and cloned the cDNA into the pCEP4 mammalian expression vector. Next, B. mori dpp was expressed in C3H10T1/2 cells. The target cells transfected with the pCEP4-Bm dpp plasmid showed biological functions similar to those of osteogenic differentiation induction growth factors such as hBMPs. We determined the relative mRNA expression rates of Runt-related transcription factor 2 (RUNX2), osterix, osteocalcin, and alkaline phosphatase (ALP) to validate the osteoblast-specific differentiation effects of B. mori dpp by performing quantitative real-time RT-PCR. Interestingly, mRNA expression levels of the 3 marker genes except RUNX2, in cells expressing B. mori dpp were much higher than those in control cells and C3H10T1/2 cells transfected with pCEP4. These results suggested that B. mori dpp signaling regulates osterix expression during osteogenic differentiation via RUNX2-independent mechanisms.