• International Journal of Oral Biology
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• 제36권4호
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• pp.173-178
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• 2011

Tumor necrosis factor alpha ($TNF{\alpha}$) is a multifunctional cytokine that is elevated in inflammatory diseases such as atherosclerosis, diabetes and rheumatoid arthritis. Recent evidence has suggested that ${\beta}2$ adrenergic receptor (${\beta}2AR$) activation in osteoblasts suppresses osteogenic activity. In the present study, we explored whether $TNF{\alpha}$ modulates ${\beta}AR$ expression in osteoblastic cells and whether this regulation is associated with the inhibition of osteoblast differentiation by $TNF{\alpha}$. In the experiments, we used C2C12 cells, MC3T3-E1 cells and primary cultured mouse bone marrow stromal cells. Among the three subtypes of ${\beta}AR$, ${\beta}2$ and ${\beta}3AR$ were found in our analysis to be upregulated by $TNF{\alpha}$. Moreover, isoproterenol-induced cAMP production was observed to be significantly enhanced in $TNF{\alpha}$-primed C2C12 cells, indicating that $TNF{\alpha}$ enhances ${\beta}2AR$ signaling in osteoblasts. $TNF{\alpha}$ was further found in C2C12 cells to suppress bone morphogenetic protein 2-induced alkaline phosphatase (ALP) activity and the expression of osteogenic marker genes including Runx2, ALP and osteocalcin. Propranolol, a ${\beta}2AR$ antagonist, attenuated this $TNF{\alpha}$ suppression of osteogenic differentiation. $TNF{\alpha}$ increased the expression of receptor activator of NF-${\kappa}B$ ligand (RANKL), an essential osteoclastogenic factor, in C2C12 cells which was again blocked by propranolol. In summary, our data show that $TNF{\alpha}$ increases ${\beta}2AR$ expression in osteoblasts and that a blockade of ${\beta}2AR$ attenuates the suppression of osteogenic differentiation and stimulation of RANKL expression by $TNF{\alpha}$. These findings imply that a crosstalk between $TNF{\alpha}$ and ${\beta}2AR$ signaling pathways might occur in osteoblasts to modulate their function.

• Biomolecules & Therapeutics
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• 제24권2호
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• pp.123-131
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• 2016

Osteoporosis is a bone pathology leading to increased fracture risk and challenging the quality of life. The aim of this study was to evaluate the effect of an anthraquinone glycoside, aloin, on osteogenic induction of MC3T3-E1 cells. Aloin increased alkaline phosphatase (ALP) activity, an early differentiation marker of osteoblasts. Aloin also increased the ALP activity in adult human adipose-derived stem cells (hADSC), indicating that the action of aloin was not cell-type specific. Alizarin red S staining revealed a significant amount of calcium deposition in cells treated with aloin. Aloin enhanced the expression of osteoblast differentiation genes, Bmp-2, Runx2 and collagen 1a, in a dose-dependent manner. Western blot analysis revealed that noggin and inhibitors of p38 MAPK and SAPK/JNK signals attenuated aloin-promoted expressions of Bmp-2 and Runx2 proteins. siRNA mediated blocking of Wnt-5a signaling pathway also annulled the influence of aloin, indicating Wnt-5a dependent activity. Inhibition of the different signal pathways abrogated the influence of aloin on ALP activity, confirming that aloin induced MC3T3-E1 cells into osteoblasts through MAPK mediated Wnt and Bmp signaling pathway.

• 치위생과학회지
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• 제21권4호
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• pp.243-250
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• 2021

Background: Endodontic sealers or their toxic components may become inflamed and lead to delayed wound healing when in direct contact with periapical tissues over an extended period. Moreover, an overfilled sealer can directly interact with adjacent tissues and may cause immediate necrosis or further resorption. Therefore, the treatment outcome conceivably depends on the endodontic sealer's biocompatibility and osteogenic potential. This study aimed to evaluate the cell viability and osteogenic effects of four different sealers in osteoblastic cells. Methods: AH Plus (resin-based sealer), Pulp Canal Sealer EWT (zinc oxide-eugenol sealer), BioRoot RCS (calcium silicate-based sealer), and Well-Root ST (MTA-based calcium silicate sealer) were mixed strictly according to the manufacturer's instructions, and dilutions of sealer extracts (1/2, 1/5 and 1/10) were determined. Cell viability was measured using the water-soluble tetrazolium-8 (WST-8) assay. Differentiation was assessed by alkaline phosphatase (ALP) activity and mineralized nodule formation by Alizarin Red S staining. Results: The cell viability of the extracts derived from the sealers excluding Well-Root ST was concentration dependent, with sealer extracts having the least viability at a 1/2 dilution. At sealer extract dilution of 1/10, the test groups showed the same survival rate as that control group, with the exception of BioRoot RCS. Among all experimental groups, BioRoot RCS showed the highest cell viability after 48 hours. The ALP activity was significantly higher in a concentration-dependent manner. Furthemore, all four materials promoted ALP activity and mineralized nodule formation compared to the control at 1/10 dilutions. Conclusion: This is the first study to highlight the differences in biological activity of these four materials. These results suggest that the composition of root canal sealers appears to alter the form of biocompatibility and osteoblastic differentiation.

• 생명과학회지
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• 제23권4호
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• pp.471-478
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• 2013

인체 중간엽 줄기세포는 다양한 세포로의 분화 및 자가증식 할 수 있는 능력뿐만 아니라 질병치료에 대한 치료적 잠재력을 가지고 있다. 줄기세포 분화의 분자 기작에 대한 이해는 줄기세포 이식의 치료 효능을 향상시킨다. 본 연구에는 인체 중간엽 줄기세포의 골분화에서 NFAT5의 역할을 밝혔다. 특이적 siRNA의 transfection으로 인한 NFAT5의 억제는 인체 중간엽 줄기세포의 골분화를 현저히 감소시켰으며, NF-${\kappa}B$ promoter 활성화 또한 세포의 증식이나 지방 세포로의 분화에 영향 없이 감소 시켰다. NFAT5의 발현 억제는 기본적으로 유도되는 NF-${\kappa}B$의 활성화와 TNF-${\alpha}$에 의해서 유도되는 NF-${\kappa}B$의 활성화를 감소시켰으나, TNF-${\alpha}$에 의해서 유도되는 NF-${\kappa}B$의 분해에는 아무런 영향을 주지 않았다. 이번 연구를 통해 NFAT5가 NF-${\kappa}B$ 경로를 조절함으로써 인체 중간엽 줄기 세포의 골분화에 아주 중요한 역할을 하는 것을 확인 할 수 있었다.

• BMB Reports
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• 제47권8호
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• pp.463-468
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• 2014

Osteoblasts are specialized mesenchymal cells that are responsible for bone formation. In this study, we examine the role of GATA4 in osteoblast differentiation. GATA4 was abundantly expressed in preosteoblast cells and gradually down-regulated during osteoblast differentiation. Overexpression of GATA4 in osteoblastic cells inhibited alkaline phosphatase activity and nodule formation in osteogenic conditioned cell culture system. In addition, overexpression of GATA4 attenuated expression of osteogenic marker genes, including Runx2, alkaline phosphatase, bone sialoprotein, and osteocalcin, all of which are important for osteoblast differentiation and function. Overexpression of GATA4 attenuated Runx2 promoter activity, whereas silencing of GATA4 increased Runx2 induction. We found that GATA4 interacted with Dlx5 and subsequently decreased Dlx5 binding activity to Runx2 promoter region. Our data suggest that GATA4 acts as a negative regulator in osteoblast differentiation by downregulation of Runx2.

• Preventive Nutrition and Food Science
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• 제16권4호
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• pp.291-298
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• 2011

Yam extracts (Dioscorea batatas) have been reported to possess a variety of functions. However, studies on its osteogenic properties are limited. In this study, we investigated the effect of ethanol and water extracts on osteoblast proliferation and bone matrix protein synthesis, type I collagen and alkaline phosphatase (ALP), using osteoblastic MC3T3-E1 cell model. MC3T3-E1 cells were cultured with yam ethanol and water extracts (0~30 mg/L) within 39 days of osteoblast differentiation period. Cell proliferation was measured by MTT assay. Bone matrix proteins were assessed by the accumulation of type I collagen and ALP activity by staining the cell layers for matrix staining. Also, the secreted (media) matrix protein concentration (type I collagen) and enzyme activity (ALP) were measured colorimetrically. Yam ethanol and water extracts stimulated cell proliferation within the range of 15~30 mg/L at 15 day treatment. The accumulation of type I collagen in the extracellular matrix, as well as secreted collagen in the media, increased with increasing doses of yam ethanol (3~15 mg/L) and water (3~30 mg/L) extracts. ALP activity was not affected by yam ethanol extracts. Our results demonstrated that yam extracts stimulated osteoblast proliferation and enhanced the accumulation of the collagenous bone matrix protein type I collagen in the extracellular matrix. These results suggest that yam extracts may be a potential activator for bone formation by increasing osteoblast proliferation and increasing bone matrix protein type I collagen. Before confirming the osteogenic action of yam, further studies for clarifying how and whereby yam extracts can stimulate this ostegenesis action are required.

• 치위생과학회지
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• 제19권4호
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• pp.254-260
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• 2019

Background: The primary aims of periodontal disease treatment is to remove dental plaque and calculus, the main causes of tooth loss, and restore periodontal tissue destroyed by inflammation. Periodontal disease treatment should also help maintain the alveolar bone, alleviate inflammation, and promote periodontal ligament cell proliferation, which is essential for tissue regeneration. Conventional antibiotics and anti-inflammatories have adverse side effects, especially during long-term use, so there is a need for adjunct treatment agents derived from natural products. The purpose of this study was to investigate whether the herbal flavone baicalein has the osteogenic activity under inflammatory conditions, and assess the involvement of osteoblast immediate early response 3 (IER3) expression. Methods: Human osteoblastic MG-63 cells were cultured with the pro-inflammatory cytokines tumor necrosis factor α and interleukin 1β in the presence and absence of baicalein. Proliferation was assessed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, and expression of IER3 mRNA was assessed using real-time polymerase chain reaction. The expression of IER3 protein levels and activation of associated signal transduction pathways were assessed using western blotting. Results: Baicalein increased IER3 mRNA and protein expression synergistically. In addition, baicalein reversed the suppression of cell proliferation, and the downregulation of osteogenic transcription factor runt-related transcription factor 2 and osterix induced by pro-inflammatory cytokines. Baicalein also upregulated the phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK 1/2). The upregulation of IER3 by pro-inflammatory cytokines was blocked by pretreatment with inhibitors of AKT, p38, JNK, and ERK 1/2. Conclusion: Baicalein mitigates the deleterious responses of osteoblasts to pro-inflammatory cytokines. Further, IER3 enhanced the effect of baicalein via activation of AKT, p38, JNK, and ERK pathways.

• BMB Reports
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• 제49권3호
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• pp.179-184
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• 2016

Bone morphogenetic protein 9 (BMP9) is a potent inducer of osteogenic differentiation of mesenchymal stem cells. The Wnt antagonist Dickkopf-1 (Dkk1) is involved in skeletal development and bone remodeling. Here, we investigated the role of Dkk1 in BMP9-induced osteogenic differentiation of MSCs. We found that overexpression of BMP9 induced Dkk1 expression in a dose-dependent manner, which was reduced by the P38 inhibitor SB203580 but not the ERK inhibitor PD98059. Moreover, Dkk1 dramatically decreased not only BMP9-induced alkaline phosphatase (ALP) activity but also the expression of osteocalcin (OCN) and osteopontin (OPN) and matrix mineralization of C3H10T1/2 cells. Furthermore, exogenous Dkk1 expression inhibited Wnt/β-catenin signaling induced by BMP9. Our findings indicate that Dkk1 negatively regulates BMP9-induced osteogenic differentiation through inhibition of the Wnt/β-catenin pathway and it could be used to optimize the therapeutic use of BMP9 and for bone tissue engineering.

• Journal of Ginseng Research
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• 제42권2호
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• pp.199-207
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• 2018

Background: Ginseng is a well-known traditional Chinese medicine that has been widely used in a range of therapeutic and healthcare applications in East Asian countries. Microbial transformation is regarded as an effective and useful technology in modification of nature products for finding new chemical derivatives with potent bioactivities. In this study, three minor derivatives of ginsenoside compound K were isolated and the inducing effects in the Wingless-type MMTV integration site (Wnt) signaling pathway were also investigated. Methods: New compounds were purified from scale-up fermentation of ginsenoside Rb1 by Paecilomyces bainier sp. 229 through repeated silica gel column chromatography and high pressure liquid chromatography. Their structures were determined based on spectral data and X-ray diffraction. The inductive activities of these compounds on the Wnt signaling pathway were conducted on MC3T3-E1 cells by quantitative real-time polymerase chain reaction analysis. Results: The structures of a known 3-keto derivative and two new dehydrogenated metabolites were elucidated. The crystal structure of the 3-keto derivative was reported for the first time and its conformation was compared with that of ginsenoside compound K. The inductive effects of these compounds on osteogenic differentiation by activating the Wnt/b-catenin signaling pathway were explained for the first time. Conclusion: This study may provide a new insight into the metabolic pathway of ginsenoside by microbial transformation. In addition, the results might provide a reasonable explanation for the activity of ginseng in treating osteoporosis and supply good monomer ginsenoside resources for nutraceutical or pharmaceutical development.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제36권1호
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• pp.7-15
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• 2010

Complex human tissues harbor stem cells and precursor cells, which are responsible for tissue development or repair. Recently, dental tissues such as dental pulp, periodontal ligament (PDL), dental follicle have been identified as easily accessible sources of undifferentiated cells. These tissues contain mesenchymal stem cells that can be differentiate into bone, cartilage, fat or muscle by exposing them to specific growth conditions. In this study, the authors procured the stem cell from pulp, PDL, and dental follicle and differentiate them into osteoblast and examine the bone induction capacity. Dental pulp stem cell (DPSC), periodontal ligament stem cell (PDLSC), and dental follicle precursor cell (DFPC) were obtained from human 3rd molar and cultured. Each cell was analyzed for presence of stem cell by fluorescence activated cell sorter (FACs) against CD44, CD105 and CD34, CD45. Each stem cell was cultured, expanded and grown in an osteogenic culture medium to allow formation of a layer of extracellular bone matrix. Osteogenic pathway was checked by alizarin red staining, alkaline phosphatase (ALP) activity test and RT-PCR for ALP and osteocalcin (OCN) gene expression. According to results from FACs, mesenchymal stem cell existed in pulp, PDL, and dental follicle. As culturing with bone differentiation medium, stem cells were differentiated to osteoblast like cell. Compare with stem cell from pulp, PDL and dental follicle-originated stem cell has more osteogenic effect and it was assumed that the character of donor cell was able to affect on differential potency of stem cell. From this article, we are able to verify the pulp, PDL, and dental follicle from extracted tooth, and these can be a source of osteoblast and stem cell for tissue engineering.