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정영단,양원경,고현정,김미리 대한치과보존학회 2014 Restorative Dentistry & Endodontics Vol.39 No.3
Objectives: The effects of bone morphogenetic protein-2 (BMP-2) and enamel matrix derivative (EMD) respectively with mineral trioxide aggregate (MTA) on hard tissue regeneration have been investigated in previous studies. This study aimed to compare the osteogenic effects of MTA/BMP-2 and MTA/EMD treatment in MC3T3-E1 cells. Materials and Methods: MC3T3-E1 cells were treated with MTA (ProRoot, Dentsply), BMP-2 (R&D Systems), EMD (Emdogain, Straumann) separately and MTA/BMP-2 or MTA/ EMD combination. Mineralization was evaluated by staining the calcium deposits with alkaline phosphatase (ALP, Sigma-Aldrich) and Alizarin red (Sigma-Aldrich). The effects on the osteoblast differentiation were evaluated by the expressions of osteogenic markers, including ALP, bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN) and osteonectin (OSN), as determined by reverse-transcription polymerase chain reaction analysis (RT-PCR, AccuPower PCR, Bioneer). Results: Mineralization increased in the BMP-2 and MTA/BMP-2 groups and increased to a lesser extent in the MTA/ EMD group but appeared to decrease in the MTA-only group based on Alizarin red staining. ALP expression largely decreased in the EMD and MTA/EMD groups based on ALP staining. In the MTA/BMP-2 group, mRNA expression of OPN on day 3 and BSP and OCN on day 7 significantly increased. In the MTA/EMD group, OSN and OCN gene expression significantly increased on day 7, whereas ALP expression decreased on days 3 and 7 (p < 0.05). Conclusions: These results suggest the MTA/BMP-2 combination promoted more rapid differentiation in MC3T3-E1 cells than did MTA/EMD during the early mineralization period.
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강호인,정영단,Paul Verma,노상호 한국조직공학과 재생의학회 2013 조직공학과 재생의학 Vol.10 No.6
The generation of cartilaginous tissues using pluripotent stem cells is one possible solution for cartilage regeneration, as the mass production of cartilaginous cells is required to cure defects and diseases of cartilage. Parthe-nogenetic ESCs (PESCs) are a useful stem cell source. The defects in full-term development of this cell type enable researchers to avoid ethical concerns. Moreover, in female patients, if the PESCs are derived from the patient’s own oocytes, the cells will have that patient's genetic information. Here we present data demonstrating that PESCs can be differentiated into chondrogenic cells with induction medium containing multiple factors such as ascorbic acid, dexam-ethasone, bone morphogenetic protein-2 (BMP-2), and transforming growth factor- beta (TGF-β), and the induction can be promoted by exogenous insulin-like growth factor 2 (IGF-2), which is silenced in PESCs. Before chondrogenic induction of the PESCs, floating aggregates called embryoid bodies (EBs) were formed in suspension culture for 5days. In the first series of experiments, the EBs were transferred to chondrogenic induction medium that contained serum, ascorbic acid, dexamethasone, BMP-2, BMP-4 and TGF-β, and the cells were cultured for an additional 21 days using either a two-dimensional (2D) or three-dimensional (3D) culture system. In the second series of experiments, we investigated the effects of different concentrations of IGF-2 supplementation (0, 1, 10 or 100ng/mL) in a 3D culture system. After induction, the gene expression of the chondrocyte-specific markers, CHORDIN-LIKE 1, COLLAGEN-2, AGGRECAN, DECORIN, MMP13 and PAX-1, was analyzed real-time PCR. In experiment 1, the cells from all experimental groups after chondrogenic induction exhibited morphology characteristic of murine chondrocytes as con-firmed by Alcian blue and Safranin O staining. The 3D culture system showed increased induction of chondrogenic differentiation in comparison to the 2D system. In experiment 2, the group supplemented with 100ng/mL IGF-2showed the highest induction of chondrogenic differentiation in comparison to other groups. The expression levels of all genes were 7-to 35-fold higher in the group treated with 100ng/mL IGF-2 in comparison to the IGF-2-free control. The result showed that supplementation with the IGF-2 improves the efficiency of chondrogenic differentiation of murine PESCs in a 3D culture environment. The addition of IGF-2 may re-activate imprinted genes in PESCs that are only expressed in the paternal genome and are normally silent in PESCs. Our findings suggest that compensative sup-plementation of imprinting factor(s) may improve the efficiency of differentiation to specific lineages in monogenic stem cells.
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