• Journal of Acupuncture Research
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• v.29 no.6
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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.

• Imaging Science in Dentistry
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• v.38 no.3
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• pp.125-132
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• 2008

Purpose : To investigate the effects of irradiation on transforming growth factor ${\beta}_1$ (TGF-${\beta}_1$) mRNA expression and calcific nodule formation in MC3T3-E1 osteoblastic cell line. Materials and Methods : Cells were cultured in alpha-minimum essential medium ($\alpha$-MEM) supplemented with 10% fetal bovine serum and antibiotics. When the cells reached the level of 70-80% confluence, culture media were changed with $\alpha$-MEM supplemented with 10% FBS, 5 mM $\beta$-glycerol phosphate, and $50\;{\mu}g/mL$ ascorbic acid. Thereafter the cells were irradiated with a single dose of 2, 4, 6, 8 Gy at a dose rate of 1.5 Gy/min. The expression pattern of TGF-${\beta}_1$ mRNA, calcium content and calcific nodule formation were examined on day 3, 7, 14, 21, 28, respectively, after the irradiation. Results : The amount of TGF-${\beta}_1$ mRNA expression decreased significantly on day 7 after irradiation of 4, 6, 8 Gy. It also decreased on day 14 after irradiation of 6, 8 Gy. and decreased on day 21 after irradiation of 8 Gy. The amount of calcium deposition decreased significantly on day 7 after irradiation of 4, 8 Gy (P < 0.01) and showed a decreased tendency on day 14, 21 after irradiation of 4, 6, 8 Gy. The number of calcific nodules was decreased on day 7 after irradiation of 4, 8 Gy. Conclusion: Irradiation with a single dose of 4, 6, 8 Gy influences negatively the bone formation at the molecular level by affecting the TGF-${\beta}_1$ mRNA expression that was associated with proliferation and the production of extracellular matrix in MC3T3-E1 osteoblastic cell line.

• Journal of dental hygiene science
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• v.23 no.4
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• pp.282-295
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• 2023

Background: Secretory leukocyte protease inhibitor (SLPI) protects tissues from proteases and promotes cell proliferation and healing. SLPI also reduces periodontal inflammation and alveolar bone resorption by inhibiting proinflammatory cytokine expression in rat periodontal tissues and osteoblasts. However, little is known of the role of SLPI in the expression of osteoclast regulatory factors from osteoblasts, which are crucial for the interaction between osteoblasts and osteoclasts. Therefore, we aimed to determine the effects of SLPI on the regulation of osteoclasts and osteoblasts in LPS-treated alveolar bone and osteoblasts. Methods: Periodontitis was induced in rats using LPS. After each LPS injection, SLPI was injected into the same area. Immunohistochemical analysis was performed with antibodies against SLPI, RANKL, OPG, and Runx2 in the periodontal tissue. RT-PCR and western blotting were performed to determine the expression levels of SLPI, RANKL, OPG, and Runx2 in LPS- and SLPI/LPS-treated MC3T3-E1 cells. SLPI/LPS-treated MC3T3-E1 cells were also stained with Alizarin Red S. Results: Immunohistochemical analysis showed that the expression levels of SLPI, OPG, and Runx2 were higher while that of RANKL was lower in the LPS/SLPI group relative to those in the LPS group. The mRNA and protein expression of SLPI, OPG, and Runx2 was higher in SLPI/LPS/MC3T3-E1 cells than in LPS/MC3T3-E1 cells, and RANKL expression was lower. During differentiation, OPG and Runx2 protein levels were higher whereas RANKL levels were lower in SLPI/LPS/MC3T3-E1 than in LPS/MC3T3-E1 cells on days 0, 4, 7, and 10. In addition, mineralization and matrix deposition were higher in SLPI/LPS/MC3T3-E1 than in LPS/MC3T3-E1 on days 7 and 10. SLPI decreased RANKL expression in LPS-treated alveolar bone and osteoblasts but increased the expression of OPG and Runx2. Conclusion: SLPI can be considered as a regulatory molecule that indirectly regulates osteoclast activation via osteoblasts and promotes osteoblast differentiation.

• International Journal of Oral Biology
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• v.33 no.3
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• pp.105-111
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• 2008

Thrombospondins (TSP-1, TSP-2) are secretory extracellular glycoproteins that are involved in a variety of physiological processes such as tumor cell adhesion, invasion, and metastasis. The present study was undertaken to elucidate the involvement of thrombospondins in the adhesion of osteoblast-like cells using the TSP-1 or TSP-2 antisense MG63 and MC3T3-E1 cell lines. For downregulation of TSPs expression, we prepared antisense constructs for TSP-1 and TSP-2 using the pREP4 an episomal mammalian expression vector, which be able to produce the specific antisense oligonucleotides around chromosome. MG63 and MC3T3-E1 osteoblast-like cells were transfected with the antisense constructs and nonliposomal Fugene 6, and then selected under hygromycin B (50 ${\mu}g/ml$) treatment for 2 weeks. Western blot analysis revealed that expression of the TSP proteins was downregulated in the antisense cell lines. The cell adhesion assay showed that adhesive properties of TSP-1 and TSP-2 antisense MG63 cells on the polystyrene culture plate were reduced to 17% and 21% of the control cells, respectively, and those of the TSP-1 and TSP-2 antisense MC3T3-E1 cells also decreased to 19% and 27% of control, respectively. Adhesion of TSP-1 and TSP-2 antisense MC3T3-E1 cells on Type I collagen-coated culture plate decreased to 27% and 76%, respectively. These results indicate that TSP-1 and TSP-2 proteins may have an important role in adhesion of osteoblast-like cells to extracellular matrix.

• Food Science and Biotechnology
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• v.16 no.5
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• pp.807-811
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• 2007

Diabetes is marked by high glucose levels and is associated with decreased bone mass and increased fracture rates. To determine if [6]-gingerol could influence osteoblast dysfunction induced by 2-deoxy-D-ribose (dRib), osteoblastic MC3T3-E1 cells was treated with dRib and [6]-gingerol and markers of osteoblast function and oxidized protein were examined. [6]-Gingerol ($10^{-7}\;M$) significantly increased the growth of MC3T3-E1 cells in the presence of 30 mM dRib (p<0.05). [6]-Gingerol ($10^{-7}\;M$) caused a significant elevation of alkaline phosphatase (ALP) activity, collagen content, and osteocalcin secretion in the cells. We then examined the effect of [6]-gingerol on the production of osteoprotegerin and protein carbonyl in osteoblasts. Treatment with [6]-gingerol ($10^{-9}$ and $10^{-7}\;M$) increased osteoprotegerin secretion in osteoblastic cells. Moreover, [6]-gingerol ($10^{-9}$ and $10^{-7}\;M$) decreased protein carbonyl contents of osteoblastic MC3T3-E1 cells in the presence of 30 mM dRib. Taken together, these results demonstrate that [6]-gingerol inhibits dRib-induced damage and may be useful in the treatment of diabetes related bone diseases.

• Proceedings of the Korean Fiber Society Conference
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• 2003.10a
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• pp.41-42
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• 2003

Osteoblasts (MC3T3-E1) were cultured on polysaccharide type polyelectrolyte complex (PEC). The growth of the MC3T3-E1 on the PEC with carbxyl group (c-type) was slightly suppressed and exhibited aggregation morphology. On the other hand, cell growth on the PEC with sulfate group (s-type) was enhanced and the cell exhibited spreading form. Differentiation markers of osteoblast (ALPase activity, calcification, expression of osteocalsin) were enhanced and localized around cell aggregates on c-type PECs. These results suggest that PEC has the ability to control osteoblast proliferation and differentiation.

• Journal of Biomedical Engineering Research
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• v.25 no.4
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• pp.309-314
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• 2004

in this study, hybrid bioreactor was used to apply physical stimuli in cell culture. Effect of the applied physical stimuli on the growth and differentiation of MC3T3-El cell in a three-dimensional Chitosan scaffold were studied by using the hybrid bioreactor. The hybrid bioreactor for physical stimulus was specially designed to apply uniaxial cyclic compressive and shear strain. Physical stimulus was applied over a period of 14 days with 150 cycles per day at a frequency of 0.5Hz. Strain magnitude was 2.5% of the scaffold size. Control group and physically stimulated group of the MC3T3-El tell were incubated and harvested at the indicated times (Day 6, 8, 10, 12, 14). The total amount of protein, which obtained information of cell growth, was determined by Lowey method. Alkaline phosphatase activity was examined by ELISA. Physically stimulated group using the hybrid bioreactor was increased in alkaline phosphatase activity comparing with control group. The nodule formation and calcium deposit of the physical stimuli group which resulted in cell differentiation was faster than that of control group.

• Toxicological Research
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• v.17 no.2
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• pp.123-129
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• 2001

This study were carried out to investigate cytotoxicity of paraquat and bentazon that is scattering to farm products were essensial for human diet and compensatory effects of 3-methylcholanthrene (3-MC) in vitro and in vivo. In vitro, The 5.0$\times$$10^4$ cell/ml of NIH 3T3 fibroblast in each well of 24 multidish were cultured. After 24 hours, the cells were treated with solution of paraquat and bentazon (1, 25, 50, 100 pM respectively). After the NIH 3T3 fibroblast of all groups were cultured in same condition for 48 hours, Sulfohordamin B Protein (SRB) assay were performed to evaluate the cytotoxicity of cell organelles. Paraquat and bentazon $SRB_50$ were 1860.73 $\mu\textrm{M}$, 1913.38 $\mu$M respectively. In vivo, Sprague Dawley male rats divided into paraquat and bentazon only administered group and simultaneous application group of paraquat and bentazon and 3-MC. At 30 min. and 1, 3, 6, 12, 24, 48 and 96 hrs. interval after each treatment, the animals were sacrificed by decapitation and kidney were immediately removed, immersed in fixatives, and processed with routine method for light microscopic study. Paraffin sections were stained with H-E, PAM, and PAS. Under the light microscope, atrophic change of renal corpuscles were frequently observed from 3 hrs after paraquat and bentazon treatment. The increase of the mesangium was apparent from 12 hrs later after paraquat and bentazon treatment. Necrotic changes of the epithelium and loss of brush border of proximal tubules were most severe at 48 hrs after paraquat and bentazon treatment, respectively. In contrast there were no evidences of the toxic effects on renal tissues at 48hrs in paraquat and bentazon plus 3-MC treated groups.

• 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.

• Nutrition Research and Practice
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• v.10 no.2
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• pp.148-153
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• 2016

BACKGROUND/OBJECTIVES: Bone formation and bone resorption continuously occur in bone tissue to prevent the accumulation of old bone, this being called bone remodeling. Osteoblasts especially play a crucial role in bone formation through the differentiation and proliferation. Therefore, in this study, we investigated the effects of Scytosiphon lomentaria extract (SLE) on osteoblastic proliferation and differentiation in MC3T3-E1 cells. MATERIALS/METHODS: A cell proliferation assay, alkaline phosphatase (ALP) activity assay, alizarin red staining and protein expression analysis of osteoblastic genes were carried out to assess the osteoblastic proliferation and differentiation. RESULTS: The results indicated that treatment of SLE promoted the proliferation of MC3T3-E1 cells and improved ALP activity. And, SLE treatment significantly promoted mineralized nodule formation compared with control. In addition, cells treated with SLE significantly upregulated protein expression of ALP, type 1 collagen, bone morphogenetic protein 2, runt-related transcription factor 2, osterix, and osteoprotegerin. CONCLUSIONS: The results demonstrate that SLE promote differentiation inducement and proliferation of osteoblasts and, therefore may help to elucidate the transcriptional mechanism of bone formation and possibly lead to the development of bone-forming drugs.