• Proceedings of the KSME Conference
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• 2008.11a
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• pp.1700-1703
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• 2008

The purpose of this study was to examine the effects of various mechanical stimuli for MC3T3-E1 cells. Among the several mechanical stimulations, we focused on compressive stain and ultrasound. In this study, we developed a bioreactor capable of applying controlled stimuli to scaffolds. PLLA/PCL scaffold was fabricated by using salt-leaching method. We performed dynamic cell culture using preosteoblasts MC3T3-E1 cells with 1MHz, 30mW/cm2 ultrasound and 10% of compressive strain. Result of CCK-8 analysis at 1, 4, 7, 10 days showed that mechanical stimuli had no significant effect for cell proliferation. However, those stimuli influenced ALP(Alkaline phopatase) activity, which is one of differentiation marker.

• 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.

• International Journal of Oral Biology
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• v.34 no.1
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• pp.37-42
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• 2009

The attachment and adhesion of RAW 264.7 and MC3T3-E1 cells to titanium (Ti) discs with various degrees of roughness was investigated. The attachment, adhesion, and proliferation of these cells were evaluated after 4 hr, 24 hr and 7 day incubations. Both RAW 264.7 and MC3T3-E1 cells showed a time-dependant correlation between attachment and adhesion on the surface of the titanium discs. Both types of cells tended to have higher survival rate on these discs as the surface roughness increased. The percentage of adherent inflammatory RAW 264.7 cells was greater than MC3T3-E1 cells at 24 hr, but this was reversed at 7 days in culture. The morphology of osteoblastic MC3T3-E1 cells at 24 hr, determined using a surface emission microscope (SEM), appeared flattened and spread out while inflammatory RAW 264.7 cells were predominantly spherical in shape. The adhesion of both cell types on the titanium discs was dependant on the levels of fibronectin adsorbed on the disc surface, indicating that serum constituents modulate the efficient adhesion of these cells. Our data indicate that the cellular response to the titanium surface is dependent on the types of cells, surface roughness and serum constituents.

• Molecules and Cells
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• v.41 no.3
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• pp.234-243
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• 2018

In recent years, the interest towards the relationship between incretins and bone has been increasing. Previous studies have suggested that glucagon-like peptide-1 (GLP-1) and its receptor agonists exert beneficial anabolic influence on skeletal metabolism, such as promoting proliferation and differentiation of osteoblasts via entero-osseous-axis. However, little is known regarding the effects of GLP-1 on osteoblast apoptosis and the underlying mechanisms involved. Thus, in the present study, we investigated the effects of liraglutide, a glucagon-like peptide-1 receptor agonist, on apoptosis of murine MC3T3-E1 osteoblastic cells. We confirmed the presence of GLP-1 receptor (GLP-1R) in MC3T3-E1 cells. Our data demonstrated that liraglutide inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as detected by Annexin V/PI and Hoechst 33258 staining and ELISA assays. Moreover, liraglutide upregulated Bcl-2 expression and downregulated Bax expression and caspase-3 activity at intermediate concentration (100 nM) for maximum effect. Further study suggested that liraglutide stimulated the phosphorylation of AKT and enhanced cAMP level, along with decreased phosphorylation of $GSK3{\beta}$, increased ${\beta}-catenin$ phosphorylation at Ser675 site and upregulated nuclear ${\beta}-catenin$ content and transcriptional activity. Pretreatment of cells with the PI3K inhibitor LY294002, PKA inhibitor H89, and siRNAs GLP-1R, ${\beta}-catenin$ abrogated the liraglutide-induced activation of cAMP, AKT, ${\beta}-catenin$, respectively. In conclusion, these findings illustrate that activation of GLP-1 receptor by liraglutide inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation through $cAMP/PKA/{\beta}-catenin$ and $PI3K/Akt/GSK3{\beta}$ signaling pathways.

• 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.

• The Journal of Korean Obstetrics and Gynecology
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• v.31 no.4
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• pp.1-15
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• 2018

Objectives: Osteoporosis is considered a serious human disease. We developed an extract of mixed herbs containing root of Platycodon grandiflorum (ExMH-PGR), which is expected to be effective in preventing or treating osteoporosis. The aim of this study was to investigate the anti-osteoporotic effect of ExMH-PGR in osteoblastic MC3T3-E1 cells and osteoclastic RAW 264.7 cells. Methods: To examine the anti-osteoporotic effect of ExMH-PGR, osteoblast and osteoclast differentiation were induced and cultured with various concentrations of ExMH-PGR. Alkaline phosphatase (ALP) activity, collagen synthesis, osteocalcin production, and mineralization in MC3T3-E1 cells were analyzed. Tartrate-resistant acid phosphatase (TRAP) activity and the formation of actin ring in RAW 264.7 cells were analyzed. Results: ExMH-PGR at concentration up to $25{\mu}g/mL$ significantly increased ALP activity, collagen synthesis, osteocalcin production, and mineralization in MC3T3-E1 cells. ExMH-PGR at 50 to $200{\mu}g/mL$ significantly inhibited TRAP activity and the formation of actin ring in RAW 264.7 cells. Conclusions: These results demonstrate that ExMH-PGR stimulates osteoblastic activities and inhibits osteoclastic activities in in vitro systems, suggesting that ExMH-PGR might be considered as an anti-osteoporotic candidate for treatment of osteoporosis disease.

• Imaging Science in Dentistry
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• v.39 no.3
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• pp.121-132
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• 2009

Purpose : To investigate the effects of 2-deoxy-D-glucose (2-DG) and quercetin (QCT) on gene expression of bone sialoprotein (BSP) and osteocalcin (OC) during the differentiation in irradiated MC3T3-E1 osteoblastic cells. Materials and Methods : When MC3T3-E1 osteoblastic cells had reached 70-80% confluence, cultures were transferred to a differentiating medium supplemented with 5 mM 2-DG or $10{\mu}M$ QCT, and then irradiated with 2, 4, 6, and 8 Gy. At various times after irradiation, the cells were analyzed for the synthesis of type I collagen, and expression of BSP and OC. Results : The synthesis of type I collagen in cells exposed to 2 Gy of radiation in the presence of 2-DG or QCT showed no significant difference compared with the control group within 15 days post-irradiation. When the cells were irradiated with 8 Gy, 2-DG facilitated the irradiation mediated decrease of type I collagen synthesis, whereas such decrease was inhibited by treating with QCT. During MC3T3-E1 osteoblastic cell differentiation, the mRNA expression of BSP and OC showed the peak value at 14 days and 21 days, respectively. 2-DG or QCT treatment alone decreased the level of BSP mRNA, but increased the OC mRNA level only at early time of differentiation (day 7). In the cells irradiated with 2, 4, 8 Gy, the mRNA expression of BSP and OC decreased at 7 days after the irradiation. The cells were treated with various dose of radiation in the presence of 2-DG or QCT, the mRNA level of both BSP and OC increased although this increase was observed at low dose of radiation (2 Gy) and at the early stage of differentiation. However, when the cells were exposed to 4, 6, or 8 Gy, the increase of BSP and OC mRNAs was detected only in cells co-incubated with QCT. Conclusion : This study demonstrates that 2-DG and QCT affect differently the expression of bone formation related factors, type I collagen, BSP, and OC in the irradiated MC3T3-E1 osteoblasic cells, according to the dose of radiation and the times of differentiation. Overall, the present findings suggest that 2-DG and QCT could have the regulatory roles as radiation-sensitizer and -protector, respectively.

• Natural Product Sciences
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• v.11 no.3
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• pp.139-144
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• 2005

Bioflavone quercetin is believed to play an important role preventing bone loss by affecting osteoclastogenesis and regulating many systemic and local factors including hormones and cytokines. This study examined how quercetin acts on tumor necrosis factor-alpha ($TNF-{\alpha}$)-mediated apoptosis in MC3T3-E1 osteoblastic cells. Apoptosis assays revealed the dose-dependent acceleration of quercetin on $TNF-{\alpha}-induced$ apoptosis in MC3T3-E1 cells, which was demonstrated by the increased number of positively stained cells in the trypan blue staining and TUNEL assay, and the migration of many cells to the $sub-G_0/G_1$ phase in flow cytometric analysis. In particular, quercetin treatment alone increased the expression of p53 and p21 proteins in the cells. Consequently, this study showed that quercetin accelerates the $TNF-{\alpha}-induced$ apoptosis in MC3T3-E1 osteoblastic cells.

• Natural Product Sciences
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• v.15 no.4
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• pp.192-197
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• 2009

Chemical investigation of the aerial parts of Artemisia iwayomogi has afforded five glycoside compounds. Their chemical structures were characterized by spectroscopic methods to be turpinionoside A (1), (Z)-3-hexenyl O-${\alpha}$-arabinopyranosyl-(1${\rightarrow}$6)-O-${\beta}$-D-glucopyranoside (2), (Z)-5'-hydroxyjasmone 5'-O-${\beta}$-Dglucopyranoside (3), (-)-syringaresinol-4-O-${\beta}$-D-glucopyranoside (4), and methyl 3,5-di-O-caffeoyl quinate (5). All of them were isolated for the first time from Artemisia species. The effect of compounds 1 - 5 on the function of osteoblastic MC3T3-E1 cells was examined by checking the cell viability, alkaline phosphatase (ALP) activity, collagen synthesis, and mineralization. Turpinionoside A (1) significantly increased the function of osteoblastic MC3T3-E1 cells. Cell viability, ALP activity, collagen synthesis, and mineralization were increased up to 117.2% (2 ${\mu}M$), 110.7% (0.4 ${\mu}M$), 156.0% (0.4 ${\mu}M$), and 143.0 % (2 ${\mu}M$), respectively.

• International Journal of Oral Biology
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• v.39 no.3
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• pp.145-151
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• 2014

During bone remodeling, there is requirement of differentiation of osteoblastic cells. Previously, we identified proteins differentially expressed in soft tissue during bone healing. Of these proteins, we focused the effect of LTF on differentiation of osteoblast. In order to analyze the osteogenic ability of LTF, we treated conditioned media collected from human LTF-stably transfected HEK293T cells into osteoblastic MC3T3-E1. The results showed that the activity and expression of alkaline phosphatase were increased in MC3T3-E1 cells treated with conditioned media containing LTF in dose- and time-dependent manner. At the same time, we observed the significant increase of the expression of osteoblastic genes, such as ALP, BSP, COL1A1, and OCN, and along with matrix mineralization genes, such as DMP1 and DMP2, in LTF conditioned media-treated groups. Moreover, the result of treating recombinant human LTF directly into osteoblastic MC3T3-E1 showed the same pattern of treating conditioned media containing LTF. Our study demonstrated that LTF constitutively enhances osteoblastic differentiation via induction of osteoblastic genes and activation of matrix mineralization in MC3T3-E1 cells.