增强型绿色荧光蛋白标记骨髓间充质干细胞与表面置换珊瑚人工骨的培养

Culture of surface exchanging coralline hydroxyapatite and enhanced green fluoresecent protein labeled human bone mesenchymal stem cells

目的通过增强型绿色荧光蛋白(eGFP)标记的人骨髓间充质干细胞(MSC)与表面置换珊瑚羟基磷灰石(SCHA)培养,研究SCHA与细胞黏附、增殖的情况。方法将海南天然滨珊瑚在特定温度和压力下部分水热反应,制成SCHA。将SCHA薄片(SCHA组)和盖玻片(玻片组)分别与eGFP标记的人骨髓MSC体外培养,分别于4、8、12、16d进行死活细胞染色和Alamar Blue实验,用荧光显微镜观察细胞活性,荧光分光光度计测量细胞增殖。结果人骨髓MSC在SCHA的表面和孔道内生长良好,第8天、第16天与玻片组达同样水平,之后超过玻片组并保持稳定状态。结论SCHA无细胞毒性,具有较好的细胞相容性,是一种良好的骨组织工程支架材料。

Objective To observe the cell attachment, proliferation, and differentiation of enhanced green fluoresecent protein （eGFP） labeled human bone marrow mesenchymal stem eells（MSC） on surface exchanging coralline hydroxyapatite （SCHA） in in vitro culture. Methods The Hainan coralline was treated into SCHA by ＂Hydrothermal exchange＂ at specific temperature and pressure. The coralline hydroxyapatite （CHA） was carefully cut into 1.5 mm （thickness） × 5 mm × 5 mm（square） slices, eGFP labeled MSC were used for cellular assay. The MSC was seeded at 2 × 10^5 cells/slice on coral; same number of cells was also seeded on 10 mm diameter glass cover slips in 24-well cell culture plates as control. Alamar Blue assay was carried out at day 4, 8, 12, 16 for cell proliferation and dead-live stain was used to assess cell viability. Results The cell number on coral were not as high as those on glass cover slips at day 4. However, cell number on SCHA continuously increased during the culture period and reached the same level as those on cover slips at day 8 and day 12. At day 16, while the ceil number on cover slips decreased with more dead cells, the cell number on SCHA remained stable. Conclusion It is demonstrated that SCHA has better cell compatibility and no cytotoxieity on MSC in vitro. It is a good osseous tissue engineering scaffold material.