• 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.

• International Journal of Oral Biology
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• 제33권1호
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• pp.33-43
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• 2008

In the process of bone remodeling, mineral phase of bone is dissolved by osteoclasts, resulting in elevation of calcium concentration in micro-environment. This study was performed to explore the effect of high extracellular calcium ($Ca{^{2+}}_e$) on mineralized nodule formation and on the expression of progressive ankylosis (Ank), plasma cell membrane glycoprotein-1 (PC-1) and osteopontin by primary cultured mouse calvarial cells. Osteoblastic differentiation and mineralized nodule formation was induced by culture of mouse calvarial cells in osteoblast differentiation medium containing ascorbic acid and ${\beta}$-glycerophosphate. Although Ank, PC-1 and osteopontin are well known inhibitors of mineralization, expression of these genes were induced at the later stage of osteoblast differentiation during when expression of osteocalcin, a late marker gene of osteoblast differentiation, was induced and mineralization was actively progressing. High $Ca{^{2+}}_e$(10 mM) treatment highly enhanced mRNA expression of Ank, PC-1 and osteopontin in the late stage of osteoblast differentiation but not in the early stage. Inhibition of p44/42 MAPK activation but not that of protein kinase C suppressed high $Ca{^{2+}}_{e^-}$induced expression of Ank, PC-1 and osteopontin. When high $Ca{^{2+}}_e$(5 mM or 10 mM) was present in culture medium during when mineral deposition was actively progressing, matrix calcifiation was significantly increased by high $Ca{^{2+}}_e$. This stimulatory effect was abolished by pyrophosphate (5 mM) or levamisole (0.1-0.5 mM), an alkaline phosphatase inhibitor. In addition, probenecid (2mM), an inhibitor of Ank, suppressed matrix calcification in both control and high $Ca{^{2+}}_{e^-}$treated group, suggesting the possible role of Ank in matrix calcification by osteoblasts. Taken together, these results showed that high $Ca{^{2+}}_e$ stimulates expression of Ank, PC-1 and osteopontin as well as matrix calcification in late differentiation stage of osteoblasts and that p44/42 MAPK activation is involved in high $Ca{^{2+}}_{e^-}$induced expression of Ank, PC-1 and osteopontin.

• 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.

• 한국식품조리과학회지
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• 제28권3호
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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.

• International Journal of Oral Biology
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• 제40권4호
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• pp.197-204
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• 2015

MicroRNA (miRNA, miR) is essential in regulating cell differentiation either by inhibiting mRNA translation or by inducing its degradation. However, the role of miRNA in odontoblastic cell differentiation is still unclear. In this study, we examined the molecular mechanism of miR-27-mediated regulation of odontoblast differentiation in MDPC-23 mouse odontoblastic cells derived from mouse dental papilla cells. The results of the present study demonstrated that the miR-27 expression increases significantly during MDPC-23 odontoblastic cell differentiation. Furthermore, miR-27 up-regulation promotes the differentiation of MDPC-23 cells and accelerates mineralization without cell proliferation. The over-expression of miR-27 significantly increased the expression levels of Wnt1 mRNA and protein. In addition, the results of target gene prediction revealed that Wnt1 mRNA has an miR-27 binding site in its 3'UTR, and is increased by miR-27. These results suggested that miR-27 promotes MDPC-23 odontoblastic cell differentiation by targeting Wnt1 signaling. Therefore, miR-27 is a critical odontoblastic differentiation molecular target for the development of miRNA based therapeutic agents in dental medicine.

• 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.

• 약학회지
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• 제58권5호
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• pp.307-313
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• 2014

Rumex crispus (curled dock), which is a perennial wild plant, has long been used as a laxative, astringent, and medicine to treat blood and skin diseases. We recently reported that the roots of R. crispus are an effective nutraceutical for bone. This study prepared ethanol extracts of the leaves and roots of R. crispus, and analyzed the major constituents using liquid chromatography and mass spectrometry. In addition, their effects on the proliferation and differentiation of human osteoblast-like MG-63 cells, such as cell viability, alkaline phosphatase (ALP) activity, collagen content, and mineralization, were compared. The chromatograms of the chemical constituents of the two extracts exhibited quite different profiles: quercetin and quercitrin were identified as major peaks in the leaf extract, whereas cinnamtannin B1 and procyanidin isomers were the major peaks for the root extract. Neither extract was cytotoxic at concentrations of < $25{\mu}g/ml$. ALP activity and collagen synthesis-which are markers of the early stage of osteogenesis-in MG-63 cells were significantly increased upon the addition of the root extract compared with the addition of the leaf extract. In contrast, the leaf extract had a more stimulatory effect on mineralization-which is marker of the late stage of osteogenesis-in MG-63 cells than did the root extract. In conclusion, extracts of both leaves and roots of R. crispus stimulated the bone-forming activity of osteoblasts; in particular, the root extract was more effective in the early stage of osteoblast differentiation, while the leaf extract was more effective in the late stage. This difference in anabolic activity may be due to differences in the constituents of the leaves and roots. The leaves and roots of R. crispus appear to complement each other as stimulators of bone formation.

• Journal of Periodontal and Implant Science
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• 제35권2호
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• pp.345-357
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• 2005

Prostaglandin plays a significant role in the local control of bone metabolism associated with periodontal disease. ${\Delta}^{12}-PGJ_2$ is a natural $PGD_2$ metabolite that is formed in vivo in the presence of plasma. It is known for ${\Delta}^{12}-PGJ_2$ to stimulate calcification in osteoblastic cells. Bone morphogenetic protein(BMP) stimulated osteoblastic differentiation in various types of cells and greatly enhanced healing of bony defects. The purpose of this study was to evaluate the effect of rhEMP-2 on ${\Delta}^{12}-PGJ_2$ induced osteoblastic differentiation and mineralization in vitro. A human osteosarcoma cells line Saos-2 were cultured. In the test groups, 10-7M of ${\Delta}^{12}-PGJ_2$ or mixture of 10-8M of ${\Delta}^{12}-PGJ_2$ and 100ng/ml of rhBMP-2 or 100ng/ml of rhEMP-2 were added to culture media. After 1 day, 2 days and 4 days of culture period, the cell number was measured. Alkaline phosphatase activity was measure at 3 days. Reverse transcription polymerase chain reaction(RT-PCR) was performed to determine the expression of mRNA of bone matrix protein at 8 hours, 1 day and 7 days. The ability to produce mineralized nodules in rat osteoblasts(MC3T3-E1) was evaluated at 21 days. The results were as follows : 1. rhEMP-2 or mixture of rhBMP-2 and ${\Delta}^{12}-PGJ_2$ inhibited cell proliferation of human osteosarcoma cells. 2. rhEMP-2 or mixture of rhBMP-2 and ${\Delta}^{12}-PGJ_2$ stimulated alkaline phosphatase activity significantly higher than ${\Delta}^{12}-PGJ_2$ alone. 3. rhBMP-2 or mixture of rhEMP-2 and ${\Delta}^{12}-PGJ_2$ stimulated mineralization compared to ${\Delta}^{12}-PGJ_2$ alone. 4. mRNA of alkaline phosphatase, BMP-2, cbfa 1, Type I collagen were detected in the group treated with ${\Delta}^{12}-PGJ_2$/rhBMP-2, rhBMP-2 alone, ${\Delta}^{12}-PGJ_2$ alone. These results show that mixture of ${\Delta}^{12}-PGJ_2$ and rhBMP-2 causes more bone formation than ${\Delta}^{12}-PGJ_2$ alone while the bone formation effects of mixture of ${\Delta}^{12}-PGJ_2$ and rhBMP-2 are less than those of rhBMP-2 alone. Further researches would be necessary to clarify the interactions of these agents.

• 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.

• Nutritional Sciences
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• 제9권4호
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• pp.233-239
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• 2006

Porous matrices of bioactive polymers such as polyglycolic acid (PGA) or polylactic acid (PLA) can be used as scaffolds in bone tissue growth during bone repair process. These polymers are highly porous and serve as a template for the growth and organization of new bone tissues. We evaluated the effect of PGA and PLA polymers on osteoblastic MC3T3-E1 cell extracellular mineralization. MC3T3-E1 cells were cultured in a time-dependent manner -1, 15, 25d as appropriate - for the period of bone formation stages in one of the five culture circumstances, such as normal osteogenic differentiation medium, PGA-plated, fetal bovine serum (FBS)-plated, PGA/FBS-coplated, and PLA-plated For the evaluation of bone formation, minerals (Ca, Mg, Mn) and alkaline phosphatase activity, a marker for osteoblast differentiation, were measured Alizarin Red staining was used for the measurement of extracellular matrix Ca deposit During the culture period, PGA-plated one was reabsorbed into the medium more easily and faster than the PLA-plated one. At day 15, at the middle stage of bone formation, cellular Ca and Mg levels showed higher tendency in PGA- or PLA-plated treatments compared to non-plated control and at day 25, at the early late stage of bone formation, all three cellular Ca, Mg or Mn levels showed higher tendency as in order of PGA-related treatments and PLA-plated treatments, compared to control even without significance. Medium Ca, Mg or Mn levels didn't show any consistent tendency. Cellular ALP activity was higher in the PGA- or PLA-plated treatments compare to normal osteogenic medium treatment PGA-plated and PGA/FBS-plated treatments showed better Ca deposits than other treatments by measurement of Alizarin Red staining, although PLA-plated treatment also showed reasonable Ca deposit. The results of the present study suggest that biodegradable material, PGA and also with less extent for PLA, can be used as a biomaterial for better extracellular matrix mineralization in osteoblastic MC3T3-E1 cells.