인간 BMP-2-생리기능 복합 바이오활성 세라믹스를 이용한 조골 세포 분화 유도

Abstract

Purpose: To assess the properties and the osteogenic potency of the calcium phosphate-recombinant human morphogenetic protein-2 (CaP-rhBMP-2 composite) on glass-ceramics. Materials and Methods: Bioactive glass-ceramics, as a scaffold, and a calcium phosphate (CaP) solution (pH7.4) were prepared. Recombinant human bone morphogenetic protein-2 (rhBMP-2) was purified from CHO-K1 cells by transfecting the cells with BMP-2 cDNA. The glass-ceramics were soaked for 3 days at room temperature in saline, a CaP only solution, and a CaP solution containing rhBMP-2. Scanning electron microscopy (SEM), Fourier transform infrared reflection spectroscopy (FT-IR), thin film X-ray diffraction (TF-XRD) and immunofluorescent staining (IF) of the anti-human BMP-2 tot composite-coated scaffold were performed to verify the characterization of the scaffold surface. In addition, RT-PCR of osteogenic marker gene and SEM photography were performed after adhering the mouse preosteoblast MC3T3-E1 cells in order to assess the osteoinductivity. Results: CaP-rhBMP-2 composite was coated on the surface of glass-ceramics, as confirmed by SEM, FT-IR, TF-XRD spectrum, and IF. The CaP-rhBMP-2 composite on the glass-ceramic showed a globular shape covered with fine spikes while the CaP on the glass-ceramic showed a fine spike structure on the flat glass surface. The expression of collagen type Ⅰ and alkaline phosphatase mRNAs had increased 4 hours after cell seeding. In addition, the level of osteocalcin mRNA ex-pression had increased significantly by 3 days in the CaP-rhBMP-2 composite compared with the control and CaP group. The SEM photographs showed more active filopodia formation in the CaP-rhBMP-2 composite than the other groups. There was extensive newly synthesized extracellular matrix around the osteoblasts and CaP-rhBMP-2 composite nodule. Conclusion: The application of Cap-rhBMP-2 composite-surface coating technique on bioactive glass-ceramic is a powerful tool for osteoinduction.