载VEGF/万古霉素的多层缓释微球对人胎盘源间充质干细胞增殖与成骨分化的影响

摘要

目的:观察载血管内皮生长因子(VEGF)/万古霉素的多层海藻酸盐壳聚糖缓释微球对人胎盘源间充质干细胞(HPMSCs)增殖与成骨分化的影响,为其在组织工程修复骨缺损中的临床应用提供理论基础.方法:根据前期基础制备缓释微球,将从人胎盘组织中分离培养的一部分HPMSCs分为载药微球组(载药微球+HPMSCs)、空载微球组(空载微球+HPMSCs)和无球组(仅HPMSCs).采用CCK-8试剂盒检测HPMSCs增殖率.取另一部分HPMSCs分为载药微球诱导组(载药微球+HPMSCs+成骨诱导液)、空载微球诱导组(空载微球+HPMSCs+成骨诱导液)、无球诱导组(HPMSCs+成骨诱导液)和非诱导组(HPMSCs+PBS).孵育21 d后分别采用茜素红染色及碱性磷酸酶(ALP)试剂盒检测HPMSCs钙盐沉积与ALP活性.%Objective:To investigate the influence of multilayer alginate chitosan sustained-release microspheres loading vascular endothelial growth factor(VEGF) and vancomycin in the proliferation and osteogenic differentiation of human plaacenta-derived mesen chymal stem cells(HPMSCs),and to provide theoretical basis for its clinical application in the repair of bone defect.Methods:The microspheres were prepared based on the previous research and HPMSCs were co-cultured with drug (VEGF/vancomycin)-loaded microspheres (drug-loaded microspheres+HPMSCs group), non-drug loaded microspheres (microspheres+HPMSCs group) and without any microspheres (HPMSCs group).Then the proliferation rate of HPMSCs was identified by CCK-8 kit.The osteogenic differentiation potential of HPMSCs was detected by Alizarin red staining and alkaline phosphatase (ALP) kit when the HPMSCs had been co-cultured with drug loaded microspheres in osteogenic medium (HPMSCs+drug-loaded microspheres+induction group), non-drug loaded microspheres in osteogenic medium (HPMSCs+microspheres+induction group), without any microspheres in osteogenic medium (HPMSCs+induction group) and without any micropheres in normal medium (HPMSCs+PBS group) for 21 d.Results:Compared with HPMSCs group,the proliferation rates of HPMSCs in drug-loaded microspheres+HPMSCs and microspheres+HPMSCs groups had no significant changes (P>0.05).The calcium deposition in HPMSCs+drug-loaded microspheres+induction group was more than those in microspheress+HPMSCs+indution group and HPMSCs+induction group after Aalizarin red staining;the ALP activity in drug-loaded microspheres+HPMSCs+indution group was higher than those in microspheres+HPMSCs+indution group and HPMSCs+induction group (P<0.05),and the ALP activity in microspheres+HPMSCs+induction group was higher than that in HPMSCs+induction group(P<0.05).Conclusion:The sustained-release microspheres loading VEGF and vancomycin have no significant effect on the proliferation activity of HPMSCs and the microspheres could stimulate the osteogenic differentiation of HPMSCs.