• Journal of Periodontal and Implant Science
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• 제38권1호
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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.

• 디지털융복합연구
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• 제13권5호
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• pp.357-363
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• 2015

본 연구는 치아 치료를 위한 다양한 구강제품의 성분들을 천연물에서 발굴하여 부작용이 적고 저렴한 구강제품을 만들에 삶의 질적인 부분에서 웰니스를 위한 융복합 연구를 하고자함이다. 사람 골육종세포(MG-63)에 오배자(Galla Rhois) 추출물을 이용하여 구강염증질환에 있어서 항염증효과를 항산화작용기전의 지표로 사용하는 NO생성과 DPPH radical 소거능으로 확인하였다. 오배자 80% 메탄올추출물에서 62%, n-헥산 추출물에서 68%의 강한 NO생성 억제 활성을 나타냈다. DPPH radical 소거능을 살펴보면 오배자 80% 메탄올추출물은 27.8%, n-헥산 추출물은 30.5%, 디에틸에테르 추출물은 51.7%, 클로로포름 추출물은 66.4% 그리고 에틸아세테이트 추출물은 62.4%의 자유라디칼 소거억제능력을 확인할 수 있었다. 이에 본 연구는 오배자 메탄올 추출물이 항산화 작용에 있어서 탁월한 효과가 있음을 확인할 수 있었고, 이러한 천연물 유래성분들을 적용하여 비용부담이 줄여서 삶의 질적 수준을 높여보고자 한다.

• Journal of Periodontal and Implant Science
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• 제36권2호
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• pp.449-459
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• 2006

DFDBA(Decalcified freeze-dried bone allograft) is one of the allograft materials for periodontal bone regeneration. DFDBA provides an osteoconductive surface and osteoinductive factors. Therefore, DFDBA have been used successfully to regenerate the attachment apparatus during periodontal treatment. But recent studies was reported that wide variations in commercial bone bank preparations of DFDBA do exist, including the ability to induce new bone formation. DFDBA was experimental materials that was recovered, processed, tested, shipped and invoiced through Musculoskeletal Transplant Foundation. MTF(Musculoskeletal Transplant Foundation) is the world largest, non-profit, AATB(American Association of Tissue Banks) accredited tissue bank. The objective of this study was to determine the effects of serial dilutions of a DFDBA on human fetal osteoblastic cell proliferation and their potential to form and mineralize bone nodules. Human fetal osteoblastic cell line(hFOB 1.19) was cultured with DMEM and SSE($1{\mu}g/m{\ell}$,$10{\mu}g/m{\ell}$, $100{\mu}g/m{\ell}$, $1mg/m{\ell}$) at $34^{\circ}C$ with 5% CO2 in 100% humidity. Cell proliferation was significantly increased at $1mg/m{\ell}$, $100{\mu}g$, $10{\mu}g/m{\ell}$, $1{\mu}g/m{\ell}$, $100ng/m{\ell}$, $10ng/m{\ell}$, $1ng/m{\ell}$ of DFDBA after 5 days incubation (p<0.05). Alkaline Phosphatase(ALP) level was significantly increased in $100ng/m{\ell}$, $10ng/m{\ell}$, $1ng/m{\ell}$ of DFDABA(p<0.05). A quantified calcium accumulation was significantly increased at $1ng/m{\ell}$, $10ng/m{\ell}$ of MTF(p<0.05). These results indicated that DFDBA has an inductive effect on bone formation in vitro.

• Molecules and Cells
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• 제41권6호
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• pp.575-581
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• 2018

Postmenopausal osteoporosis (PMOP) is a common systemic skeletal disease characterized by reduced bone mass and microarchitecture deterioration. Although differentially expressed SOX5 has been found in bone marrow from ovariectomized mice, its role in osteogenic differentiation in human mesenchymal stem cells (hMSCs) from bone marrow in PMOP remains unknown. In this study, we investigated the biological function of SOX5 and explore its molecular mechanism in hMSCs from patients with PMOP. Our findings showed that the mRNA and protein expression levels of SOX5 were upregulated in hMSCs isolated from bone marrow samples of PMOP patients. We also found that SOX5 overexpression decreased the alkaline phosphatase (ALP) activity and the gene expression of osteoblast markers including Collagen I, Runx2 and Osterix, which were increased by SOX5 knockdown using RNA interference. Furthermore, $TNF-{\alpha}$ notably upregulated the SOX5 mRNA expression level, and SOX5 knockdown reversed the effect of $TNF-{\alpha}$ on osteogenic differentiation of hMSCs. In addition, SOX5 overexpression increased Kruppel-like factor 4 (KLF4) gene expression, which was decreased by SOX5 silencing. KLF4 knockdown abrogated the suppressive effect of SOX5 overexpression on osteogenic differentiation of hMSCs. Taken together, our results indicated that $TNF-{\alpha}$-induced SOX5 upregulation inhibited osteogenic differentiation of hMSCs through KLF4 signal pathway, suggesting that SOX5 might be a novel therapeutic target for PMOP treatment.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제44권6호
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• pp.259-268
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• 2018

Objectives: The purpose of this study was to evaluate the synergic effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) and low-level laser therapy (LLLT) on bisphosphonate-treated osteoblasts. Materials and Methods: Human fetal osteoblast cells (hFOB 1.19) were cultured with $100{\mu}M$ alendronate. Low-level Ga-Al-As laser alone or with 100 ng/mL rhBMP-2 was then applied. Cell viability was measured with MTT assay. The expression levels of receptor activator of nuclear factor kappa-B ligand (RANKL), macrophage colony-stimulating factor (M-CSF), and osteoprotegerin (OPG) were analyzed for osteoblastic activity inducing osteoclastic activity. Collagen type and transforming growth factor beta-1 were also evaluated for bone matrix formation. Results: The results showed that rhBMP-2 and LLLT had a synergic effect on alendronate-treated osteoblasts for enhancing osteoblastic activity and bone matrix formation. Between rhBMP-2 and LLLT, rhBMP-2 exhibited a greater effect, but did not show a significant difference. Conclusion: rhBMP-2 and LLLT have synergic effects on bisphosphonate-treated osteoblasts through enhancement of osteoblastic activity and bone formation activity.

• Journal of Periodontal and Implant Science
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• 제32권2호
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• pp.389-402
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• 2002

BMP can induce ectopic bone formation when implanted into sites such as rat muscle and can greatly enhance healing of bony defects when applied exogenously. In addition, BMP stimulated osteoblastic differentiation in vitro in various types of cells. The aim of this study was to investigate the effect of recombinant human bone morphogenetic protein(rhBMP-2) on the proliferation and osteoblastic differentiation of human periodontal ligament cells and gingival fibroblasts. The cell number and alkaline phosphatase activity were measured in 3 experimental groups of human periodontal ligament cells and gingival fibroblasts (control group, rhBMP-2 50ng/ml group, and rhBMP-2 100ng/ml group) at 1 and 2 weeks after culture. At the same time, total RNA of cultured cells were extracted and reverse trascription polymerase chain reaction(RT-PCR) was performed to determine the expression of mRNA of bone matrix protein. RhBMP-2 had no effect on the cell proliferation of human periodontal ligament cells and gingival fibroblasts. Alkaline phosphatase activity was elevated significantly by rhBMP-2 in both cells. And periodontal ligament cells showed significantly higher alkaline phosphatase activity than gingival fibroblasts. ${\beta}$-actin, type I collagen, alkaline phosphatase, BMP-2 mRNA were expressed in all of the samples. Osteopontin, osteocalcin mRNA were expressed in all periodontal ligament cell groups, and rhBMP-2 50ng/ml group, rhBMP-2 100ng/ml group of 2 week culture period of gingival fibroblasts. Bone sialoprotein mRNA was only expressed in rhBMP-2 50ng/ml group and rhBMP-2 100ng/ml group of 2-week culture period. These results suggest that rhBMP-2 stimulates osteoblastic differentiation in human periodontal ligament cells and gingival fibroblasts in vitro.

• Journal of Korean Dental Science
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• 제4권1호
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• pp.6-13
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• 2011

Purpose: Craniosynostosis (CS), one of the most common congenital craniofacial deformities, is the premature closure of cranial sutures. NELL-1 is a novel molecule overexpressed during premature cranial suture closure in human CS. From a functional perspective, NELL-1 has been reported to accelerate chondrocyte maturation and modulate calvarial osteoblast differentiation and apoptosis pathways. The mechanism through which NELL-1 induces these phenomena, however, remains unclear. The purpose of this study is to identify the NELL-1 binding protein(s) through which the biologic mechanism of NELL-1 can be further investigated. Materials and Methods: Far-Western and Immunoprecipitation (IP) assays were performed, independently and in sequence, followed by mass spectrometry to identify the NELL-1 binding proteins. Reverse IP was used to verify and confirm candidate binding protein. Results: The only confirmative protein from current experimentation was vimentin. Vimentin is the major structural component of the intermediate filaments. Conclusion: The present study identified and confirmed vimentin as a NELL-1 binding protein, which opened up a new window to mechanistically facilitate studies on this CS-associated molecule.

• 셀메드
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• 제8권3호
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• pp.13.1-13.8
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• 2018

In the field of osteoporosis, there has been growing interest in anabolic agents that enhance bone formation. The purpose of this study was to examine the effects of NNMBS 246 osteoblastic differentiation with associated signaling pathways. NNMBS 246 markedly increased alkaline phosphatase (ALP) activity and calcium nodule formation. Stimulation with NNMBS 246 not only increased the differentiation markers (ALP, OPN, OCN) level and transcription markers (RUNX2, Osterix) mRNA expression but also upregulated the ECM molecules and OPG mRNA expression. Treatments of NNMBS 246 downregulated MMPs (MMP-1, MMP-2, MMP-9), but RANKL mRNA expression. Furthermore, NNMBS 246 activated osteoblastic differentiation markers and formed calcium nodules in human periodontal ligament cells (hPDLCs) and cementoblast cells. NNMBS 246 induced phosphorylation of MAPKs, Akt, nuclear p65 and IkB-${\alpha}$. BMP-2/Smad and ${\beta}$-catenin signaling pathways were activated by NNMBS 246. Sirtinol (SIRT1 inhibitor) inhibited NNMBS 246-induced osteoblastic differentiation markers mRNA expression. These results suggested that NNMBS 246 has the potential to enhance osteoblastogenesis probably through the activation of BMP/Smad and ${\beta}$-catenin signal pathways, and SIRT1 plays as critical mediator in bone anabolic effect of NNMBS 246.

• Journal of Periodontal and Implant Science
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• 제27권2호
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• pp.291-303
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• 1997

Periodontal ligament cells may have a role in the regulation of hard and soft periodontal tissues, but their specific function has not yet to be determined. To evaluate further their role in periodontal regeneration, they were examined for osteoblast-like behavior. Periodontal ligament cells and gingival fibroblasts were primarily cultured from extracted premolar with non-periodontal diseases. Cells were cultured with DMEM at $37^{\circ}C$, 5% $CO_2$, 100% humidity incubator, and as a measure of cell characterization, it was examined that the morphology, alkaline phosphatase activity, collagen synthesis, and immunocytochemistry for osteonectin, osteocalcin, and collagen type I. Healthy periodontal ligament cells has more osteoblastic-like cell property in alkaline phosphatase activity. and collagen synthesis than gingival fibroblast. Immunocytochemistry localization explained that calcitonin were expressed in periodontal ligament cells only, and osteonectin and type I collagen were produced in both cells simultaneously. This results indicate that the growth characteristics of periodontal ligament cells and gingival fibroblasts exhibit some differences in proliferative rates and biochemical synthesis. The differences may help to calrify the role such cells play in the regenearation of periodontal tissues.

• International Journal of Oral Biology
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• 제31권4호
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• pp.119-125
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• 2006

The importance of phytoestrogens to human health is currently being actively investigated. Hesperidin, abundantly found in citrus fruits, is known to possess antioxidant, anticancer, and anti-inflammatory effects. Recently, it has been reported that hesperidin inhibits bone loss and decreases serum and hepatic lipids in ovariectomized mice. In our study, to determine the possible role of hesperidin in the regulation of bone metabolism, we observed the effects of hesperidin on the proliferation and activity of osteoblasts, as well as the effects of hesperidin on osteoclast generation and activity. We observed that, when treated with hesperidin, the number and viability of osteoblastic cells increased, alkaline phosphatase (ALP) activity of osteoblastic cells increased, and osteoprotegerin (OPG) secretion from MG63 cells decreased. Hesperidin treatment had no effect on the osteoclast generation and activity in the bone marrow cell culture, but decreased the number and resorptive activity of osteoclasts generated from RAW/264.7 cells. Taken together, these results indicate that hesperidin increases the proliferation and activity of osteoblasts, while inhibiting generation and activity of osteoclasts. Although the precise role of hesperidin remains to be elucidated, our study suggests that it is one of the important modulators of bone metabolism.