中国修复重建外科杂志

中国修复重建外科杂志

载 VEGF165 多孔聚己内酯支架促进脂肪来源干细胞体内外成骨分化的实验研究

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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 7 d 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 7 d 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。

ObjectiveTo 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).MethodsThe 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.ResultsThe 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).ConclusionThe VEGF165-loaded scaffolds can significantly improve the osteogenic differentiation of ADSCs both in vitro and in vivo.

关键词: 骨组织工程; VEGF; 脂肪干细胞; 聚己内酯; 支架; 成骨分化; 大鼠

Key words: Bone tissue engineering; vascular endothelial growth factor; adipose-derived stem cells; poly (ε-caprolactone); scaffold; osteogenic differentiation; rat

引用本文: 徐万林, 卢浩, 叶金海, 杨雯君. 载 VEGF165 多孔聚己内酯支架促进脂肪来源干细胞体内外成骨分化的实验研究 . 中国修复重建外科杂志, 2018, 32(3): 270-275. doi: 10.7507/1002-1892.201710064 复制

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