• The korean journal of orthodontics
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• v.23 no.3 s.42
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• pp.415-425
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• 1993

Fluoride is one of the most potent stimulators of bone formation in vivo. But its direct effects on osteoblast is not yet clear This study was to investigate the effects of Sodium fluoride on alkaline phosphatase(ALP) activity, cAMP formation responsive to parathormone(PTH) and type I $\alpha$ 2 collagen ribonucleic acid (mRNA) level in Murin osteoblast-like (MC3T3-E1) cells. The cells were cultured in $\alpha-Minimal$ essential medium $(\alpha-MEM)$ supplemente with $10\%$ fetal bovine serum (FBS) and then changed to $0.1\%$ FBS with various concentration of Sodium fluoride. The ALP activity was assayed by the method of Lowry with disodium phenyl phosphated as substrate. cAMP formation was measured by Radioimmuno Assay(RIA). Type I $\alpha$ 2 collagen ribonucleic acid(mRNA) expression was studied by Nothern blot analysis. The results were as follows: 1. cAMP level was increased by PTH in MC3T3-E1 cells. 2. Sodium fluoride showed the tendency of inhibitory effects on cAMP responsiveness to PTH in MC3T3-E1 cells. 3. Sodium fluoride increased ALP activity at cocentration of $2{\mu}M,\;4{\mu}M,\;and\;10{\mu}M$ significantly different from control at the 0.001 level. ALP activity revealed maximum value at $10{\mu}M$ in this study. 4. Nothern blot analysis of Sodium fluoride treated cells, using Type I $\alpha$ 2 collagen prove, revealed significant increase at $10{\mu}M$ in MC3T3-E1 cells.

• Journal of Periodontal and Implant Science
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• v.27 no.4
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• pp.669-684
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• 1997

Polypeptide growth factors belong to a class of potent biologic mediators which regulate cell differentiation, proliferation, migration and metabolism. IGF-I is polypeptides secreted by skeletal cells and is considered as regulators of bone formation. The purpose of this study is to evaluate the effects of IGF-I on bone nodule formation and alkaline phosphatase activity of MC3T3-E1 cells. MC3T3-E1 cells were seeded at $1{\times}10^4$ cells/well, $1{\times}10^5$ cells/well in alpha-modified Eagle medium containing 10% fetal bovine serum, 10 mM ${\beta}-glycerophosphate$ and $5O{\mu}g/ml$ of ascorbic acid. Before 48 hours of indicated time, medium were changed with serum free medium. After 24 hours, 0.1, 1, 10 ng/ml IGF-I were added to the cells and cultured for 3, 7, 14, 21, 28 days. And histochemical analysis was done and ALP activity was measured and was expressed as nmol/min/mg of protein. The bone nodule formation in MC3T3-E1 cells of IGF-I was seen at 21, 28 days, but there were no difference between control group and experimental groups. The ALP activity decreased when it is compare to control 2 group except for 1 ng/ml, 10 ng/ml IGF-I of 21-day-groups and 1 ng/ml IGF-I of 28-day-groups. Dose response effects of IGF-I of ALP activity in MC3T3-E1 cells were seen the highest ALP activity at 1ng/ml until 21days and the highest ALP activity at 10 ng/ml of 28 daygroups. The peak times were seen at 7-day group, 14-day group on control group and experimental group respectively, and 1 ng/ml group was the highest ALP activity, From the above results, IGF-I was not seen notable effect on bone nodule formation and decreased ALP activity of MC3T3-E1 cells but the use of IGF-I to mediate biological stimulation of MC3T3-E1 cells shows promise for future therapeutic application.

• 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.

• Journal of Nutrition and Health
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• v.51 no.4
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• pp.316-322
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• 2018

Purpose: The Glycyrrhiza uralensis species (Leguminosae) as a medicinal biocompound, and one of its root components, isoliquritigenin (ISL), which is a flavonoid, has been reported to have anti-tumor activity in vitro and in vivo. However, its function in bone formation has not been studied yet. In this study, we tested the effect of Glycyrrhiza uralensis (ErLR) and baked Glycyrrhiza uralensis (EdLR) extracts on osteoblast proliferation, alkaline phosphatase (ALP) activity, and bone-related gene expression in osteoblastic MC3T3-E1 cells. Methods: MC3T3-E1 cells were cultured in various levels of ErLR (0, 5, 10, 15, $20{\mu}g/mL$), EdLR (0, 5, 10, 15, $20{\mu}g/mL$), or ISL (0, 5, 10, 15, $20{\mu}M$) in time sequences (1, 5, and 20 days). Also, isoliquritigenin (ISL) was tested for comparison to those two biocompound extracts. Results: MTT assay results showed that all three compounds (ErLR, EdLR, and ISL) increased osteoblastic-cell proliferation in a concentration-dependent manner for one day. In addition, both ErLR and EdLR compounds elevated the osteoblast proliferation for 5 or 20 days. Extracellular ALP activity was also increased as ErLR, EdLR, and ISL concentration increased at 20 days, which implies the positive effect of Glycyrrhiza species on osteoblast mineralization. The bone-related marker mRNAs were upregulated in the ErLR-treated osteoblastic MC3T3-E1 cells for 20 days. Bone-specific transcription factor Runx2 gene expression was also elevated in the ErLR- and EdLR-treated osteoblastic MC3T3-E1 cells for 20 days. Conclusion: These results demonstrated that Glycyrrhiza uralensis extracts may be useful for preventing osteoporosis by increasing cell proliferation, ALP activity, and bone-marker gene expression in osteoblastic cells.

• 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.

• Korean journal of food and cookery science
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• v.28 no.3
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• pp.311-318
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• 2012

The correlation between osteoporosis and reactive oxygen species (ROS)-induced oxidative stress was investigated. Thus, interest in food and plants with antioxidant effects that can reduce damage caused by ROS during bone metabolism is heightening. In this study, the antioxidant effect of Taraxacum mongolicum on proliferation and differentiation of MC3T3-E1 cells under H2O2-induced oxidative stress was studied to investigate its protective effect against oxidative stress and its availability as an antioxidant material related to bone diseases. As a result, total polyphenol and total flavonoid contents of T. mongolicum were 33.65 mg/g and 4.45 mg/g, respectively. The T. mongolicum extract increased proliferation of both MC3T3-E1 cells and differentiated osteoblasts under $H_2O_2$-induced oxidative stress conditions. In addition, two differentiation markers, alkaline phosphatase activity and mineralization level in the T. mongolicum extract, tended to increase. These results indicate that T. mongolicum extract suppressed the damage to osteoblasts under oxidative stress and that it is potential antioxidant materials for preventing bone diseases.

• The korean journal of orthodontics
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• v.30 no.2 s.79
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• pp.205-214
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• 2000

Recently, the magnetic force has been considered as a method for a more efficient tooth movement. The purpose of this study was to evaluate the effects of different static magnetic fields of Nd-Fe-B magnet on MC3T3-E1 cells by measuring the alkaline phosphatase activity and observing the amount of stained alkaline phosphatase. For measuring of alkaline phosphatase activity, MC3T3-E1 cells were seeded in first and third row of 12 well culture plates. And Nd-Fe-B magnets were positioned under the first column of first and third row to apply different static magnetic fields(first column:100mT ; second column:4.6mT ; third column:0.5mT ; forth column:0.0mT) to the cells for 7, 13, 19, and 25 days. For staining of alkaline phosphatase, MC3T3-E1 cells were seeded in 100mm culture plates. And Nd-Fe-B magnets were positioned under the corner of plates to apply different static magnetic fields(magnet side:100mT : the opposite side:0.5mT) to the cells for 7, 13, 19, and 25 days. The results were as follows : 1. ALP activity was increased until day 19 in biochemical determination as well as in histochemical staining, 2. The application of higher magnetic field(100mT) suppressed ALP activity at day 13, 19, 25. On the contrary, the application of the lower magnetic field(4.6mT, 0.5mT) significantly enhanced the ALP activity. 3. Consistent with enzyme assay, histochemical staining of ALP also demonstrated that higher magnetic field(100mT) suppressed ALP activity, lower one(0.5mT) enhanced.

• 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 Life Science
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• v.27 no.5
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• pp.501-508
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• 2017

Bone morphogenetic proteins (BMPs) are multifunctional cytokines that play important roles in a variety of cellular functions. Among BMP family members, BMP2 efficiently promotes osteoblast differentiation through Smad-mediated runt-related transcription factor 2 (Runx2) expression. Several recent studies suggest that BMPs are associated with clock genes, in particular Bmal1. Bmal1 protein heterodimerizes with Clock protein and then induces period 1 (Per1) expression. However, the role of Per1 on osteoblast differentiation remains unclear. In this study, we investigated whether Per1 is involved in osteoblast differentiation. MC3T3-E1 cells were treated with BMP2 for induction of osteoblastic differentiation. Osteogenic maker gene and Per1 mRNA expression were measured using real-time PCR. Interestingly, BMP2 treatment induced Per1 mRNA expression in MC3T3-E1 cells. To further investigate the function of Per1 on osteoblast differentiation, MC3T3-E1 cells were transiently transfected with pCMV-Per1. Per1 overexpression increased Runx2 mRNA and protein levels. Also, mRNA expression and promoter activity of osteocalcin were upregulated by Per1 overexpression. To investigate the effect of interaction between Per1 and osteogenic condition, MC3T3-E1 cells were cultured in osteogenic medium containing ascorbic acid and ${\beta}$-glycerophosphate. Osteogenic medium-induced ALP staining level and mineralization were synergistically increased by overexpression of Per1. Taken together, these results demonstrate that Per1 is a positive regulator of osteoblast differentiation.

• 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.