纳米羟基磷灰石／细菌纤维素对兔骨髓间充质干细胞黏附及成骨分化的影响

Effects of nano -hydroxyapatite/bacterial cellulose on the adhesion and osteoblastic differentiation of rabbit bone marrow mesechymal stem cells

目的观察纳米羟基磷灰石／细菌纤维素（nHA／BC）复合支架对兔骨髓间充质干细胞（BMSCs）的黏附及成骨分化的影响，以探讨其在骨组织工程领域潜在的应用价值。方法培养及鉴定兔BMSCs，将BMSCs与nHA／BC体外复合，倒置相差显微镜及扫描电镜观察BMSCs与nHA／BC的复合情况。利用诱导培养基诱导细胞成骨分化，观察细胞分化过程中的形态变化、扫描电镜观察矿化基质沉积。结果第3代BMSCs呈均匀一致的长梭形，流式细胞仪检测其表面抗体CD44阳性，CIM5阴性。倒置相差显微镜下观察BMSCs能够在表面有效的黏附生长，保持细胞形态，至7天时细胞融合成片。扫描电镜观察：nHA／BC呈三维立体多孔结构，BMSCs与nHA／BC复合后，BMSCs伸出伪足黏附在表面及孔隙内，细胞相互连接、融合。细胞诱导后呈现明显的成骨细胞特征，可见钙结节生成。扫描电镜观察可见nHA／BC膜上明显的矿物质沉积，并与nHA／BC膜紧密相连，结节中可见大量粗大的颗粒。结论nHA／BC能促进兔BMSCs黏附，为成骨分化提供结构支撑。

Objective To observe the effects of nano -hydroxyapatite/bacterial cellulose （nHA/BC） on the adhe- sion and osteoblastic differentiation of bone marrow mesechymal stem cells （BMSCs） , and explore the feasibility and po- tential applications of nHA/BC in the field of bone tissue engineering. Methods Rabbit BMSCs were isolated and iden- tified before adhesion on nHA/BC for phase contrast microscopy and scanning electron microscopy （SEM）. Then, BM- SCs were induced for osteoblastic differentiation. The morphological changes of BMSCs and the deposition of mineralized matrix on nHA/BC were observed. Results BMSCs became long fusiform - shaped at the third generation. According to flow cytometry, they were CIM4 positive and CD45 negative. Under the phase contrast microscope, BMSCs grew on the surface of nHA/BC with the same cell morphology until attachment of each cell on Day 7. The SEM images showed that nHA/BC was in a three - dimensional porous structure, while BMSCs attached to and fused with each other＇through adhe- sion on the surface and within the pores of nHA/BC through pseudopodia. After inducement, BMSCs showed obvious pro- files of osteoblasts, and calcium nodules were found. According to SEM images, deposited minerals were clearly seen on the surface of nHA/BC membrane, and closely linked with the membrane, with a large number of coarse particles in the nodules. Conclusions nHA/BO can stimulate the adhesion of rabbit BMSCs, and provide structural evidence for osteo- genic differentiation.