다양한 rhBMP-2 코팅 임플란트 표면에서의 인간 간엽 줄기세포의 부착, 증식, 분화반응 분석
Analysis of attachment, proliferation and differentiation response of human mesenchymal stem cell to various implant surfaces coated with rhBMP-2
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연구 목적: 본 실험은 거친 표면으로 유의성 있는 줄기세포반응을 나타냈던 4 가지의 티타늄 표면 위에 rhBMP-2를 코팅했을 때 어떤 유의한 줄기세포반응(세포부착, 증식, 분화)이 나타나는지 비교 분석함으로 rhBMP-2 코팅을 위한 가장 적절한 표면을 평가하기 위해 시행되었다. 연구 재료 및 방법: 대조군인 기계절삭표면(machined surface)과 실험군인 양극산화(anodized), RBM, SLA 표면에 rhBMP-2를 코팅한 후 코팅하지 않은 표면과 같이 8가지 표면 위에 인간줄기세포를 배양하였다. 배양 후 24시간 후SEM을 통해 줄기세포의 부착을 평가하였고 배양 3, 7, 14일후MTT와 ALP 검사를 통해 줄기세포의 증식과 분화반응을 평가하였다. 그리고 배양 7일후RT-PCR 검사를 통해 Type I collagen, osteocalcin, osteopontin의 유전자 발현의 변화를 평가하였다. 결과:SEM 평가에서 4가지 rhBMP-2 표면이 코팅하지 않은 표면에 비해 세포부착 면적이 넓고 긴밀하며 세포돌기가 더 많이 관찰되었다. 양극산화 rhBMP-2코팅표면에서 가장 두드러지게 관찰되었다. MTT 검사에서 크게 의미 있는 차이는 나타나지 않았다. ALP검사에서 양극산화 rhBMP-2코팅 표면은 대조군과 비교해서 (3, 14일) 또 RBM rhBMP-2 코팅 표면과 비교해서 (14일) 유의성 있는 ALP 활성도의 증가를 나타내었다(P<.05). RT-PCR 검사에서 osteocalcin과 osteopontin의 유전자 발현은 양극산화 rhBMP-2코팅 표면에서 높게 나타났다. 결론: 양극산화 rhBMP-2코팅표면이 줄기세포의 부착과 분화실험에서 대조군표면과 rhBMP-2를 코팅한 기계절삭표면이나 RBM 표면에 비해 유의성 있는 증가를 나타냈다(P<.05).
Abstract
Purpose: In this paper we tried to evaluate the most appropriate surface for rhBMP-2 coating among 4 rough titanium surfaces. Materials and methods: We used machined surface as a control group and anodized, RBM and SLA surfaces as test groups. We coated rhBMP-2 on the 4 surfaces and with uncoated surfaces for each case, we cultured human mesenchymal stem cells on all 8 surfaces. 24 hours after we measured the stem cell’attachment with SEM, and on 3rd, 7th, and 14th days, we checked the cell proliferation and differentiation by using MTT and ALP activity assay. And on the 7th day after the culture, we performed RT-PCR assay to determine whether the expression levels of Type I collagen, osteocalcin, osteopontin were changed. Results: We observed with SEM that 4 rhBMP-2 coated surfaces exhibited wider and tighter cell attachment and more cell process spreading than uncoated surfaces. The anodized rhBMP-2 surface caused robustest effects. In MTT assay we could not find any meaningful difference. In ALP assay there was a significant increase (P<.05) in the ALP activity of anodized rhBMP-2 coated surface compared with that of the control (3rd and 14th days) and with that of the RBM rhBMP-2 coated surface (14th day). In RT-PCR assay there was increased expressions in the anodized rhBMP-2 coated surface for osteocalcin, and osteopontin. Conclusion: We found that the anodized rhBMP-2 coated surface were most prominent stem cell attachment and differentiation in compared to control and Machined rhBMP-2 coated, RBM rhBMP-2 coated surface.
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