• Journal of Periodontal and Implant Science
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• v.38 no.1
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• pp.41-50
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• 2008

Purpose: Bone morphogenetic protein-2(BMP-2) has been shown to possess significant osteoinducitve potential. There have been attempts to overcome a limitation of mass production, and economical efficiency of BMP. The aim of this study was to produce recombinant human BMP-2(rhBMP-2) from E. coli in a large scale and evaluate its biological activity. Materials and Methods: The E.coli strain BL21(DE3) was used as a host for rhBMP-2 production. Dimerized rhBMP-2 was purified by affinity chromatography using Heparin column. To determine the physicochemical properties of the rhBMP-2 expressed in E. coli, we examined the HPLC profile and performed Western blot analysis. The effect of the purified rhBMP-2 dimer on osteoblast differentiation was examined by alkaline phosphatase (ALP) activity and representing morphological change using C2C12 cell. Results: E. coli was genetically engineered to produce rhBMP-2 in a non-active aggregated form. We have established a method which involves refolding and purifying a folded rhBMP-2 dimer from non-active aggregates. The purified rhBMP-2 homodimer was characterized by SDS-PAGE as molecular weight of about 28kDa and eluted at 34% acetonitrile, 13.27 min(retention time) in the HPLC profile and detected at Western blot. The purified rhBMP-2 dimer stimulated ALP activity and induced the transformation from myogenic differentiation to osteogenic differentiation. Conclusion: rhBMP-2 was produced in E. coli using genetic engineering. The purified rhBMP-2 dimer stimulated ALP activity and induced the osteogenic differentiation of C2C12 cells.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.33 no.4
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• pp.331-339
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• 2007

Background: Distraction osteogenesis(DO) is a useful method for treating cases demanding the generation of new bone. During DO, the angiogenic activity is crucial factor in the new bone formation. The aim of this study was to detect the autocrine growth activity in the cellular components of the distracted bone with observation of the co-expression of vascular endothelial growth factor(VEGF) and its receptors following the mandibular DO. Materials and methods: Unilateral mandibular distraction(0.5 mm twice per day for 10 days) was performed in six mongrel dogs. Two animals were killed at 7, 14, and 28 days after completion of distraction, respectively. Immediately after the animals were killed, the right mandibles were harvested en block. Immunohistochemical staining was processed for observation of the VEGF expression, and double immunofluorescent staining was also processed for detection of the co-expression of osteocalcin and VEGF's two distinct receptors(VEGFR-1 and VEGFR-2). Results: At 7 and 14 days after distraction, the expressions of VEGF were significantly increased in the osteogenic cells of the distracted bone. Up to 28 days after distraction, VEGF was still expressed moderate in the osteoblastic cells of distracted bone. The co-expressions of osteocalcin/VEGFR-1 and osteocalcin/VEGFR-2 were observed in the distracted bone at 7 and 14 days after distraction. In the double immunofluorescent staining, the co-expression' s level of osteocalcin/VEGFR-1 was more than that of osteocalcin/VEGFR-2. Conclusion: Taken together, this study suggested that VEGF plays an important role in the osteogenesis, and these osteoblastic cell-derived VEGF might act as autocrine growth factor during distraction osteogenesis. In the other word, the cellular components, such as osteoblasts and immature fibroblast-like cellsor mesenchymal cells in the distracted bone, might have autocrine growth activity during distraction osteogenesis.

• Development and Reproduction
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• v.15 no.2
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• pp.87-98
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• 2011

The present experiment was performed to evaluate the osteogenic differentiation of human adipose tissue derived mesenchymal stem cells (ATMSCs) seeded in bioceramic-poly D,L-latic-co-glycolic acid (PLGA) scaffold. Osteogenic differentiation of ATMSCs were induced using the osteogenic induction (OI) medium. ATMSCs were cultured with OI medium during 28 days in well plate. The proliferation of ATMSCs in OI medium group was significantly increased for 14 days of plate culture but slowed after 21 days. On the other hand, proliferation in the control group showed constant increase for 28 days of culturing. The alkaline phosphatase (ALP) activity of ATMSCs in OI medium group increased during the 21 days of culture but decreased on 28 days. However, in control group ALP activity of ATMSCs was continuously decreased as time goes. Nodule was observed at 21 days of culture in OI medium group and confirmed accumulation of calcium in cell by alizarin red staining. ATMSCs were seeded in PLGA scaffold or in Bioceramic-PLGA scaffold, and cultured with OI medium. ALP activity of ATMSCs by osteoblast differentiation in each scaffold increased on 21 days of culture and decreased rapidly on 28 days. ALP activity of ATMSCs was increased highly in Bioceramic-PLGA scaffold compared to PLGA scaffold on 21 days of culturing. SEM-EDS analysis demonstrated that calcium and phosphate content and Ca/P ratio in Bioceramic-PLGA scaffold increased higher than in PLGA scaffold. Biodegradability of scaffold at 56 days after implantation showed that Bioceramic-PLGA scaffold was more biodegradable than PLGA scaffold. The results demonstrated that the differentiation of ATMSCs to osteoblast were more effective in scaffold culture than well plate culture. Bioceramic increased cell adhesion rate on scaffold and ALP activity by osteoblast differentiation. Also, bioceramic was considered to increase the calcium and phosphate in scaffold when ATMSCs was mineralized by osteogenic differentiation. Bioceramic-PLGA scaffold enhanced the osteogenesis of seeded ATMSCs compared to PLGA scaffold.

• 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.

• Biomolecules & Therapeutics
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• v.24 no.2
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• pp.123-131
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• 2016

Osteoporosis is a bone pathology leading to increased fracture risk and challenging the quality of life. The aim of this study was to evaluate the effect of an anthraquinone glycoside, aloin, on osteogenic induction of MC3T3-E1 cells. Aloin increased alkaline phosphatase (ALP) activity, an early differentiation marker of osteoblasts. Aloin also increased the ALP activity in adult human adipose-derived stem cells (hADSC), indicating that the action of aloin was not cell-type specific. Alizarin red S staining revealed a significant amount of calcium deposition in cells treated with aloin. Aloin enhanced the expression of osteoblast differentiation genes, Bmp-2, Runx2 and collagen 1a, in a dose-dependent manner. Western blot analysis revealed that noggin and inhibitors of p38 MAPK and SAPK/JNK signals attenuated aloin-promoted expressions of Bmp-2 and Runx2 proteins. siRNA mediated blocking of Wnt-5a signaling pathway also annulled the influence of aloin, indicating Wnt-5a dependent activity. Inhibition of the different signal pathways abrogated the influence of aloin on ALP activity, confirming that aloin induced MC3T3-E1 cells into osteoblasts through MAPK mediated Wnt and Bmp signaling pathway.

• Journal of dental hygiene science
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• v.21 no.4
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• pp.243-250
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• 2021

Background: Endodontic sealers or their toxic components may become inflamed and lead to delayed wound healing when in direct contact with periapical tissues over an extended period. Moreover, an overfilled sealer can directly interact with adjacent tissues and may cause immediate necrosis or further resorption. Therefore, the treatment outcome conceivably depends on the endodontic sealer's biocompatibility and osteogenic potential. This study aimed to evaluate the cell viability and osteogenic effects of four different sealers in osteoblastic cells. Methods: AH Plus (resin-based sealer), Pulp Canal Sealer EWT (zinc oxide-eugenol sealer), BioRoot RCS (calcium silicate-based sealer), and Well-Root ST (MTA-based calcium silicate sealer) were mixed strictly according to the manufacturer's instructions, and dilutions of sealer extracts (1/2, 1/5 and 1/10) were determined. Cell viability was measured using the water-soluble tetrazolium-8 (WST-8) assay. Differentiation was assessed by alkaline phosphatase (ALP) activity and mineralized nodule formation by Alizarin Red S staining. Results: The cell viability of the extracts derived from the sealers excluding Well-Root ST was concentration dependent, with sealer extracts having the least viability at a 1/2 dilution. At sealer extract dilution of 1/10, the test groups showed the same survival rate as that control group, with the exception of BioRoot RCS. Among all experimental groups, BioRoot RCS showed the highest cell viability after 48 hours. The ALP activity was significantly higher in a concentration-dependent manner. Furthemore, all four materials promoted ALP activity and mineralized nodule formation compared to the control at 1/10 dilutions. Conclusion: This is the first study to highlight the differences in biological activity of these four materials. These results suggest that the composition of root canal sealers appears to alter the form of biocompatibility and osteoblastic differentiation.

• Journal of Life Science
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• v.23 no.4
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• pp.471-478
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• 2013

Human adipose tissue-derived mesenchymal stem cells (hADSCs) have therapeutic potential, including the ability to self-renew and differentiate into multiple lineages. Understanding of molecular mechanisms of stem cell differentiation is important for improving the therapeutic efficacies of stem cell transplantation. In this study, we determined the role of nuclear factor of activated T cells (NFAT5) in the osteogenic differentiation of hADSCs. The down-regulation of NFAT5 expression by the transfection of a specific siRNA significantly inhibited osteogenic differentiation of hADSCs and decreased the activity of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-${\kappa}B$) promoter without affecting their proliferation and adipogenic differentiation. The inhibition of NFAT5 expression inhibited the basal and Tumor Necrosis Factor ${\alpha}$ (TNF-${\alpha}$) induced activation of NF-${\kappa}B$, but it did not affect TNF-${\alpha}$-induced degradation of the $I{\kappa}B$ protein. These findings indicate that NFAT5 plays an important role in the osteogenic differentiation of hADSCs through the modulation of the NF-${\kappa}B$ pathway.

• 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.

• Preventive Nutrition and Food Science
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• v.16 no.4
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• pp.291-298
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• 2011

Yam extracts (Dioscorea batatas) have been reported to possess a variety of functions. However, studies on its osteogenic properties are limited. In this study, we investigated the effect of ethanol and water extracts on osteoblast proliferation and bone matrix protein synthesis, type I collagen and alkaline phosphatase (ALP), using osteoblastic MC3T3-E1 cell model. MC3T3-E1 cells were cultured with yam ethanol and water extracts (0~30 mg/L) within 39 days of osteoblast differentiation period. Cell proliferation was measured by MTT assay. Bone matrix proteins were assessed by the accumulation of type I collagen and ALP activity by staining the cell layers for matrix staining. Also, the secreted (media) matrix protein concentration (type I collagen) and enzyme activity (ALP) were measured colorimetrically. Yam ethanol and water extracts stimulated cell proliferation within the range of 15~30 mg/L at 15 day treatment. The accumulation of type I collagen in the extracellular matrix, as well as secreted collagen in the media, increased with increasing doses of yam ethanol (3~15 mg/L) and water (3~30 mg/L) extracts. ALP activity was not affected by yam ethanol extracts. Our results demonstrated that yam extracts stimulated osteoblast proliferation and enhanced the accumulation of the collagenous bone matrix protein type I collagen in the extracellular matrix. These results suggest that yam extracts may be a potential activator for bone formation by increasing osteoblast proliferation and increasing bone matrix protein type I collagen. Before confirming the osteogenic action of yam, further studies for clarifying how and whereby yam extracts can stimulate this ostegenesis action are required.

• Journal of dental hygiene science
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• v.19 no.4
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• pp.254-260
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• 2019

Background: The primary aims of periodontal disease treatment is to remove dental plaque and calculus, the main causes of tooth loss, and restore periodontal tissue destroyed by inflammation. Periodontal disease treatment should also help maintain the alveolar bone, alleviate inflammation, and promote periodontal ligament cell proliferation, which is essential for tissue regeneration. Conventional antibiotics and anti-inflammatories have adverse side effects, especially during long-term use, so there is a need for adjunct treatment agents derived from natural products. The purpose of this study was to investigate whether the herbal flavone baicalein has the osteogenic activity under inflammatory conditions, and assess the involvement of osteoblast immediate early response 3 (IER3) expression. Methods: Human osteoblastic MG-63 cells were cultured with the pro-inflammatory cytokines tumor necrosis factor α and interleukin 1β in the presence and absence of baicalein. Proliferation was assessed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, and expression of IER3 mRNA was assessed using real-time polymerase chain reaction. The expression of IER3 protein levels and activation of associated signal transduction pathways were assessed using western blotting. Results: Baicalein increased IER3 mRNA and protein expression synergistically. In addition, baicalein reversed the suppression of cell proliferation, and the downregulation of osteogenic transcription factor runt-related transcription factor 2 and osterix induced by pro-inflammatory cytokines. Baicalein also upregulated the phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK 1/2). The upregulation of IER3 by pro-inflammatory cytokines was blocked by pretreatment with inhibitors of AKT, p38, JNK, and ERK 1/2. Conclusion: Baicalein mitigates the deleterious responses of osteoblasts to pro-inflammatory cytokines. Further, IER3 enhanced the effect of baicalein via activation of AKT, p38, JNK, and ERK pathways.