• Molecules and Cells
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• 제22권2호
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• pp.182-188
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• 2006

Dlx5 and Osx are master regulatory proteins essential for initiating the cascade leading to osteoblast differentiation in mammals, but the mechanism of osteoblast-specific expression is not fully understood. DNA methylation at CpG sequences is involved in tissue and cell type-specific gene expression. We investigated the methylation status of Dlx5 and Osx in osteogenic and nonosteogenic cell lines by methylationspecific PCR (MSP). The CpG dinucleotides of the Dlx5 and Osx promoter regions were unmethylated in osteogenic cell lines transcribing these genes but methylated in nonosteogenic cell lines. Treatment of C2C12 cells with 5-AzadC induced dose- and timedependent expression of Dlx5 and Osx mRNA by demethylating the corresponding promoters. Furthermore the mRNAs for the osteoblast markers ALP and OC, which were undetectable in untreated cells, gradually increased after 5-AzadC treatment. In addition, BMP-2 stimulation induced Dlx5 expression by hypomethylating its promoter. These findings suggest that DNA methylation plays an important role in cell type-specific expression of Dlx5 and Osx.

• Journal of Acupuncture Research
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• 제29권6호
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• pp.11-21
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• 2012

Objectives : BHH10 is traditional medicine herb used for enhancing body resistance against various diseases. The aim of this study was to identify BHH10 extract induces osteogenic activity in human osteoblast-like MC3T3-E1 cells. Methods : MC3T3-E1, pre-osteoblast cell line, were treated with BHH10 of various concentrations($0.1{\mu}g/mL$, $1{\mu}g/mL$, $10{\mu}g/mL$). And then, the effect of BHH10 on osteoblast differentiation was examined by alkaline phosphatase(ALP) activity, von Kossa staining and RT-PCR for osteoblast differentiation markers such as osteocalcin(OCN), osteopontin(OPN). Results : BHH10 had dose-dependent effect on the viability of osteoblastic cells, and dose-dependently increased alkaline phosphatase(ALP) activity. BHH10 markedly increased mRNA expression for OCN, OPN in MC3T3-E1 cells. Also, BHH10 significantly induced mineralization in the culture of MC3T3-E1 cells. Conclusions : In conclusion, these results propose that BHH10 can play an important role in osteoblastic bone formation, osteogenesis, and may possibly lead to the development of bone-forming drugs.

• Preventive Nutrition and Food Science
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• 제19권3호
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• pp.194-203
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• 2014

Yam (Dioscorea batatas) is widely consumed as functional food for health promotion mainly in East Asia countries. We assessed whether yam root (tuber) or bark (peel) extracts stimulated the activity of osteoblasts for osteogenesis. MC3T3-E1 cells (mouse osteoblasts) were treated with yam root extracts (water or methanol) (study I) or bark extracts (water or hexane) (study II) within $0{\sim}10{\mu}g/mL$ during the periods of osteoblast proliferation (5~10 day), matrix maturation (11~15 day) and mineralization (16~20 day) as appropriate. In study I, both yam root water and methanol extracts increased cell proliferation as concentration-dependent manner. Cellular collagen synthesis and alkaline phosphatase (ALP) activity, both the indicators of bone matrix protein and inorganic phosphate production for calcification respectively, were also increased by yam root water and methanol extract. Osteoblast calcification as cell matrix Ca and P accumulation was also increased by the addition of yam root extracts. In study II, yam bark extracts (water and hexane) increased osteoblast proliferation and differentiation, as collagen synthesis and ALP activity and osteoblast matrix Ca and P deposition. The study results suggested that both yam root and bark extracts stimulate osteogenic function in osteoblasts by stimulating bone matrix maturation by increasing collagen synthesis, ALP activity, and matrix mineralization.

• 대한한의학회지
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• 제28권4호
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• pp.158-167
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• 2007

Background & Objective : Uncaria rhynchophylla is traditional medicine herb used for enhancing body resistance against various diseases. The aim of this study was to identify if Uncaria rhynchophylla extracts induce osteogenic activity in human osteoblast-like SaOS-2 cells. Methods : The osteogenic activity of Uncaria rhynchophylla was evaluated on cell proliferation assay by WST-8, and osteoblast-specific genes, such as VEGF, type I collagen (Col I), osteocalcin (OCN), and osteopontin (OPN) by RT-PCR analysis and ELISA assay in osteoblasts-like SaOS-2 cells. Bone mineralization was stained with Alizalin red method. Results : Uncaria rhynchophylla had significantly increased cell proliferation at a dose dependent manner in human osteoblast-like SaOS-2 cells. Uncaria rhynchophylla markedly increased alkaline phosphatase (ALP), vascular endothelial growth factor (VEGF) mRNA expression at 7 days and dose dependently increased ALP activity and VEGF secretion in human osteoblast-like SaOS-2 cells. Also, Uncaria rhynchophylla time-dependently increased type I collagen (Col I), osteopontin (OPN), and osteocalcin (OCN) mRNA in SaOS-2 cells. Extracellular accumulation of proteins such as Col I and OCN was maximal increased by Uncaria rhynchophylla at 10 ${\mu}g/ml$. Also, Uncaria rhynchophylla significantly induced mineralization in the culture of SaOS-2 cells. Conclusion : This study showed that Uncaria rhynchophylla had enhanced proliferation, ALP activity, VEGF, bone matrix proteins such as OCN, OPN, and Col I, and mineralization in SaOS-2 cells. These results propose that Uncaria rhynchophylla can play an important role in osteoblastic bone formation, osteogenesis, and may possibly lead to the development of bone-forming drugs.

• Journal of Periodontal and Implant Science
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• 제48권1호
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• pp.34-46
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• 2018

Purpose: The purpose of this study was to evaluate the effects of 1,25-dihydroxyvitamin $D_3$ on the proliferation, differentiation, and matrix mineralization of MC3T3-E1 osteoblast-like cells in vitro. Methods: MC3T3-E1 osteoblastic cells and 1,25-dihydroxyvitamin $D_3$ were prepared. Cytotoxic effects and osteogenic differentiation were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, alkaline phosphatase (ALP) activity assay, ALP staining, alizarin red S staining, and reverse transcription-polymerase chain reaction (RT-PCR) for osteogenic differentiation markers such as ALP, collagen type I (Col-I), osteocalcin (OCN), vitamin D receptor (VDR), and glyceraldehyde 3-phosphate dehydrogenase. Results: The MTT assay showed that 1,25-dihydroxyvitamin $D_3$ did not inhibit cell growth and that the rate of cell proliferation was higher than in the positive control group at all concentrations. ALP activity was also higher than in the positive control group at low concentrations of 1,25-dihydroxyvitamin $D_3$ ($10^{-10}$, $10^{-12}$, and $10^{-14}M$). RT-PCR showed that the gene expression levels of ALP, Col-I, OCN, and vitamin D receptor (VDR) were higher at a low concentration of 1,25-dihydroxyvitamin $D_3$ ($10^{-12}M$). Alizarin red S staining after treatment with 1,25-dihydroxyvitamin $D_3$ ($10^{-12}M$) showed no significant differences in the overall degree of calcification. In contrast to the positive control group, formation of bone nodules was induced in the early stages of cell differentiation. Conclusions: We suggest that 1,25-dihydroxyvitamin $D_3$ positively affects cell differentiation and matrix mineralization. Therefore, it may function as a stimulating factor in osteoblastic bone formation and can be used as an additive in bone regeneration treatment.

• Biomolecules & Therapeutics
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• 제24권6호
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• pp.589-594
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• 2016

Insulin is a peptide hormone of the endocrine pancreas and exerts a wide variety of physiological actions in insulin sensitive tissues, such as regulation of glucose homeostasis, cell growth, differentiation, learning and memory. However, the role of insulin in osteoblast cells remains to be fully characterized. In this study, we demonstrated that the insulin (100 nM) has the ability to stimulate the phosphorylation of protein kinase B (Akt/PKB) and extracellular signal-regulated kinase (ERK) and the levels of inhibin-${\beta}E$ in the osteoblast-like UMR-106 cells. This insulin-stimulated activities were abolished by the PI3K and MEK1 inhibitors LY294002 and PD98059, respectively. This is the first report proving that insulin is a potential candidate that enables the actions of inhibin-${\beta}E$ subunit of the TGF-${\beta}$ family. The current investigation provides a foundation for the realization of insulin as a potential stimulator in survival signaling pathways in osteoblast-like UMR-106 cells.

• 대한치과보철학회지
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• 제43권6호
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• pp.751-763
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• 2005

Statement of problem. To improve a direct implant fixation to the bone, various strategies have been developed focusing on the surface of materials. The surface quality of the implant depends on the chemical, physical, mechanical and topographical properties of the surface. The different properties will interact with each other and a change in thickness of the oxide layer may also result in a change in surface energy, the surface topography and surface, chemical composition. However, there is limited the comprehensive study with regard to changed surface and biologic behavior of osteoblast by anodization. Purpose of study. The aim of this study was to analyze the characteristics of an oxide layer formed and to evaluate the cellular biologic behaviors on titanium by anodic oxidation (anodization) by cellular proliferation, differentiation, ECM formation and gene expression. And the phospholipase activity was measured on the anodized surface as preliminary study to understand how surface properties of Ti implant are transduced into downstream cellular events. Methods and Materials. The surface of a commercially pure titanium(Grade 2) was modified by anodic oxidation. The group 1 samples had a machined surface and other three experimental specimens were anodized under a constant voltage of 270 V(Group 2), 350 V(Group 3), and 450 V(Group 4). The specimen characteristics were inspected using the following five categories; the surface morphology, the surface roughness, the thickness of oxide layer, the crystallinity, and the chemical composition of the oxide layer. Cell numbers were taken as a marker for cell proliferation. While the expression of alkaline phosphatase and Runx2 (Cbfa1) was used as early differentiation marker for osteoblast. The type I collagen production was determined, which constitutes the main structural protein of the extracellular matrix. Phospholipase $A_2$ and D activity were detected. Results. (1) The anodized titanium had a porous oxide layer, and there was increase in both the size and number of pores with increasing anodizing voltage. (2) With increasing voltage, the surface roughness and thickness of the oxide film increased significantly (p<0.01), the $TiO_2$phase changed from anatase to rutile. During the anodic oxidization, Ca and P ions were more incorporated into the oxide layer. (3) The in vitro cell responses of the specimen were also dependant on the oxidation conditions. With increasing voltage, the ALP activity, type I collagen production, and Cbfa 1 gene expression increased significantly (p<0.01), while the cell proliferation decreased. (4) In preliminary study on the relation of surface property and phospholipase, PLD activity was increased but $PLA_2$ activity did not changed according to applied voltage. Conclusion. The anodized titanium shows improved surface characteristics than the machined titanium. The surface properties acquired by anodization appear to give rise more mature osteoblast characteristics and might result in increased bone growth, and contribute to the achievement of a tight fixation. The precise mechanism of surface property signaling is not known, may be related to phospholipase D.

• 한국식품영양과학회지
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• 제39권2호
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• pp.203-209
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• 2010

본 연구를 통하여 여주 추출물은 세포 증식을 제외하고는 조골세포에 긍정적인 영향을 미치지 못하였지만 머위 추출물은 세포의 증식, ALP 활성, bone nodule의 형성이 대조군과 비슷한 결과를 나타내거나 높은 경향을 나타냄으로써 조골세포의 골 형성 과정인 증식, 기질의 성숙, 기질의 석회화의 세 단계에서 유효성을 증명하였다. 또한 OPG mRNA의 2배 이상의 증가는 조골세포의 골 형성에 주요 매개 물질로서 가능성이 있음을 밝혔다. 따라서 머위 추출물은 골수의 미세 환경에서 세포의 조절작용을 하는 물질로 여겨지며, 골다공증을 포함한 각종 골 결손 질환의 예방과 치료약 개발에 긍정적인 가능성을 제시할 것이라 사료된다.

• 대한한의학방제학회지
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• 제25권4호
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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.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제32권6호
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• pp.524-529
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• 2006

For the repairing of bone defect, autogenous or allogenic bone grafting remains the standard. However, these methods have numerous disadvantages including limited amount, donor site morbidity and spread of diseases. Tissue engineering technique by culturing stem cells may allow for a smart solution for this problem. Adipose tissue contains mesenchymal stem cells that can be differentiate into bone, cartilage, fat or muscle by exposing them to specific growth conditions. In this study, the authors procured the stem cell from buccal fat pad and differentiate them into osteoblast and are to examine the bone induction capacity. Buccal fat-derived cells (BFDC) were obtained from human buccal fat pad and cultured. BFDC were analyzed for presence of stem cell by immunofluorescent staining against CD-34, CD-105 and STRO-1. After BFDC were differentiated in osteogenic medium for three passages, their ability to differentiate into osteogenic pathway were checked by alkaline phosphatase (ALP) staining, Alizarin red staining and RT-PCR for osteocalcin (OC) gene expression. Immunofluorescent and biochemical assays demonstrated that BFDC might be a distinguished stem cells and mineralization was accompanied by increased activity or expression of ALP and OC. And calcium phosphate deposition was also detected in their extracelluar matrix. The current study supports the presence of stem cells within the buccal fat pad and the potential implications for human bone tissue engineering for maxillofacial reconstruction.