• Journal of Periodontal and Implant Science
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• v.31 no.4
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• pp.833-846
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• 2001

Recently, use of natural medicine is getting more attention, and some of them are believed to be effective in　the treatment of periodontitis. Among them, the seeds of safflower(Carthamus tinctrorius L.) have been proven to be effective through its use in bone diseases such as fracture and osteoporosis. During the last few years, studies using the seeds of safflower gown in Korea have been active, and it has been reported that safflower seed extract increase the proliferation and the alkaline phosphatase(ALP) activity of human periodontal ligament fibroblast(hPDLF), osteoblast, and that they promote the mineralization process. In animal studies, when safflower seed extract were administered orally new bone formation was promoted. Recently, in an effort to find out the most effective osteogenic components, among many components of the safflower seed, various safflower seed fraction extracts were obtained by multistep extraction of the safflower components using various solvents. Among these, saf-M-W fraction extracted by methanol and water was most effective in increasing osteogenic potential of osteoblasts. In this study, the effect of safflower seed fraction extract, saf-M-W, on the growth and differentiation of hPDLF and MC3T3-E1 cell was investigated. The toxicity of saf-M-W on both cells was measured using M'IT(3-(4,5dimethylthiazol-2-y1)-2,5-diphenyl tetrazolium bromide) test, and ALP activity was measured using the colorimetric assay of hPDLF. In addition, in MC3T3-El cells, the expression of ALP, bone sialoprotein(BSP) mRNA was observed using Northern blot, and the mineralized nodule formation Was observed using von Kossa stain and phase-contrast microscope. 1. In concentrations below $10{\mu}g/ml$, saf-M-W didn't show any toxicity on hPDLF and MC3T3-El cell. 2. The change in saf-M-W concentration had no effect on the ALP activity of hPDLF. 3. In MC3T-E1 cells, mRNA expressions of ALP and BSP were greater in the experimental group treated with $10{\mu}g/ml$ concentration of saf-M-W compared with the control group. 4. In MC3T3-El cells, abundance of mineralized nodules were formed in the experimental group treated with $10{\mu}g/ml$ Concentration of saf-M-W, while no mineralized nodule was formed in the control group. These results suggest that safflower seed fraction extract, saf-M-W. didn't show any toxicity on hPDLF and MC3T3-E1 cell at concentrations below $10{\mu}g/ml$ and effectively enhanced the differentiation and osteogenic potential of MC3T3-El cell.

• 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.

• Herbal Formula Science
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• v.25 no.4
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• pp.527-536
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• 2017

Objectives : The present study, to confirm the osteoblast differentiation effects of Acer tegmentosum Maxim. (AT) extract. Methods : In this experiment, cell viability, Alizarin red S assay, and Alkaline phosphatase (ALP) activity with AT extract (50, $100{\mu}g/m{\ell}$). Also, we studied the expression of differentiation regulator with AT extract in primary calvarial osteoblasts cells (pOB). Results : As a result of AT treatment, we determined that AT extract stimulates ALP activity and alizarin red activities in the pOB cells for mineralization for 18 days. Moreover, these factors increasing osteogenic markers such as Runt-related transcription factor2 ($Run{\times}2$), osteocalcin (OC), osteopontin, osterix, smad1, smad5, activating transcription factor4 (ATF4) and collagen type I alpha 1. Conclusions : These results indicate that AT extract have effect on bone through the promotion of osteoblastic differentiation, suggesting that it could be used for the treatment of bone diseases.

• 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.

• International Journal of Oral Biology
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• v.45 no.2
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• pp.42-50
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• 2020

Lysophosphatidic acid (LPA) is a lipid messenger mediated by G protein-coupled receptors (LPAR1-6). It is involved in the pathogenesis of certain chronic inflammatory and autoimmune diseases. In addition, it controls the self-renewal and differentiation of stem cells. Recent research has demonstrated the close relationship between periodontitis and various diseases in the human body. However, the precise role of LPA in the development of periodontitis has not been studied. We identified that LPAR1 was highly expressed in human periodontal ligament stem cells (PDLSCs). In periodontitis-mimicking conditions with Porphyromonas gingivalis-derived lipopolysaccharide (Pg-LPS) treatment, PDLSCs exhibited a considerable reduction in the cellular viability and osteogenic differentiation potential, in addition to an increase in the inflammatory responses including tumor necrosis factor-α and interleukin-1β expression and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. Of the various LPAR antagonists, pre-treatment with AM095, an LPAR1 inhibitor, showed a positive effect on the restoration of cellular viability and osteogenic differentiation, accompanied by a decrease in NF-κB signaling, and action against Pg-LPS. These findings suggest that the modulation of LPAR1 activity will assist in checking the progression of periodontitis and in its treatment.

• Restorative Dentistry and Endodontics
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• v.39 no.3
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• pp.187-194
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• 2014

Objectives: The effects of bone morphogenetic protein-2 (BMP-2) and enamel matrix derivative (EMD) respectively with mineral trioxide aggregate (MTA) on hard tissue regeneration have been investigated in previous studies. This study aimed to compare the osteogenic effects of MTA/BMP-2 and MTA/EMD treatment in MC3T3-E1 cells. Materials and Methods: MC3T3-E1 cells were treated with MTA (ProRoot, Dentsply), BMP-2 (R&D Systems), EMD (Emdogain, Straumann) separately and MTA/BMP-2 or MTA/EMD combination. Mineralization was evaluated by staining the calcium deposits with alkaline phosphatase (ALP, Sigma-Aldrich) and Alizarin red (Sigma-Aldrich). The effects on the osteoblast differentiation were evaluated by the expressions of osteogenic markers, including ALP, bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN) and osteonectin (OSN), as determined by reverse-transcription polymerase chain reaction analysis (RT-PCR, AccuPower PCR, Bioneer). Results: Mineralization increased in the BMP-2 and MTA/BMP-2 groups and increased to a lesser extent in the MTA/EMD group but appeared to decrease in the MTA-only group based on Alizarin red staining. ALP expression largely decreased in the EMD and MTA/EMD groups based on ALP staining. In the MTA/BMP-2 group, mRNA expression of OPN on day 3 and BSP and OCN on day 7 significantly increased. In the MTA/EMD group, OSN and OCN gene expression significantly increased on day 7, whereas ALP expression decreased on days 3 and 7 (p < 0.05). Conclusions: These results suggest the MTA/BMP-2 combination promoted more rapid differentiation in MC3T3-E1 cells than did MTA/EMD during the early mineralization period.

• 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 Microbiology and Biotechnology
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• v.33 no.9
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• pp.1197-1205
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• 2023

Osteoporosis, Greek for "porous bone," is a bone disease characterized by a decrease in bone strength, microarchitectural changes in the bone tissues, and an increased risk of fracture. An imbalance of bone resorption and bone formation may lead to chronic metabolic diseases such as osteoporosis. Wolfiporia extensa, known as "Bokryung" in Korea, is a fungus belonging to the family Polyporaceae and has been used as a therapeutic food against various diseases. Medicinal mushrooms, mycelium and fungi, possess approximately 130 medicinal functions, including antitumor, immunomodulating, antibacterial, hepatoprotective, and antidiabetic effects, and are therefore used to improve human health. In this study, we used osteoclast and osteoblast cell cultures treated with Wolfiporia extensa mycelium water extract (WEMWE) and investigated the effect of the fungus on bone homeostasis. Subsequently, we assessed its capacity to modulate both osteoblast and osteoclast differentiation by performing osteogenic and anti-osteoclastogenic activity assays. We observed that WEMWE increased BMP-2-stimulated osteogenesis by inducing Smad-Runx2 signal pathway axis. In addition, we found that WEMWE decreased RANKL-induced osteoclastogenesis by blocking c-Fos/NFATc1 via the inhibition of ERK and JNK phosphorylation. Our results show that WEMWE can prevent and treat bone metabolic diseases, including osteoporosis, by a biphasic activity that sustains bone homeostasis. Therefore, we suggest that WEMWE can be used as a preventive and therapeutic drug.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.27 no.1
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• pp.16-23
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• 2005

It is commonly acknowledged that bone morphogenic protein (BMP-2) functions as a potential osteogenic inducer in bone formation. Recently, several papers reported that bone marrow-derived stem cell (BMSC) from human is not responsive to BMP-2 in comparison to high capacity of BMP-2 in the osteoinduction of stromal cell derived from bone marrow of rodent animals such as rat or mouse. In this study, we characterized BMSC derived from 11 years old donor for the responsiveness to rhBMP-2, dexamethasone (Dex) and 1,25-dihydroxyvitamin D (vitamin D), in order to analyze their function in the early osteogenesis. The effect of over mentioned agents was evaluated by means of assessing alkaline phosphatase (ALP) activity/staining, RT-PCR analysis and von Kossa staining. In addition, we analyzed the meaning of expressed several osteoblastic markers such as alkaline phosphatase, collagen typeI, osteopontin, bone sialoprotein and osteocalcin with relation to either differentiation or mineralization. Only in the presence of Dex, human BMSC could commit osteoblastic differentiation and matrix mineralization, and either BMP-2 or vitamin D treatment was not able to induce. But BMP-2 or Vitamin D showed potential synergy effect with Dex. ALP and bone sialoprotein were clearly expressed in response of Dex treatment compared to weak expression of osteopontin in early osteogenesis. Therefore, we expect that this study will contribute partly to elucidiating early osteogenesis mechanism in human, but variations among bone marrow donors must be considered through further study.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.35 no.1
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• pp.31-37
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• 2013

Purpose: Maxilla and mandible have different patterns of cortical and trabecular bone and different bone mineral densities, even though both are components of the jaw bone. However, cellular differences between maxilla- and mandible derived osteoblasts (OBs) have rarely been studied. We hypothesize that maxilla- and mandible-derived OBs show different responses to $17{\beta}$-estradiol (E2), which is one of the critical factors for bone formation. This study compares skeletal site-specific cell responses between maxilla- and mandible-derived human OBs to E2. Methods: Maxilla- and mandible-derived OBs derived from an identical donor were separately isolated from a total of five normal healthy subjects aged 18~44 years old, cultured with a treatment of 100 nM estrogen. The responses between maxilla- and mandible-derived OBs to E2 were evaluated and compared using cell proliferation, alkaline phosphatase (ALP) activity and gene expression of osteoprotegerin (OPG), ALP, insulin-like growth factor-1 (IGF-1), and estrogen receptor ${\alpha}$ ($ER{\alpha}$). Results: E2 did not have any distinct effects on the proliferation of both types of OBs. Mandible-derived OBs exhibited higher ALP activity than maxilla-derived OBs in the non-treated condition, which was common in all tested individuals. ALP activities of both types of OBs showed a minor increasing tendency with the treatment of E2, even though there was no statistical significance in some specimens. The gene expression of OB by E2 was diverse, depending on the individuals. There was increased expression of OPG, IGF-1, or $ER{\alpha}$ gene in the part of subjects, which was more repeated in maxilla-derived OBs. In particular, OPG or ALP induction by E appeared less frequently in mandible-derived OBs. Conclusion: Current results revealed that E2 affects maxilla- and mandible-derived OBs into facilitating the osteogenic process despite individual differences. Mandible-derived OBs are less sensitive to bone-forming gene expression by E2.