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

국문 초록 (Abstract)

목적: Glass-ceramics 표면에 코팅된 인산칼슘-재조합 인간 BMP-2 (CaP-rhBMP-2) 복합체의 특성과 조골 세포 분화능 검증을 위함이다.
대상 및 방법: 기판으로는 바이오활성 glass-ceramics를, 기판 함침을 위한 인산칼슘용액(CaP, pH7.4)을 준비하였다. 재조합 인간 골 형태형성 단백질-2 (rhBMP-2)은 BMP-2 cDNA가 전이된 CHO-Kl 세포로부터 분리 정제하였다. Glass-ceramics은 생리식염수, CaP 단독 용액, rhBMP-2가 포함된 CaP 용액에 3일 동안 상온에서 함침하였다. 복합체가 코팅된 기판 표면의 특성을 확인하고자, 주사 전자현미경, 적외선 반사 분광학, 박막 x-선 회절과 항-인간 BMP-2에 대한 면역 형광 염색을 시행하였다. 또한, 조골세포 분화능을 평가하고자, 마우스 조골아세포인 MC3T3-El 세포를 흡착시킨 후, 조골세포 표식 유전자에 대한 RT-PCR과 SEM을 시행하였다.
결과: CaP-rhBMP-2 복합체가 glass-ceramics 표면에 코팅되어 있음을 SEM, FT-IR 및 TF-XRD, IF 결과를 통해 확인할 수 있었다. Glass-ceramics 위의 CaP-rhBMP-2 복합체는 미세한 침상 구조로 덮인 구형을 나타낸 반면, CaP 군에서는 glass-ceramics 표면에 미세한 침상 구조가 평평하게 퍼져있었다. 제1형 교원질과 알칼리 인산화 효소 mRNA의 발현은 세포 흡착 후 4시간째에, 또한 osteocalcin mRNA의 발현은 3일째에 CaP-rhBMP-2 복합체군에서 표준군, CaP군과 비교하여 뚜렷하게 증가되어 있었다. SEM 촬영 결과, 실험군에 흡착된 MC3T3-El세포에서 다른 군과 비교하여 활성화된 filopodia 형성이 관찰되었다. 조골세포와 CaP-rhBMP-2 복합체 주변에는 신생 세포기질의 합성도 관찰되었다.
결론: 바이오 활성 glass-ceramic에서의 CaP-rhBMP-2 복합체-표면 코팅 기술의 적용은 조골 분화 유도에 있어서 매우 강력한 기법이다.

다국어 초록 (Multilingual 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.

목차 (Table of Contents)

• 서론
• 대상 및 방법
• 결과
• 고찰
• 결론
• 참고문헌
• 국문초록

참고문헌 (Reference)

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