羟基磷灰石／壳聚糖复合微球的制备及其生物学作用的体外研究

目的：通过探究羟基磷灰石（HAp）壳聚糖（CS）复合微球支架对骨髓间充质干细胞体外生物学行为的影响，评估其作为骨组织工程支架的可行性。方法纳米 HAp 和CS 复合物通过微流体技术自组装成微球支架，显微镜下形态学观察。将 P2代骨髓间充质干细胞（BMSCs）与微球行体外共培养，计算前6 h 黏附率。培养1、3、6、9 d，计算增殖率并用 GraphPad Prism 6软件对数据进行处理；行扫描电镜及共聚焦扫描式显微镜检测，观察细胞黏附及分布。将细胞微球复合物填入自制模具中培养14～21 d，进行形态观察。结果显微镜镜下微球为完整的圆形，大小一致。BMSCs 与微球体外培养6 h，黏附率达90％以上。6 d 时，BMSCs 的增殖率达到最高。扫描电镜结果显示微球上有大量 BMSCs 黏附定植并分泌大量胞外基质将微球连接成整体；共聚焦扫描式显微镜结果可见明显的细胞骨架微丝蛋白。细胞微球体外模具培养18 d 后，形成了结构完整的组织块。结论HAp-CS 微球是一种良好的促BMSCs 种子细胞黏附定植的支架材料，是促进细胞生长的有效支撑载体，与共培养细胞形成的复合组织块有望应用于体内动物实验修复标准缺损。

Fabrication of hydroxyapatite-chitosan composite microspheres and their biological effect study in vitro

Objective To investigate the influence of HAp-CS composite microsphere scaffold on the in vitro cell behaviors of mesenchymal cells and evaluate its potential application for bone tissue engineering.Methods Nano-hydroxyap-atite (HAp)and chitosan (CS)composites solution were assembled into microsphere scaffold through microfluidic and observed by inverted microscope.Rat bone marrow mesenchymal stem cells were co-cultured in vitro with the microspheres for calculating the adhesion rate for the first 6h.Proliferation rate was measured by cell counting in the next 1,3,6,9 d,respectively,and GraphPad Prism 6 software was used for statistical analysis.The morphology of BMSCs on the surface of HAp-CS composite microsphere was observed by scanning electron micros-copy (SEM)and confocal scanning microscopy.The cells and HAp-CS microspheres were filled into a disc mold and co-cultured for 14 ~21 d to observe the morphology.Results HAp-CS microspheres were observed to be round and with uniform size by microscope.The adhesion rate of BMSCs reached 80% after cultured for 6 h,and proliferation rate reached the highest value when cultured for 6 d.SEM observations showed that BMSCs adhered compactly to the surface of the microspheres,and the microspheres could be connected together through BMSCs.Af-ter co-culturing BMSCs with microspheres for 14 ~21 d,a complete tissue constructs could be formed.Conclusion HAp-CS microspheres are proved to be good scaffolds for promoting BMSCs adhesion and proliferation.Large a-mount of extracellular matrix can be formed to connect microspheres after co-cultured for a certain time,which paves the way for HAp-CS microspheres to be applied for bone regeneration in animal experiments.