丝素蛋白对可注射磷酸钙骨水泥细胞相容性的影响

Effect of silk fibroin on cell compatibility of injectable calcium phosphate cement

背景:已有的研究证实在磷酸钙骨水泥中添加适量的丝素蛋白能增加其抗压强度同时仍保持其可注射性,但制备的丝素蛋白/磷酸钙骨水泥复合材料是否仍具良好的生物相容性尚不确切。目的:将人骨髓间充质干细胞与丝素蛋白/磷酸钙骨水泥复合材料共培养,评价该材料的细胞相容性。设计、时间及地点:对比观察体外实验,于2007-09/2008-03在苏州大学附属第一医院骨科实验室完成。材料:磷酸钙骨水泥由上海瑞邦公司提供。丝素蛋白由苏州大学材料学院提供。方法:应用密度梯度离心法分离人骨髓间充质干细胞,条件培养基优化分离培养。浸提法制备丝素蛋白/磷酸钙骨水泥复合材料及磷酸钙骨水泥的浸提液。用MTT法测定丝素蛋白/磷酸钙骨水泥及磷酸钙骨水泥浸提液培养人骨髓间充质干细胞的活力,绘制生长曲线、计算第1,3,5,7天的相对增殖率并进行毒性分级;流式细胞仪检测丝素蛋白/磷酸钙骨水泥及磷酸钙骨水泥浸提液培养第4天的人骨髓间充质干细胞的细胞周期及倍体情况;扫描电镜观察丝素蛋白/磷酸钙骨水泥复合材料与人骨髓间充质干细胞共培养后第1,3,5,7天细胞的黏附及增殖情况。主要观察指标:①细胞毒性分级。②细胞周期。③细胞黏附、增殖情况。结果:丝素蛋白/磷酸钙骨水泥及磷酸钙骨水泥浸提液培养的人骨髓间充质干细胞具有良好的活力,其毒性分级均为0或1级;流式细胞仪检测见两组细胞均为二倍体细胞,未见异倍体细胞,两组G0/G1期、S期、G2/M期及细胞增殖指数差异无显著性意义(P>0.05);扫描电镜见人骨髓间充质干细胞在丝素蛋白/磷酸钙骨水泥复合材料上能黏附、生长,并随时间的推移材料表面的细胞数呈增长趋势。结论:人骨髓间充质干细胞与丝素蛋白/磷酸钙骨水泥具有良好的体外细胞相容性。

BACKGROUND： Calcium phosphate cement （CPC） added with silk fibroin （SF） can improve compressive strength. However, whether cell compatibility of injectable calcium phosphate cement reinforced by silk fibroin is not confirmed. OBJECTIVE： To evaluate the cell compatibility of the injectable CPC reinforced by SF. DESIGN, TIME AND SETTING： The control observation was performed in the Orthopaedic Laboratory of the First Affiliated Hospital of Soochow University from September 2007 to March 2008. MATERIALS： CPC was supplied by Shanghai Rebone Biomaterials Co., Ltd. SF was offered by Material Engineering Institute of Soochow University. METHODS： Human bone marrow mesenchymal stem cells （hBMSCs） were isolated by combining density gradient centrifugation with plastic adherence and cultured in specified culture medium, hBMSCs cultured with extracted liquid of SF/CPC and CPC. MTT assay was used to calculate the relative growth rate and to evaluate the cytotoxicity at days 1,3, 5, 7. Cell cycle and ploidy status was detected at 4 days by flow cytomety. The adhesion and growth of cell was assayed by scanning electron microscope at days 1, 3, 5, 7. MAIN OUTCOME MEASURES： Cell cytotoxicity, cell cycle, adhesion and proliferative ability of cells. RESULTS; hBMSCs cultured with extracted liquid of SF/CPC and CPC had excellent activities. The classification of cytotoxicity of SF/CPC and CPC were grade 0 to 1. No abnormal DNA contents were observed by flow cytomety. The G0/G1 stage, S stage, G2/M stage and proliferation index （PI） of hBMSCs cultured with extracted liquid of SF/CPC and CPC had no difference （P〉 0.05） h3MSCs seeded on SF/CPC had good adhesion and proliferative ability. CONCLUSION： The injectable calcium phosphate cement reinforced by silk fibroin has good cell compatibility in vitro.