包载血管内皮生长因子的乙二胺/聚己内酯微囊的制备及其生物活性的研究

Study on the preparation and biological activity of polycaprolactone microcapsule scaffoldmodified with ethylenediamine encapsulating VEGF

目的采用乳化溶剂挥发法制备一种新型的包载血管内皮生长因子(vascular endothelial growth factor,VEGF)的经过乙二胺(ethylenediamine,ED)修饰的聚己内酯(polycaprolactoe,PCL)微囊支架(VEGF/ED-PCL),测试其表征,并研究其对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)的粘附、增殖和成血管作用。方法运用乳化溶剂挥发法制备PCL微囊,以激光粒度分析仪测试其粒度分布;通过二氯甲烷蒸汽熏蒸微囊,扫描电镜扫描支架表面形态,选取融合度最适宜的支架;体外释放实验测试VEGF微囊的体外释放性能;使用乙二胺浸泡法修饰PCL微囊支架,利用接触角测试仪测试其亲水性;将HUVEC细胞接种于微囊支架上,扫描电镜观察细胞在支架上的形态,并通过CCK-8实验测试实验组和对照组细胞生长情况;同时将实验组和对照组支架植入小鼠皮下,2周后处死小鼠,取得支架周围组织,行免疫组化染色,光学显微镜下比较两组微血管形成数目。结果成功制备VEGF/ED-PCL微囊支架;VEGF/ED-PCL微囊支架细胞增殖高于VEGF/PCL微囊支架组、PCL微囊支架组;植入VEGF/ED-PCL微囊支架的小鼠皮下微血管生成高于PCL支架组,结果具有统计学差异(P<0.05)。结论通过制备乙二胺修饰的包载VEGF的PCL微囊支架有很好的体外缓释性能,表面适宜成血管细胞粘附及增殖,在小鼠体内促进血管的形成。

Objective To prepare a novel Polycaprolactoe （PCL） microcapsule scaffold encapsulating vascular endothelial growth factor（VEGF） modified with aethylenediamine for bone tissue engineering, to characterize the scaffold, and to detect its influence on the proliferation and adhesion of Human Umbilical Vein Endothelial Cells （HUVEC） and neovascularization. Methods PCL mierocapsules were prepared by W/O/W method and the size distribution was analyzed by laser particle size analyzer. Then dichlo- romethane vapor was used to suffocate PCL microeapsules. Further sintering helped to create a three-dimensional microspheres scaffold, then modified with aethylenediamine to improve its hydrophilicity. The structure of mierospheres scaffold was examined by scanning e-lectron microscopy, and the most suitable fusion of microcapsules was chosen. Hemangioblast cell line HUVEC was chosen to seed on the scaffolds,and its adhesive status was observed by SEM.The ELISA method was used to establish sustained-release profile in vitro. CCK-8 kit was used to analyze the cell proliferation of the groups with scaffolds and to compare with the group without VEGF scaffolds. The VEGF/ED-PCL scaffolds were implanted subcutaneously in mice for 2 weeks. After 2 weeks, the vaseularization within scaffolds loaded with VEGF or scaffolds without VEGF was observed. Results Polycaprolaetoe mieroeapsule scaffolds modified with aethylenedi- amine encapsulating VEGF were successfully prepared,and SEM showed that PCL scaffolds had porous structure with porosity.The in vitro study demonstrated that excellent cellular attachment was throughout the polyeaprolactoe microcapsule scaffolds modified with aeth- ylenediamine.The in vitro and in vivo studies demonstrated that excellent cellular attachment was throughout the microspheres scaffolds. It stimulated the angiogenie potential of HUVEC. Conclusion According to the above results, it is demonstrated that the PCL micro-capsule scaffold has made contribution to highlighting the efficacy of cell adhesion and proliferation. It is also demonstrated that sustained release of VEGF from microsphere has extended angiogenie stimulation capacity.