• Journal of Nutrition and Health
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• 제53권4호
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• pp.347-355
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• 2020

Purpose: Zinc is known to be associated with osteoblast proliferation and differentiation. Osterix as zinc-finger transcription factor is also related to osteoblast differentiation and bone formation. In the present study, we aimed to investigate whether zinc modulates osterix gene and protein expression in osteoblastic MC3T3-E1 cells. Methods: MC3T3-E1 cells were cultured in zinc-dependent concentrations (0, 0.5, 1, 5, or 15 µM Zn), along with osteogenic control (normal osteogenic medium) for 1 and 3 days. The gene and protein expression levels of osterix were analyzed by real-time reverse transcription polymerase chain reaction and Western blotting, respectively. Results: Zinc increased osteoblast proliferation in a concentration-dependent manner at day 1 and 3. Similarly, zinc increased the activity of osteoblast marker enzyme alkaline phosphatase in cells and media in a zinc concentration-dependent manner. Moreover, our results showed that the pattern of osterix gene expression by zinc was down-regulated within the low levels of zinc treatments (0.5-1 µM) at day 1, but it was up-regulated after extended culture period at day 3. Osterix protein expression by zinc showed the similar pattern of gene expression, which down-regulated by low zinc levels at day 1 and up-regulated back at day 3 as the early stage of osteoblast differentiation. Conclusion: Our results suggest that zinc modulates osterix gene and protein expression in osteoblasts, particularly in low level of zinc at early stage of osteoblast differentiation period.

• Journal of Dental Anesthesia and Pain Medicine
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• 제19권2호
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• pp.91-99
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• 2019

Background: The imbalance between osteoblasts and osteoclasts can lead to pathological conditions such as osteoporosis. It has been reported that opioid adversely affect the skeletal system, but it is inconsistent. Remifentanil is currently used as an adjuvant analgesic drug in general anesthesia and sedation. The aim of the present study was to investigate the effect of remifentanil on the osteoblast differentiation and mechanism involved in this effect. Methods: The C2C12 cells (mouse pluripotent mesenchymal cell line) were used as preosteoblast. Osteoblastic differentiation potency was determined by alkaline phosphatase (ALP) staining. C2C12 cell migration by remifentanil was evaluated using Boyden chamber migration assay. The expression of Runx2 and osterix was evaluated by RT-PCT and western blot analysis to investigate the mechanism involved in remifentanil-mediated osteoblast differentiation. Results: ALP staining showed that remifentanil increased significantly osteoblast differentiation. In Boyden chamber migration assay, C2C12 cell migration was increased by remifentanil. RT-PCR and western blot analysis showed that the expression of Runx2 and osterix was upregulated by remifentanil. Conclusions: We demonstrated that remifentanil increased osteoblast differentiation in vitro by upregulation of Runx2 and osterix expression. Therefore, remifentanil has the potential for assisting with bone formation and bone healing.

• Journal of Periodontal and Implant Science
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• 제36권4호
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• pp.839-847
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• 2006

The aim of this study is to monitor reporter gene expression under osteocalcin gene promoter, using a real-time molecular imaging system, as tool to investigate osteoblast differentiation. The promoter region of mouse osteocalcin gene 2 (mOG2), the best-characterized osteoblast-specific gene, was inserted in promoterless luciferase reporter vector. Expression of reporter gene was confirmed and relationship between the reporter gene expression and osteoblastic differentiation was evaluated. Gene expression according to osteoblstic differentiation on biomaterials, utilizing a real-time molecular imaging system, was monitored. Luciferase was expressed at the only cells transduced with pGL4/mOGP and the level of expression was statistically higher at cells cultured in mineralization medium than cells in growth medium. CCCD camera detected the luciferase expression and was visible differentiation-dependent intensity of luminescence. The cells produced osteocalcin with time-dependent increment in BMP-2 treated cells and there was difference between BMP-2 treated cells and untreated cells at 14days. There was difference at the level of luciferase expression under pGL4/mOGP between BMP-2 treated cells and untreated cells at 3days. CCCD camera detected the luciferase expression at cells transduced with pGL4/mOGP on Ti disc and was visible differentiation-dependent intensity of luminescence This study shows that 1) expression of luciferase is regulated by the mouse OC promoter, 2) the CCCD detection system is a reliable quantitative gene detection tool for the osteoblast differentiation, 3) the dynamics of mouse OC promoter regulation during osteoblast differentiation is achieved in real time and quantitatively on biomaterial. The present system is a very reliable system for monitoring of osteoblast differentiation in real time and may be used for monitoring the effects of growth factors, drug, cytokines and biomaterials on osteoblast differentiation in animal.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제35권4호
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• pp.205-212
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• 2009

Purpose : The purpose of this study was to examine the expression of various angiogenic factors during osteoblastic differentiation of periostealderived cells and the effects of osteogenic inductive medium of periosteal-derived cells on the proliferation of endothelial progenitor cells. Materials and methods : Periosteal-derived cells were obtained from mandibular periosteums and introduced into the cell culture. After passage 3, the cells were divided into two groups and cultured for 21 days. In one group, the cells were cultured in the DMEM supplemented with osteogenic inductive agent, including 50g/ml L-ascorbic acid 2-phosphate, 10 nM dexamethasone and 10 mM -glycerophosphate. In the other group, they were cultured in DMEM supplemented without osteogenic inductive agent. VEGF isoforms, VEGFR-1, VEGFR-2, and neuropilin-1 mRNA expression was observed. Human umbilical cord blood-derived endothelial progenitor cell proliferation was also observed. Results : The expression of VEGF isoforms was higher in osteogenic inductive medium than in non-osteogenic inductive medium. The expression of VEGFR-2 was also higher in osteogenic inductive medium than in non-osteogenic inductive medium. However, the expression of VEGFR-1 and neuropilin-1 was similar in both osteogenic inductive medium and non-osteogenic inductive medium. In addition, conditioned medium from differentiated periosteal-derived cells stimulated human umbilical cord blood-derived endothelial progenitor cell numbers compared to conditioned medium from non-differentiated periosteal-derived cells. Conclusion : These results suggest that in vitro osteoblastic differentiation of periosteal-derived cells has angiogenic capacity to support endothelial progenitor cell numbers.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제39권2호
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• pp.55-62
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• 2013

Bone tissue engineering is one of the important therapeutic approaches to the regeneration of bones in the entire field of regeneration medicine. Mesenchymal stem cells (MSCs) are actively discussed as material for bone tissue engineering due to their ability to differentiate into autologous bone. MSCs are able to differentiate into different lineages: osteo/odontogenic, adipogenic, and neurogenic. The tissue of origin for MSCs defines them as bone marrow-derived stem cells, adipose tissue-derived stem cells, and, among many others, dental stem cells. According to the tissue of origin, DSCs are further stratified into dental pulp stem cells, periodontal ligament stem cells, stem cells from apical papilla, stem cells from human exfoliated deciduous teeth, dental follicle precursor cells, and dental papilla cells. There are numerous in vitro/in vivo reports suggesting successful mineralization potential or osteo/odontogenic ability of MSCs. Still, there is further need for the optimization of MSCs-based tissue engineering methods, and the introduction of genes related to osteo/odontogenic differentiation into MSCs might aid in the process. In this review, articles that reported enhanced osteo/odontogenic differentiation with gene introduction into MSCs will be discussed to provide a background for successful bone tissue engineering using MSCs with artificially introduced genes.

• 동의생리병리학회지
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• 제23권4호
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• pp.860-865
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• 2009

Osteoclasts are bone-resorbing multinucleated cells derived from the monocyte/macrophage lineage. The differentiation of osteoclasts are regulated by osteoblastic cells expressed RANKL, which is the most critical molecule for osteoclast differentiation. In this study, we found that water extracts of cuscuta inhibited RANKL-mediated osteoclast differentiation by direct action on bone marrow macrophages (BMMs) without cytotoxicity. In BMMs, water extracts of cuscuta inhibited the mRNA expression of c-Fos, NFATc1, TRAP, and OSCAR. Also, the protein expression of c-Fos and NFATc1 was inhibited by water extracts of cuscuta treatement. Water extracts of cuscuta inhibited the phosphorylation of p38, ERK, and JNK in BMMs treated with RANKL. However, water extracts of cuscuta did not inhibit RANKL-induced I-${\kappa}B$ activation. Water extract of cuscuta failed to inhibit bone resorption by osteoclasts cultured on hydroxyapatite plates. These results suggest that cuscuta may be a promising drug for use against bone disorders such as osteoporosis and rheumatoid arthritis.

• Preventive Nutrition and Food Science
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• 제19권3호
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• pp.187-193
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• 2014

This is an Open Access article distributed under the terms of the Creative Commons Attribution For decades, Spergularia marina, a local food that is popular in South Korea, has been regarded as a nutritious source of amino acids, vitamins, and minerals. While several halophytes are reported to possess distinct bioactivities, S. marina has yet to be promoted as a natural source of bioactives. In this study, the effects of S. marina on the adipogenic differentiation of 3T3-L1 fibroblasts and the osteoblastic differentiation of MC3T3-E1 pre-osteoblasts and C2C12 myoblast cells were evaluated. The anti-adipogenic effect of S. marina was assessed by measuring lipid accumulation and adipogenic differentiation marker expression. S. marina treatment significantly reduced lipid accumulation and notably decreased the gene levels of peroxisome proliferator-activated receptor ${\gamma}$, CCAAT/enhancer-binding protein ${\alpha}$, and sterol regulatory element binding protein 1c. In addition, S. marina enhanced osteoblast differentiation, as indicated by increased alkaline phosphatase activity and increased levels of osteoblastogenesis indicators, namely bone morphogenetic protein-2, osteocalcin, and type I collagen. In conclusion, S. marina could be a source of functional food ingredients that improve osteoporosis and obesity. Further studies, including activity-based fractionation, will elucidate the mechanism of action and active ingredients of S. marina, which would provide researchers with a better understanding of the nutraceutical and therapeutic applications of S. marina.

• International Journal of Oral Biology
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• 제32권3호
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• pp.85-91
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• 2007

Dlx3 is a homeodomain protein and is known to playa role in development and differentiation of many tissues. Deletion of four base pairs in DLX3 (NT3198) is causally related to tricho-dento-osseous (TDO) syndrome (OMIM # 190320), a genetic disorder manifested by taurodontism, hair abnormalities, and increased bone density in the cranium. Although the observed defects of TDO syndrome involves bone, little is known about the role of Dlx3 in bone remodeling process. In this study, we examined the effect of wild type DLX3 (wtDlx3) expression on osteoclast differentiation and compared it with that of 4-BP DEL DLX3 (TDO mtDlx3). To examine whether Dlx3 is expressed during RANKL-induced osteoclast differentiation, RAW264.7 cells were cultured in the presence of receptor activator of nuclear factor-B ligand (RANKL). Dlx3 protein level increased slightly after RANKL treatment for 1 day and peaked when the fusion of prefusion osteoclasts actively progressed. When wtDlx3 and TDO mtDlx3 were overexpressed in RAW264.7 cells, they enhanced RANKL-induced osteoclastogenesis and the expression of osteoclast differentiation marker genes such as calcitonin receptor, vitronectin receptor and cathepsin K. Since osteoclast differentiation is critically regulated by the balance between RANKL and osteoprotegerin (OPG), we examined the effect of Dlx3 overexpression on expression of RANKL and OPG in C2C12 cells in the presence of bone morphogenetic protein 2. Overexpression of wtDlx3 enhanced RANKL mRNA expression while slightly suppressed OPG expression. However, TDO mtDlx3 did not exert significant effects. This result suggests that inability of TDO mtDlx3 to regulate expression of RANKL and OPG may contribute to increased bone density in TDO syndrome patients. Taken together, it is suggested that Dlx3 playa role as a positive regulator of osteoclast differentiation via up-regulation of osteoclast differentiation-associated genes in osteoclasts, as well as via increasing the ratio of RANKL to OPG in osteoblastic cells.

• 생명과학회지
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• 제33권1호
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• pp.64-72
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• 2023

심장 발생과정에 관여하는 주요 전사인자들의 기능에 대한 규명 등의 발전에도 불구하고 줄기 세포에서 매우 효율적인 심근 세포로의 분화를 촉진하는 새로운 생체 활성 분자를 찾는 것이 여전히 필요하다. 마우스배아줄기세포(mESC) 유래 심근세포의 Illumina 발현 마이크로어레이 데이터를 분석하였다. 미분화 mESCs와 비교하여 mESC 유래 심근세포에서 4배 이상 유전자 발현이 증가한 276개 유전자가 스크리닝되었다. Secreted phosphoprotein 2 (Spp2)는 후보물질 중 하나이며 bone morphogenetic protein 2 (BMP2)에 대한 슈도수용체로서 BMP2 신호 전달을 억제하는 것으로 알려져 있다. 그러나 심근 형성과의 연관성은 알려진 바 없다. 우리는 mESC 세포주인 TC-1/Kh2와 E14를 이용하여 기능성 심근세포로 분화하는 동안 Spp2 발현이 증가함을 검증하였다. 흥미롭게도, Spp2 분비는 배아체(embryoid body, EBs) 형성 후 3일차에 일시적으로 증가했는데, 이는 Spp2의 분비가 ESCs의 심근세포로의 분화에 관여함을 시사한다. Spp2의 기능을 분석하기 위해, 우리는 BMP2를 처리하면 분화 경로를 근모세포에서 골모세포로 전환되는 특성을 가진 C2C12 마우스 근모세포 세포주를 사용하여 실험을 수행하였다. mESCs의 분화와 유사하게, Spp2의 전사는 C2C12 근모세포가 근관으로 분화됨에 따라 증가하였다. 특히, 분화 초기 단계에서 Spp2의 세포외 분비가 극적으로 증가하였다. 또한, Spp2-Flag 재조합 단백질로 처리하면 C2C12 근모세포의 근관으로의 분화가 촉진되었다. 종합하면, ESCs를 심근 세포로 분화시키는 새로운 생체 활성 단백질로 Spp2를 제안한다. 이것은 심근형성의 분자 경로를 이해하고 허혈성 심장질환에 대한 줄기세포 요법의 실험적 또는 임상적 발전을 촉진하는 역할을 할 것으로 기대한다.

• 대한치과보철학회지
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• 제53권3호
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• pp.198-206
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• 2015

목적: 다양한 크기의 마이크로그루브가 형성된 티타늄 표면에 열산화 처리를 한 복합 표면의 표면특성을 규명하고, 인간치주인대세포 배양 시 표면에 따른 다양한 세포행동들간 차이와 상관관계를 분석하고자 하였다. 재료 및 방법: Grade II 티타늄 디스크를 시편으로 제작하였다. 포토리소그라피를 이용하여 티타늄 시편의 마이크로그루브 크기를 폭/깊이 $0/0{{\mu}m}$, $15/3.5{{\mu}m}$, $30/10{{\mu}m}$, $60/10{{\mu}m}$로 각각 형성하였다. 평활한 티타늄 표면인 대조군(ST)을 제외한 모든 실험군(ST/TO, Gr15-TO, Gr30-TO, Gr60-TO)에 $700^{\circ}C$에서 3시간동안 열산화 처리하고, 주사현미경 사진을 사용하여 표면특성을 평가하였다. 인간치주인대세포를 배양한 후 BrdU (Bromdeoxyuridine) 실험, 알칼리성 인산가수분해효소 활성 실험, 세포외 칼슘 침착 실험을 통해 세포접착, 세포분화 및 골광화를 평가하였다. 통계분석으로는 일요인분산분석과 피어슨상관관계분석(SPSS version 17.0)을 사용하였다. 결과: 열산화를 동반한 마이크로그루브가 형성된 실험군(Gr15-TO, Gr30-TO, Gr60-TO)들은 평활한 대조군(ST)과 단순 열산화 처리 실험군(ST-TO)에 비하여 BrdU 실험, 알칼리성 인산가수분해효소 활성 실험, 세포 외 칼슘 침착 실험 모두에서 유의하게 증가된 활성도를 나타내었다. 특히, Gr60-TO군은 대조군 및 Gr15-TO, Gr30-TO, Gr60-TO 군 등에 비해 가장 증진된 세포접착 및 골아세포분화/골광화를 나타냈다. 결론: 본 연구의 한계 내에서, 열산화 처리 및 마이크로그루브 복합 티타늄 표면은 골아세포분화에 효과적 방법임이 확인되었다. 본 연구에서 규명 된 적정한 마이크로그루브 크기와 열산화 처리 조건은 마이크로그루브-열산화 복합 표면 티타늄 임플란트 개발의 기초 확립에 기여할 수 있을 것이다.