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目的 探讨载VEGF165多孔聚己内酯[poly (ε-caprolactone),PCL]支架对脂肪来源干细胞(adipose-derived stem cells,ADSCs)成骨分化的影响.方法 采用溶剂置换法、粒子浸出法及热致相分离法制备载VEGF165多孔PCL支架(记作Sf-g/VEGF),扫描电镜观察其形貌、测定药物释放率.取15只SD大鼠腹股沟脂肪,分离培养ADSCs并传代.取第3～4代细胞复合至Sf-g/VEGF,体外培养7d后行茜素红染色、茜素红活性测试及实时荧光定量PCR检测体外成骨效果;以明胶修饰的多孔PCL支架(记作Sf-g)与ADSCs复合培养作为对照.取SPF级SD大鼠6只,于颅骨左、右侧各制备一直径为5 mm缺损,将其随机分为3组(n=4);阴性对照组不作任何处理,Sf-g组植入细胞-Sf-g支架复合体,Sf-g/VEGF组植入细胞-Sf-g/VEGF支架复合体.8周后取出含细胞-支架复合体的颅骨分别行Micro-CT扫描及HE染色,评估体内成骨效果.结果 扫描电镜示Sf-g/VEGF支架呈多孔结构,VEGF释放曲线呈二阶段释放,120 h累积释放率为80％;提示成功制备Sf-g/VEGF.体外培养7d后茜素红染色检查示Sf-g/VEGF组茜素红活性显著高于Sf-g组(t=10.761,P=0.000).实时荧光定量PCR检测,Sf-g/VEGF组成骨特异性指标特异AT序列结合蛋白2(special AT-rich sequence protein 2,Satb2)、ALP、骨钙素(osteocalcin,OCN)和骨桥蛋白(osteopontin,OPN) mRNA相对表达量均较Sf-g组升高(P＜0.05).体内植入后8周Micro-CT扫描及组织学观察显示,Sf-g组及Sf-g/VEGF组均可见骨缺损部分修复,且Sf-g/VEGF组更显著;Sf-g/VEGF组新生骨组织体积及面积均显著优于Sf-g组(P＜0.05).结论 载VEGF165多孔PCL支架可显著提高ADSCs的体内、外成骨分化效果.“,”Objective To explore the effect of vascular endothelial growth factor 165 (VEGF165)-loaded porous poly (ε-caprolactone) (PCL) scaffolds on the osteogenic differentiation of adipose-derived stem cells (ADSCs).Methods The VEGF165-loaded porous PCL scaffolds (written,Sf-g/VEGF) were fabricated through a combination of solvent casting/salt leaching and a thermal-induced phase separation technique and then observed under scanning electron microscope (SEM).The release kinetics was determined by ELISA kit.The ADSCs were isolated from inguinal fat pads of 15 Sprague Dawley rats and cultured.The passage 3-4 ADSCs were seeded into the scaffolds,and then cultured in vitro for 7 days.The passage 3-4 ADSCs were seeded into the porous PCL scaffolds (written,Sf-g) as control.The alizarin red S (ARS) staining,ARS activity assay,and real-time quantitative PCR (RT-PCR) were performed to measure the osteogenic differentiation of ADSCs in vitro.Six Sprague Dawley rats were recruited to prepare the bilateral calvarial bone defects models (n=12).The 12 calvarial bone defects were randomly divided into 3 group (n=4).The defects of negative control group were not treated;the defects of Sf-g group and Sf-g/VEGF group were repaired with ADSCs-Sf-g scaffold complex and ADSCs-Sf-g scaffold complex,respectively.At 8 weeks after transplantation,the Micro-CT and HE staining were conducted to evaluate the osteogenic effects in vivo.Results The morphology of the Sf-g/VEGF scaffolds were porous and well-connected,and the cumulative release rate was approximately 80％ in 120 hours.The ARS staining showed that the ARS activity of Sf-g/VEGF group were stronger than that of Sf-g group (t=10.761,P=0.000).The mRNA expressions of osteogenic specific markers [special AT-rich sequence protein 2 (Satb2),alkaline phosphatase (ALP),osteocalcin (OCN),and osteopontin (OPN)] were significantly higher in Sf-g/VEGF group than in Sf-g group (P＜0.05).The results of Micro-CT and HE staining also confirmed the promotion effect of Sf-g/VEGF scaffolds.All defects of 2 groups were partially repaired by new bone tissue,especially in Sf-g/VEGF group.The volume and area of new bone tissue were significantly higher in Sf-g/VEGF group than in Sf-g group (P＜0.05).Conclusion The VEGF165-loaded scaffolds can significantly improve the osteogenic differentiation of ADSCs both in vitro and in vivo.