• Maxillofacial Plastic and Reconstructive Surgery
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• v.29 no.3
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• pp.197-205
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• 2007

Angiogenesis is a essential part for bone formation and bone fracture healing. Vascular endothelial growth factor (VEGF), one of the most important molecules among many angiogenic factors, is a specific mitogen for vascular endothelial cells. VEGF-mediated angiogenesis is required for bone formation and repair. However, the effect of VEGF on osteoblastic cells during osteogenesis is still controversial. In recent days, substantial progress have been made toward developing tissue-engineered alternatives to autologous bone grafting for maxillofacial bony defects. Periosteum has received considerable interest as a better source of adult stem cells. Periosteum has the advantage of easy harvest and contains various cell types and progenitor cells that are able to differentiate into a several mesenchymal lineages, including bone. Several studies have reported the bone formation potential of periosteal cells, however, the correlation between VEGF signaling and cultured human periosteal cell-derived osteogenesis has not been fully investigated yet. The purpose of this study was to examine the correlation between VEGF signaling and cultured human periosteal-derived cells osteogenesis. Periosteal tissues of $5\;{\times}\;20\;mm$ were obtained from mandible during surgical extraction of lower impacted third molar from 3 patients. Periosteal-derived cells were introduced into the cell culture and were subcultured once they reached confluence. After passage 3, the periosteal-derived cells were further cultured for 42 days in an osteogenic inductive culture medium containing dexamethasone, ascorbic acid, and ${\beta}-glycerophosphate$. We evaluated the alkaline phosphatase (ALP) activity, the expression of Runx2 and VEGF, alizarin red S staining, and the quantification of osteocalcin and VEGF secretion in the periosteal-derived cells. The ALP activity increased rapidly up to day 14, followed by decrease in activity to day 35. Runx2 was expressed strongly at day 7, followed by decreased expression at day 14, and its expression was not observed thereafter. Both VEGF 165 and VEGF 121 were expressed strongly at day 35 and 42 of culture, particularly during the later stages of differentiation. Alizarin red S-positive nodules were first observed on day 14 and then increased in number during the entire culture period. Osteocalcin and VEGF were first detected in the culture medium on day 14, and their levels increased thereafter in a time-dependent manner. These results suggest that VEGF secretion from cultured human periosteal-derived cells increases along with mineralization process of the extracellular matrix. The level of VEGF secretion from periosteal-derived cells might depend on the extent of osteoblastic differentiation.

• The Korean Journal of Physiology and Pharmacology
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• v.6 no.3
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• pp.165-169
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• 2002

We have previously demonstrated that phytoestrogens isolated from safflower seeds significantly attenuated bone loss in ovariectomized rats, and directly stimulated proliferation and differentiation of cultured osteoblastic cells. In an attempt to elucidate underlying cellular mechanisms, in the present study we investigated effects of $17{\beta}-estradiol\;(E_2)$ and phytoestrogens such as matairesinol and acacetin, a type of lignan and flavonoid, respectively, on activation of mitogen activated protein (MAP) kinases, extracellular signal-regulated kinase 1 (ERK1) and ERK2, in cultured osteoblastic ROS 17/2.8 cells. Western blot analysis with anti-MAP kinase antibody showed that a wide range concentrations $(10^{-14}\;to\;10^{-6}\;M)\;of\;E_2$ as well as both phytoestrogens induced rapid and transient activation of ERK1/2 through phosphorylation within minutes. Maximum activation of MAP kinases by $E_2$ and phytoestrogens were observed at 10 and 15 min, respectively. $E_2-induced$ phosphorylation of ERK1/2 returned to the control level at 30 min, whereas phytoestrogen-induced phosphorylation was maintained at high level until 30 min. PD-98059, a highly selective inhibitor of MAP kinase, prevented phosphorylation of ERK1/2 in the cells treated either with $E_2$ or phytoestrogens. To examine a possible involvement of estrogen receptor in the activation process of MAP kinase, Western blot analysis was performed in the presence and absence of the estrogen receptor antagonists, ICI 182,780 and tamoxifen. These antagonists blocked MAP kinase phosphorylation induced not only by $E_2,$ but also by the phytoestrogens. To the best our knowledge, this study is the first to demonstrate that phytoestrogens such as flavonoid and lignan extracted from safflower seeds produce a rapid activation of MAP kinase, at least partially via membrane estrogen receptor of the cultured osteoblastic cells.

• Molecules and Cells
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• v.41 no.3
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• pp.234-243
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• 2018

In recent years, the interest towards the relationship between incretins and bone has been increasing. Previous studies have suggested that glucagon-like peptide-1 (GLP-1) and its receptor agonists exert beneficial anabolic influence on skeletal metabolism, such as promoting proliferation and differentiation of osteoblasts via entero-osseous-axis. However, little is known regarding the effects of GLP-1 on osteoblast apoptosis and the underlying mechanisms involved. Thus, in the present study, we investigated the effects of liraglutide, a glucagon-like peptide-1 receptor agonist, on apoptosis of murine MC3T3-E1 osteoblastic cells. We confirmed the presence of GLP-1 receptor (GLP-1R) in MC3T3-E1 cells. Our data demonstrated that liraglutide inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as detected by Annexin V/PI and Hoechst 33258 staining and ELISA assays. Moreover, liraglutide upregulated Bcl-2 expression and downregulated Bax expression and caspase-3 activity at intermediate concentration (100 nM) for maximum effect. Further study suggested that liraglutide stimulated the phosphorylation of AKT and enhanced cAMP level, along with decreased phosphorylation of $GSK3{\beta}$, increased ${\beta}-catenin$ phosphorylation at Ser675 site and upregulated nuclear ${\beta}-catenin$ content and transcriptional activity. Pretreatment of cells with the PI3K inhibitor LY294002, PKA inhibitor H89, and siRNAs GLP-1R, ${\beta}-catenin$ abrogated the liraglutide-induced activation of cAMP, AKT, ${\beta}-catenin$, respectively. In conclusion, these findings illustrate that activation of GLP-1 receptor by liraglutide inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation through $cAMP/PKA/{\beta}-catenin$ and $PI3K/Akt/GSK3{\beta}$ signaling pathways.

• Archives of Pharmacal Research
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• v.26 no.12
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• pp.1029-1035
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• 2003

Osteoporosis is recognized as one of the major hormonal deficiency diseases, especially in menopausal women and the elderly. When estrogen is reduced in the body, local factors such as IL-1 $\beta$ and IL-6, which are known to be related with bone resorption, are increased and promote osteoclastogenesis, which is responsible for bone resorption. In the present study, we investigated whether glucosidic isoflavones (Isocal, PIII) extracted from Sophorae fructus affect the proliferation of osteoblasts and prevent osteoclastogenesis in vitro by attenuating upstream cytokines such as IL-1$\beta$ and IL-6 in a human osteoblastic cell line (MG-63) and in a primary osteoblastic culture from SD rat femurs. Interestingly, IL-1$\beta$ and IL-6 mRNA were significantly suppressed in osteoblast-like cells treated with 17$\beta$-estradiol (E2) and PIII when compared to positive control (SDB), and this suppression was more effective at $10^{-8}$% than at the highest concentration of $10^{-4}$%. In addition, these were confirmed in protein levels using ELISA assay. In the cell line, the cells showed that E2 was the most effective in osteoblastic proliferation over the whole range of concentration ($10^{-4}%-10^{-12}$%), even though PIII also showed the second greatest effectiveness at $10^{-8}$%. Nitric oxide (NO) was significantly (p<0.05) upregulated in PIII and E2 over the concentration range $10^{-6}% to 10^{-8}$% when compared to SDB, without showing any dose dependency. In bone marrow primary culture, we found by TRAP assay that PIII effectively suppressed osteoclastogenesis next to E2 in comparison with SDB and culture media (control). In conclusion, these results suggest that local bone-resorbing cytokines can be regulated by PIII at lower concentrations and that, therefore, PIII may preferentially induce anti-osteoporosis response by attenuating osteoclastic differentiation and by upregulating NO.

• International Journal of Oral Biology
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• v.38 no.3
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• pp.127-134
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• 2013

Xylitol is a sugar alcohol with a variety of functions including bactericidal and anticariogenic effects. However, the cellular mechanisms underlying the role of xylitol in bone metabolism are not yet clarified. In our present study, we exploited the physiological role of xylitol on osteoclast differentiation in a co-culture system of osteoblastic and RAW 264.7 cells. Xylitol treatment of these co-cultures reduced the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells induced by 10 nM $1{\alpha},25(OH)_2D_3$ in a dose-dependent manner. A cell viability test revealed no marked cellular damage by up to 100 mM of xylitol. Exposure of osteoblastic cells to xylitol decreased RANKL, but not OPG, mRNA expression in the presence of $10^{-8}M$ $1{\alpha},25(OH)_2D_3$ in a dose-dependent manner. Furthermore, bone resorption activity, assessed on bone slices in the coculture system, was found to be dramatically decreased with increasing xylitol concentrations. RANKL and OPG proteins were assayed by ELISA and the soluble RANKL (sRANKL) concentration was decreased with an increased xylitol concentration. In contrast, OPG was unaltered by any xylitol concentration in this assay. These results indicate that xylitol inhibits $1{\alpha},25(OH)_2D_3$-induced osteoclastogenesis by reducing the sRANKL/OPG expression ratio in osteoblastic cells.

• 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.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.32 no.6
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• pp.495-505
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• 2006

In this study we evaluate the effects of bFGF-BB and PDGF on in vitro proliferation, differentiation and mineralization of mesenchymal stem cells (MSCs) from rat. MSCs were prepared from the bone marrow of 6 or 7-week-old male rats with a technique previously described by Maniatopoulos et al. in 1988. Lineage differentiation to osteogenesis, chondrogenesis and adipogenesis were performed. At first, we characterized the cultured cell on passage 1, 3, 5, 7 with immunocytochemical staining using CD29, 44, 34, 45, ${\alpha}$-SMA and type I collagen. And to study the effects of bFGF and PDGF-BB on proliferation, differentiation and mineralization, we seeded the expanded cell at a density of 6 $6{\times}10^3\;cells/cm^2$ to 100-mm dish for evaluation of cell proliferation and MTT assay was carried out on day 2, 4, 7, 9. We also resuspended the cells with same density $(6{\times}10^3\;cells/cm^2)$ to 24 well plates for subculture. On the following day, the attached cells were exposed to 2.5ng/ml bFGF and/or 25ng/ml PDGF-BB daily during 5 days. The osteocalcin (OC) level was assessed and mineral contents were evaluated with alizarin red S staining on subculture day 2, 7, 14, 21. We identified the mesenchymal stem cell from the bone marrow derived cells of rat through their successful multi-differentiation and stable display of its phenotype. And bFGF and PDGF-BB showed the effect that inhibited osteoblastic differentiation and mineralization mildly in above concentration at in vitro culture. This study was supported by grant 04-2004-0120 from the Seoul National University Hospital Research Fund.

• Journal of the Korean Society of Food Science and Nutrition
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• v.39 no.2
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• pp.203-209
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• 2010

In this study, the effects of Petasites japonicus and Momordica charantia L. extracts on MC3T3-ET1 osteoblastic cells were investigated. Since the activity of osteoblastic cell is one of the important factors for bone formation, the cellular proliferation of osteoblast was evaluated by MTT and alkaline phosphatase (ALP) activity. Compared to control, the cell proliferation was elevated to 114% and 112% by the treatment of Petasites japonicus and Momordica charantia L. extracts, respectively at the concentration of $10\;{\mu}g/mL$. The cell differentiation was also measured by alkaline phosphatase (ALP) activity at 3, 7, 14, and 27 days treatments with one of the extracts, respectively. As results, the ALP activity was significantly increased at 3 days, compared to control (p<0.05). To evaluate the effect of Petasites japonicus and Momordica charantia L. extracts on bone nodule formation, MC3T3-E1 cells were cultured in $\alpha$-MEM for 3, 14, and 21 days and then stained by alizarin red. To determine the expression patterns of bone-related proteins during the MC3T3-E1 osteoblast-like cell differentiation, osteoblast cells were cultured in $\alpha$-MEM for 24 hr. RNA was extracted and RT-PCR analysis was performed to examine the expression of OPG, RANKL and osteocalcin. Petasites japonicus extract exhibited the significant increment of osteocalcin compared with the positive control, which suggests that Petasites japonicus may have beneficial effects on bone health through the proliferation of osteoblast cells.

• Korean Journal of Medicinal Crop Science
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• v.19 no.6
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• pp.491-500
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• 2011

Ursolic acid, triterpenoid compound has been shown to stimulate osteoblast differentiation and enhance bone formation. In the present study, we examined the effects of similar triterpenoid compounds, oleanolic acid (OA) and its derivatives, such as oleanolic acid acetate (OAA) and oleanolic acetate methyl ester (OAM) on the bone formation in MC3T3-E1 osteoblast cells. We determined cellular proliferation, alkaline phosphatase (ALP) activity, mineralization, and expression of osteoblast specific genes and mitogen activated protein kinase phosphorylation. Treatment of $0.1-10{\mu}m$ OA, OAA, and OAM increased cellular proliferation, but not significantly increased as compared with dimethyl sulfoxide (DMSO). OA, OAA, and OAM at 5uM concentration enhanced ALP expression, mineralization, and osteocalcin (OCN) mRNA level. In conclusion, OA and its derivatives stimulated the osteoblast differentiation by increasing ALP, mineralization, and OCN mRNA expression. However, there were no significantly difference on osteoblast differentiation among treatment of OA, OAA, and OAM.

• 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.