丝素蛋白多孔支架与兔髓核细胞生物相容性研究  

Evaluation of the compatibility of silk fibroin porous scaffolds with rabbit nucleus pulposus cells

作　　者：曾超[1,2] 朱美峰[3] 徐宝山[2] 杨强[2] 王连永 李秀兰[4] 张杨[4] 夏群[2] 许海委 伍耀宏[1,2] 

机构地区：[1]天津医科大学研究生院,300070 [2]天津市天津医院脊柱外科,300211 [3]南开大学研究生院,天津市300071 [4]天津市天津医院骨研所细胞工程室,300211

出　　处：《中国矫形外科杂志》2013年第23期2402-2407,共6页Orthopedic Journal of China

基　　金：国家自然科学基金课题资助项目(编号:31000432;81272046);中国博士后科学基金资助项目(编号:2011M500530;2012T50235);天津市卫生局科技基金重点资助项目(编号:2013KR16);天津市卫生局攻关课题(编号:10KG113)

摘　　要：[目的]以丝素蛋白为原料制备三维多孔髓核支架,并探讨其与兔髓核细胞的生物相容性。[方法]以丝素蛋白为原料,采用蜡球致孔结合相分离技术制成三维多孔支架;从兔椎间盘中分离出髓核细胞,培养后获取P1代细胞;四甲基偶氮唑蓝(MTT)检测支架浸提液毒性;将髓核细胞以1×107/ml密度接种在丝素支架上体外培养48 h,通过HE染色、死活细胞染色(LIVE/DEAD染色)、扫描电镜观察细胞在支架上的粘附及活性。[结果]丝素蛋白多孔支架在敷水状态下呈光滑乳白色,分离的髓核细胞呈典型的软骨细胞样形态;经MTT检测支架浸提液对髓核细胞增殖无影响;HE染色和扫描电镜可见髓核细胞呈球状或短梭形均匀地贴附在支架内部。LIVE/DEAD染色显示全部为绿色荧光(活细胞),未见红色荧光(死细胞)。[结论]丝素蛋白多孔支架与兔髓核细胞具有良好的生物相容性,可以作为髓核组织工程的支架材料。[Objective] To study the compatibility of silk fibroin porous scaffold with rabbit nucleus pulposus cells.[Methods] A silk fibroin solution was preparedby using the paraffin sphere leaching method.Porous scaffolds were constructed from this solution by using a freeze- drying technique.Nucleus pulposus cells were isolated from the lumbar intervertebral disc of a rabbit,and the P1 generation cells were subsequently obtained through tissue culture.After testing the toxicity of leaching liquor from the scaffolds by using the MTT assay,the nucleus pulposus cells were seeded onto the scaffolds at a density of 1 × 107/ml and cultured for 48h in vitro.The viability and adhesion properties of the cells on the scaffolds were evaluated by using hematoxylin andeosin(HE) staining,LIVE / DEAD staining,and scanning electron microscopy.[Results] The silk fibroin porous scaffold showed smooth,transparent,and isolated nucleus pulposus cells that had typical chondrocyte- like morphology.The difference in cell proliferation among the groups,as demonstrated by using the MTT assay,was not statistically significant(P>0.05).Under a scanning electron microscope,the cells showed spherical or short- spindle morphology and were attached to the scaffolds.A similar observation was detected through HE staining.The nucleus pulposus cells on the scaffolds secreted proteoglycan,which was easily observed by toluidine blue staining.All the cells showed green fluorescence(indicating live cells) after staining with LIVE / DEAD dye;none of the cells showed red fluorescence(indicating dead cells).[Conclusion] The silk fibroin porous scaffold can be used as nucleus pulposus tissue because it shows good cell compatibility with rabbit nucleus pulposus cells.

关 键 词：丝素蛋白 髓核细胞 生物相容性 

分 类 号：R318.08[医药卫生—生物医学工程]