• The Journal of Advanced Prosthodontics
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• 제2권1호
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• pp.18-24
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• 2010

PURPOSE. The aim of this pilot study was to investigate the effect of etched microgrooves on the hydrophilicity of Ti and osteoblast responses. MATERIAL AND METHODS. Microgrooves were applied on Ti to have 15 and $60{\mu}m$ width, and 3.5 and $10{\mu}m$ depth by photolithography, respectively. Further acid etching was applied to create Ti surfaces with etched microgrooves. Both smooth- and acid-etched Ti were used as the controls. The hydrophilicity of Ti was analyzed by determining contact angles. Cell proliferation and osteogenic activity of MC3T3 mouse preosteoblasts were analyzed by bromodeoxyuridine assay and alkaline phosphatase (ALP) activity test, respectively. One-way ANOVA, Pearson's correlation analysis and multiple regression analysis were used for statistics. RESULTS. Etched microgrooves significantly increased the hydrophilicity of Ti compared to the smooth Ti. $60{\mu}m$-wide etched microgrooves significantly enhanced cell proliferation, whereas the osteogenic activity showed statistically non-significant differences between groups. Result of the osteogenic activity significantly correlated with those of hydrophilicity and cell proliferation. Hydrophilicity was determined to be an influential factor on osteogenic activity. CONCLUSION. This study indicates that increase in hydrophilicity of Ti caused by etched microgrooves acts as an influential factor on osteogenic activity. However, statistically non-significant increase in the ALP activity suggests further investigation.

• International Journal of Oral Biology
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• 제38권3호
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• pp.111-119
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• 2013

Objective. To investigate the effects of the hypoxia inducible factor-1 (HIF-1) activation-mimicking agent cobalt chloride ($CoCl_2$) on the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and elucidate the underlying molecular mechanisms. Study design. The dose and exposure periods for $CoCl_2$ in hMSCs were optimized by cell viability assays. After confirmation of $CoCl_2$-induced HIF-$1{\alpha}$ and vascular endothelial growth factor expression in these cells by RT-PCR, the effects of temporary preconditioning with $CoCl_2$ on hMSC osteogenic differentiation were evaluated by RT-PCR analysis of osteogenic gene expression, an alkaline phosphatase (ALP) activity assay and by alizarin red S staining. Results. Variable $CoCl_2$ dosages (up to $500{\mu}M$) and exposure times (up to 7 days) on hMSC had little effect on hMSC survival. After $CoCl_2$ treatment of hMSCs at $100{\mu}M$ for 24 or 48 hours, followed by culture in osteogenic differentiating media, several osteogenic markers such as Runx-2, osteocalcin and osteopontin, bone sialoprotein mRNA expression level were found to be up-regulated. Moreover, ALP activity was increased in these treated cells in which an accelerated osteogenic capacity was also verified by alizarin red S staining. Conclusions. The osteogenic differentiation potential of hMSCs could be preserved and even enhanced by $CoCl_2$ treatment.

• Journal of Periodontal and Implant Science
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• 제53권1호
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• pp.54-68
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• 2023

Purpose: The purpose of this study was to determine whether metformin (MF) could alleviate the expresssion of reactive oxygen species (ROS) and improve the osteogenic ability of bone marrow mesenchymal stem cells derived from diabetic rats (drBMSCs) in vitro, and to evaluate the effect of MF on the ectopic osteogenesis of drBMSCs in a nude mouse model in vivo. Methods: BMSCs were extracted from normal and diabetic rats. In vitro, a cell viability assay (Cell Counting Kit-8), tests of alkaline phosphatase (ALP) activity, and western blot analysis were first used to determine the cell proliferation and osteogenic differentiation of drBMSCs that were subjected to treatment with different concentrations of MF (0, 50, 100, 200, 500 µM). The cells were then divided into 5 groups: (1) normal rat BMSCs (the BMSCs derived from normal rats group), (2) the drBMSCs group, (3) the drBMSCs + Mito-TEMPO (10 µM, ROS scavenger) group, (4) the drBMSCs + MF (200 µM) group, and (5) the drBMSCs + MF (200 µM) + H₂O₂ (50 µM, ROS activator) group. Intracellular ROS detection, a senescence-associated β-galactosidase assay, ALP staining, alizarin red staining, western blotting, and immunofluorescence assays were performed to determine the effects of MF on oxidative stress and osteogenic differentiation in drBMSCs. In vivo, the effect of MF on the ectopic osteogenesis of drBMSCs was evaluated in a nude mouse model. Results: MF effectively reduced ROS levels in drBMSCs. The cell proliferation, ALP activity, mineral deposition, and osteogenic-related protein expression of drBMSCs were demonstrably higher in the MF-treated group than in the non-MF-treated group. H₂O₂ inhibited the effects of MF. In addition, ectopic osteogenesis was significantly increased in drBMSCs treated with MF. Conclusions: MF promoted the proliferation and osteogenic differentiation of drBMSCs by inhibiting the oxidative stress induced by diabetes and enhenced the ectopic bone formation of drBMSCs in nude mice.

• Journal of Microbiology and Biotechnology
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• 제17권7호
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• pp.1113-1119
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• 2007

Human bone marrow-derived mesenchymal stem cells (hBMMSCs) must differentiate into osteogenic cells to allow for successful bone regeneration. In this study, we investigated the effects of different combinations of three soluble osteogenic differentiation-inducing factors [L-ascorbic acid (AC), ${\beta}$-glycerophosphate (${\beta}G$), and bone morphogenic protein-2 (BMP-2)] and the presence of a hydroxyapatite (HA) substrate on hBMMSC osteogenic differentiation in vitro. hBMMSCs were cultured in medium containing various combinations of the soluble factors on culture plates with or without HA coating. After 7 days of culture, alkaline phosphatase (ALP) activity, calcium deposition, and osteoprotegerin (OPG) and osteopontin (OPN) expression were measured. The effects of individual and combined factors were evaluated using a factorial analysis method. BMP-2 predominantly affected expression of early markers of osteogenic differentiation (ALP and OPG). HA had the highest positive effect on OPN expression and calcium deposition. The interaction between AC, ${\beta}G$, and HA had the second highest positive effect on ALP activity.

• Journal of Acupuncture Research
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• 제24권5호
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• pp.13-22
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• 2007

Objectives : The differentiation of osteoblasts is controlled by various growth factors and matrix protein expressed in bone. The aim of this study was to investigate the effects of many herbs medicine(KHBJs) for bone healing that induces osteogenic activity in human osteoblast-like SaOS-2 cells. Methods : The osteogenic effects of KHBJs were evaluated by using cell proliferation(WST-8) assay, alkaline phosphatase(ALP) activity assay, colorimetric analysis of vascular endothelial growth factor(VEGF) expression in human osteoblast like SaOS-2 cell. Also, osteogenic activity of KHBJ fractions(KHBJB and KHBJR) by activity guided fractionation were evaluated. Results : About 7 KHBJs had effect on the proliferation of osteoblast like SaOS-2 cells, and dose-dependently increased alkaline phosphatase(ALP) activity. KHBJs markedly increased expression for VEGF. Fractionated KHBJs(KHBJB or KHBJR) not enhanced more than KHBJs on osteogenic activity in SaOS-2 cells. Conclusions: This study found that 7 KHBJs had effect on proliferation, ALP activity, and VEGF expression in osteoblast like SaOS-2 cells. These results propose that KHBJs can play an important role in osteoblastic bone formation, and may possibly lead to the development of bone-forming drugs.

• 생명과학회지
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• 제34권10호
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• pp.701-712
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• 2024

후코이단(fucoidan)은 갈조류에서 발견되는 다당류로, 면역 증강, 항암, 항염증 등 다양한 생리 활성을 나타내는 것으로 알려져 있다. 본 연구에서는 조골세포(osteoblast)와 유사한 생물학적 특성을 지닌 MC3T3-E1 세포에서 최종당산화물에 의해 유도되는 세포와 미토콘드리아 손상, 조골세포 분화, 조골 및 파골 활동성의 변화에 미치는 후코이단의 효과를 관찰하였다. 최종당산화물로 처리한 세포군에서는 MTT 환원능의 감소와 함께 카스파아제(caspase)의 활성화, 세포 사멸의 증가가 관찰되었다. 후코이단을 사전 처리하면 최종당산화물에 의한 이들 세포 손상 지표들이 현저히 완화되었다. 후코이단은 또한 최종당산화물에 의해 유도된 미토콘드리아 막 전위 감소, 세포 내 ATP 고갈, 미토콘드리아 막 투과성 전이(mitochondrial permeability transition) 형성을 유의하게 억제하여, 미토콘드리아의 기능적 손상을 완화하는 효과를 보였다. 최종당산화물로 처리한 세포군에서는 반응성 산소종(reactive oxygen species)과 과산화 지질 및 과산화 단백의 생성이 현저히 증가하였으며, 후코이단은 이를 유의하게 억제하였다. 조골세포 분화능의 지표로 뼈형태발생단백-2(bone morphogenetic protein, BMP2), 알칼리 인산분해효소(alkaline phosphatase, ALP), 오스테오칼신 (osteocalcin, OC), 제1형 콜라젠(collagen-I, Col-I)의 mRNA 발현 정도를 정량 실시간 역전사 중합효소 연쇄반응(qRT-PCR) 검사로 분석한 결과 최종당산화물로 처리 시 이들 mRNA들의 발현이 현저히 감소하였으며, 후코이단은 이들 지표 mRNA들의 발현 감소를 유의하게 차단하였다. 조골 활동성(osteogenic activity)의 지표로 알칼리 인산분해효소 활성과 무기질 침착을 측정한 결과도 mRNA 발현의 변화와 일치하는 결과를 보였다. 반면에 파골 활동성(osteoclastic activity)을 나타내는 지표들인 인터루킨-6(interleukin-6)와 NF-kB 리간드 수용체 활성 인자(receptor activator of NF-kB ligand, RANKL)의 분비는 최종당산화물로 처리한 세포군에서 증가하였으며, 후코이단은 이를 유의하게 억제하였다. 이상의 결과를 종합하면 후코이단은 MC3T3-E1 세포에서 최종당산화물에 의해 유도되는 세포 및 미토콘드리아 손상과 조골세포 분화 저해, 조골 활동성 저하를 완화하는 효과를 나타내는 것으로 사료된다.

• Molecules and Cells
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• 제40권6호
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• pp.393-400
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• 2017

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease characterized by lack of insulin and high glucose levels. T2DM can cause bone loss and fracture, thus leading to diabetic osteoporosis. Promoting osteogenic differentiation of osteoblasts may effectively treat diabetic osteoporosis. We previously reported that Sirtuin 1 (Sirt1), a $NAD^+$-dependent deacetylase, promotes osteogenic differentiation through downregulation of peroxisome proliferator-activated receptor (PPAR) ${\gamma}$. We also found that miR-132 regulates osteogenic differentiation by downregulating Sirt1 in a $PPAR{\beta}/{\delta}$-dependent manner. The ligand-activated transcription factor, $PPAR{\alpha}$, is another isotype of the peroxisome proliferator-activated receptor family that helps maintain bone homeostasis and promot bone formation. Whether the regulatory role of $PPAR{\alpha}$ in osteogenic differentiation is mediated via Sirt1 remains unclear. In the present study, we aimed to determine this role and the underlying mechanism by using high glucose (HG) and free fatty acids (FFA) to mimic T2DM in MC3T3-E1 cells. The results showed that HG-FFA significantly inhibited expression of $PPAR{\alpha}$, Sirt1 and osteogenic differentiation, but these effects were markedly reversed by $PPAR{\alpha}$ overexpression. Moreover, siSirt1 attenuated the positive effects of $PPAR{\alpha}$ on osteogenic differentiation, suggesting that $PPAR{\alpha}$ promotes osteogenic differentiation in a Sirt1-dependent manner. Luciferase activity assay confirmed interactions between $PPAR{\alpha}$ and Sirt1. These findings indicate that $PPAR{\alpha}$ promotes osteogenic differentiation via the Sirt1-dependent signaling pathway.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제27권1호
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• pp.1-8
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• 2005

This study was aimed to characterize osteogenic potential of rat bone marrow stromal cells (BMSC) isolated with standard flushing method and investigate the plasticity of transdifferentiation between osteoblastic and adipocytic lineage of cultured BMSC. Unlike aspiration method in human, rat bone marrow was extracted by means of irrigation with culture media that elevates the possibility of co-extraction of committed osteoprogenitor, or preosteoblast or other progenitor cells of several types present inside bone marrow. The cultured stromal cells showed high ALP activity which is representative marker of osteoblast without any treatment. Osteogenic inducers such as Dex and BMP-2 were examined for the evaluation of their effect on osteogenic and adipocytic differentiation of stromal cells, because they function as osteoinductive agent in stromal cells, but simultaneously induce adipogenic differentiation. Osteogenic differentiation was evaluated by measuring alkaline phosphatase activity or mRNA expression of osteoblast markers such as osteopontin, bone sialoprotein, collagen type I and CbfaI, and in vitro matrix mineralization by von Kossa staining. Oil red staining method was used to detect adipocyte and adipocytic marker, aP2 and $PPAR{\gamma}2$ expression was examined using RT-PCR. It can be supposed that irrigation procedure resulted in high portion of already differentiation-committed osteoprogenitor cell showing elevated ALP activity and strong mineralization only under the supplement of $100{\mu}M$ ascorbic 2-phosphate and 10mM ${\beta}$-glycerophosphate without any treatment of osteogenic inducers such as Dex and BMP-2. Dex and BMP-2 seemed to transdifferentiate osteoprogenitor cells having high ALP activity into adipocytes temporarily, but continuous treatment redifferentiated into osteoblast and developed in vitro matrix mineralization. This property must be considered either in tissue engineering for bone regeneration, or in research of characterization of osteogenic differentiation, with rat BMSC isolated by the standard irrigation method.

• Journal of Industrial and Engineering Chemistry
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• 제68권
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• pp.220-228
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• 2018

In this study, we found a simple surface biofunctionalization of biphasic calcium phosphate (BCP) based on the high affinity between alendronate and the calcium ions of BCP, and the strong interaction between heparin and bone morphogenic protein-2 (BMP-2). The biofunctionalized BCP did not be precipitated well and display a remarkable enhancement of osteogenic activity of human adipose-derived stem cells by showing increased alkaline phosphatase (ALP), calcium deposition and osteogenic-related genes (i.e., Runx-2, ALP, osteocalcin, and osteopontin), and bone regeneration in the calvarial defect model. Therefore, this simple surface technique can be used to easily functionalize various calcium phosphates.

• 대한한의학회지
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• 제38권4호
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• pp.1-10
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• 2017

Objectives: The aim of the present study is to evaluate the effects of the extract of Pongamia pinnata on the morphology, viability, and differentiation potential of human stem cells derived from the gingiva. Methods: Stem cells obtained from gingivae were cultured in an osteogenic medium in the presence of methanol extract of Pongamia pinnata (PPT) at concentrations ranging from 0.001 to 1%. Evaluations of cell morphology and cellular viability were done at Day 1. Alkaline phosphatase activity assays and Alizarin red S staining were performed to evaluate the osteogenic differentiation of stem cells. Results: The morphology of stem cells in the presence of PPT at final concentrations of 0%, 0.001%, 0.01%, 0.1%, and 1% did not produce any noticeable changes when compared with the untreated control group. Application of PPT produced a significant increase in alkaline phosphatase activity when compared to the control group. The results of the Alizarin Red S staining showed a significant increase of absorbance with the 0.001% group. Conclusions: Based on these findings, it was concluded that PPT could produce beneficial effects on mesenchymal stem cells with enhanced osteogenic differentiation.