EDC-NHS交联的骨骼肌脱细胞支架对心肌细胞生物功能性的影响

Effects of EDC-NHS cross-linked skeletal muscle decellularized scaffolds on the biological function of cardiomyocytes

目的探讨利用骨骼肌脱细胞支架制备工程化心肌补片的方法,并评价其生物功能性。方法取大鼠腹直肌进行脱细胞后进行EDC和NHS交联得到工程化骨骼肌脱细胞支架;观察脱细胞情况、微观结构和测定力学性能;通过体外细胞实验和qRT⁃PCR实验等评估骨骼肌脱细胞支架的生物相容性、心肌细胞功能化及内皮细胞血管化功能。结果当EDC浓度为0.5 mol/L时,骨骼肌脱细胞支架弹性模量最适中;各组骨骼肌脱细胞支架均含VEGF,体外细胞实验中0.5 mol/L组的活细胞率最高,细胞骨架伸展较好,种植原代心肌细胞后可见细胞间肌节生成;在种植人脐静脉血管内皮细胞后可见血管生成,并通过qRT⁃PCR实验验证血管特定VEGF和eNOS蛋白。结论在交联剂EDC 0.5 mol/L时的骨骼肌脱细胞支架具有良好的力学性能,并可促进原代心肌细胞成熟和功能化,促进人脐静脉血管内皮细胞血管化。

Objective To investigate the method of preparing engineered myocardial patch using skeletal muscle decellularized scaffold,and to evaluate its biological function. Methods Rat rectus abdominis was decellularized and then cross-linked with EDC and NHS to obtain skeletal muscle decellularized scaffolds. Decellularization and microstructure observed while mechanical properties were determined. Biocompatibility,cardiomyocyte functionalization,and endothelial cell vascularization in the skeletal muscle decellularized scaffolds were evaluated by in vitro cell assay and q RT-PCR. Results As the EDC concentration was 0.5 mol/L,the most appropriate elastic modulus was found in the skeletal muscle decellularized scaffolds. The skeletal muscle decellularized scaffolds contained VEGF in each group,and the 0.5 mol/L group had the highest viable cell rate and better cytoskeleton extension in the in vitro cell assay,and the formation of intercellular sarcomere was observed after implantation of primary cardiomyocytes. Angiogenesis was seen after implantation of human umbilical vein endothelial cells,and vascular-specific VEGF and e NOS proteins were verified by qRT-PCR. Conclusions The skeletal muscle decellularized scaffolds with cross-linking agent EDC 0.5 mol/L have better mechanical properties. They can accelerate maturation and functionalization of primary cardiomyocytes,and vascularization of human umbilical vein endothelial cells.