自固化磷酸钙支架接种成骨细胞后的灌注性三维动态培养  

Flow perfusion culture of osteoblasts seeded on calcium phosphate cement scaffolds with controlled internal channel architecture

作　　者：王林[1] 王臻[1] 李祥[2] 李涤尘[2] 许宋锋[1] 卢秉恒[2] 

机构地区：[1]第四军医大学西京医院全军骨科研究所,西安710032 [2]西安交通大学机械制造系统工程国家重点实验室

出　　处：《中华创伤骨科杂志》2007年第4期357-361,共5页Chinese Journal of Orthopaedic Trauma

基　　金：国家自然科学基金资助（50235020）

摘　　要：目的探讨带有规则管道结构的自固化磷酸钙（CPC）支架作为体外成骨细胞灌注培养载体的可行性，以及规则管道结构在培养中的作用。方法利用计算机辅助设计及快速成形技术制备带有规则管道结构的CPC圆柱体支架。分离、培养的兔颅骨成骨细胞接种支架后分别行静态和灌注性三维动态培养，3、7、14d后分别利用激光共聚焦扫描显微镜、扫描电镜观察，四甲基偶氮唑蓝比色、碱性磷酸酶（ALP）测定，x射线能谱分析检测细胞的流体剪应力应答、在支架内的聚集及分布、增殖活力和成骨分化情况。结果灌注培养3d细胞内微丝及DNA表达增强，与静态培养差异有统计学意义（P〈0．05）；灌注培养14d细胞均匀分布在支架表面和内部管道内，而静态培养细胞仅分布在支架表面；灌注培养细胞数量、ALP含量显著高于静态培养，差异有统计学意义（P〈0．05）。结论规则管道结构能调节支架内灌注液流，从而使营养供应及流体剪应力均匀化，促进细胞增殖、分化及在整个支架内的均匀分布，该支架可作为深入研究支架内液流调配的研究模型。Objective To investigate the feasibility of culturing osteoblasts on calcium phosphate cement （CPC） scaffolds with controlled internal channel architecture in a flow perfusion bioreactor, and the effects of channels on fluid flow configuration inside the scaffolds. Methods The serf-hardening CPC scaffolds with controlled internal channel architecture were designed and fabricated using computer aided design and indirect rapid prototyping techniques. A flow perfusion bioreactor was developed in our lab. Osteoblasts were isolated from rabbit skulls, seeded onto CPC scaffolds, and cultured for up to 14 days in static or flow perfusion conditions. Effects of fluid shear stress on the cells were determined by confocal laser scanning microscopy and image analysis. 3 D cell organization and distribution throughout the scaffolds were observed by scanning electron microscopy. Proliferation of osteoblasts was determined by MTT assay and osteoblastic differentiation was assessed by alkaline phosphate （ALP） activity assay. Results Fluid flow enhanced the expressions of F-actin and DNA in cells compared with static culture. At all culture times, flow perfused constructs demonstrated greater proliferation and osteoblastic differentiation than statically cultured constructs as evidenced by MTT and ALP assay. The cells were distributed throughout the entire scaffold by 14 days of flow perfusion culture whereas they were located only along the scaffold perimeter in static culture. Condusions The controlled channel architecture allows well-defined fluid flow configu- ration which benefits cell proliferation, differentiation, and distribution by enhancing uniform supply of oxygen and medium components and fluid shear stress inside the scaffold. Furthermore, fluid flow inside the scaffold can be studied in detail in the future.

关 键 词：成骨细胞 支架 灌注 局部 生物反应器 规则管道 

分 类 号：R686[医药卫生—骨科学]