异种骨对骨髓间充质干细胞活性及成骨诱导的分化影响

Effects of heterogeneous bone on bone mesenchymal stem cell viability and oesteoinductivity

目的研究异种骨材料对骨髓间充质干细胞（BMSC）的增殖、迁移、细胞周期及定向分化为成骨细胞的能力，并对其生物安全性进行评价。方法取体外培养第5代骨髓间充质干细胞直接接触培养，观察经环氧化物固定制备的异种骨浸提液对骨髓间充质干细胞的增殖率、迁移率、细胞周期、碱性磷酸酶（ALP）及钙结节形成的影响，以H—DMEM培养液组作为阴性对照。结果扫描电镜观察BMSC于异种骨上生长状态饱满，细胞呈梭形或多边形；骨髓问充质干细胞在浸提液中培养，第1、3、5、7d组与对照组相比，结果差异无统计学意义，无细胞毒性；膜滤器（Transwell小室）检测细胞迁移，浸提液组为（46．03±5．00）个／视野，对照组为（37．00±3．80）个／视野，P〈0．05。浸提液组组织在G2／M期的细胞为（27．77±1．06）％，对照组为（26．60±1．05）％，P〈0．01。浸提液组的ALP活性高于对照组（P〈0．05），茜素红染色显示浸提液组于14d时钙沉积明显增多。结论所制备异种骨具有良好的细胞相容性，并对骨髓间充质干细胞的迁移和成骨分化有一定的促进作用。

Objective To study the effects of heterogeneous bone on the proliferation, adhesion, migration, cell cycle and osteoblastic differentiation of bone marrow derived stromal cells （BMSCs）, and evaluate the biosafety of heterogeneous bone. Methods The heterogeneous bones were fixed with epoxides and the extracts were prepared according to ISO 10993-12：2007. Mouse passage 5 BMSCs were cultured with the heterogeneous bone extracts for 24 hours to observe cell proliferation, migration, cell cycle, ALP activity and mineralization node formation. The morphology of BMSCs was observed by scanning electron microscopy （SEM）. Cells cultured with H-DMEM without addition of the extracts served as control. Results Morphological observation under SEM showed healthy growth of BMSCs on heterogeneous bone extracts with spindle-shape or polygonal shape. There was no obvious difference in cell proliferation after 1, 3, 5 and 7 days co-culture with the extracts when compared to the control, indicating the absence of cytotoxicity. The transwell cell migration test showed （46.00 _＋ 5.00） cells/field in the extract group and （37.00 ＋ 3.80） cells/field in the control group （P 〈 0.05）. The extract group induced cell cycle arrest at G2/M phase in about （27.77 ＋_ 1.06）% of the cells, while the control group （26.60 _＋ 1.05）% （P 〈 0.01）. ALP activity and mineralization nodes detected by Alizarin bordeaux staining were significantly higher in the extract group than in the control group after 14 days＇ co-culture （P 〈 0.05）. Conclusion The heterogeneous bone was biocompatible. It can potentially promote BMSC migration and osteoblastic differentiation.