脐带血间充质干细胞的成骨诱导及与β-磷酸3钙的生物相容性

目的探讨脐带血间充质干细胞(umbilical cord blood-derived mesenchymal stem cells,UCB-MSCs)的成骨诱导及与β-磷酸3钙(β-tricalcium phosphate,β-TCP)的生物相容性。方法在无菌条件下抽取人脐血,用密度梯度离心的方法获得脐血单个核细胞,接种到含10%胎牛血清的DMEM/F12培养基中。单个核细胞行贴壁培养后,进行细胞形态学观察,绘制细胞生长曲线,分析细胞周期,检测细胞表面抗原;应用条件培养基进行成骨诱导后,应用免疫组织化学方法检测碱性磷酸酶(alkaline phosphatase,AKP)的表达,应用放免方法分析骨钙素(osteocalcin,OCN)的含量、荧光法检测细胞内钙离子的含量;与β-TCP复合培养后,用扫描电镜检测成骨诱导后的UCB-MSCs与β-TCP的生物相容性。结果采用Percoll(1.073g/ml)分离的脐血间充质干细胞大小较为均匀,梭形或星形的成纤维细胞样细胞。细胞生长曲线测定表明接种后第5d细胞进入指数增生期,至第9d后数量减少;流式细胞检测表明50%～70%细胞为CD29、CD45和CD105阳性;成骨诱导后,UCB-MSCs内AKP、培养基中的OCN含量和细胞内钙离子的含量均明显高于对照组(p0.01);成骨诱导的细胞在β-TCP表面生长良好,细胞周围有白色的钙盐沉积。结论 UCB-MSCs成骨诱导后具有骨细胞特性,与β-TCP具有良好的生物相容性,可作为骨组织工程的种子细胞。

Biocompatibility of bony induced umbilical cord blood-derived mesenchymal stem cells with tricalcium phosphate in vitro

Objective To explore the biocompatibility of umbilical cord blood-derived mesenchymal stem cells（UCB-MSCs） induced by bony conditioned medium with the β-tricalcium phosphate（β-TCP）. Methods Human umbilical cord blood was collected in sterile condition, single nuclear cells were isolated by density gradient centrifugation and cultured in DMEM/F12 medium containing 10% fetal bovin serum. The shape of the cells were watched by microscope, the cell growth curve, the cell cycle and the cell surface antigens were determined by immunocytochemistry and flow cytometry methods. The expression of AKP was detected by immunohistochemical method, the concentration of Ca2＋ within the UCB-MSCs plasm was determined by fluorescent method and concentration of OCN in the medium were detected by radioactive immunizing method. The biocompatibility of induced-UCB-MSCs with β -TCP was observed by scanning electron microscope after induced-UCB-MSCs transplanted into the β -TCP. Results MSCs obtained by Percoll（1.073g/ml） were similar in size, spindle-shaped or star-shaped fibroblasts-liked cells. Cell growth curve analysis indicated that MSCs were in the exponential stage 5d after culture and in the stationary stages 9d after culture. Flow cytometry analysis showed that the CD29, CD44 and CD105 positive cells were about 50%～70%. Compared with the control group, AKP and Ca^2＋ contents in the medium were higher in UCB-MSCs and OCN than in the control cells （P〈0.01）. UCB-MSCs grew well on the surface of the β-TCP and white calcium particle was observed around the induced-UCBMSCs. Conclusion Induced UCB-MSCs have bony characteristics and satisfactory biocompatibility with the TCP in vitro,could be used as the seed-cells in bone tissue engineering.