| Background:Cardiovascular disease is the most important cause of death in the world and acute myocardial infarction is the first killer.In recent years,the number of myocardial infarction patients in China has obviously shown an increasing trend year by year.Clinical traditional methods for the treatment of myocardial infarction include thrombolytic drug therapy,interventional therapy,coronary artery bypass surgery,and heart transplantation.Although these methods have a certain repair effect,their wide application is limited due to the limited regeneration ability of myocardial cells,long repair cycle of drugs and surgery,and lack of sources of heart transplantation.The recovery of myocardial cells after myocardial infarction is limited,which makes the damaged part of myocardial tissue difficult to recover.After the continuous development of social medical level and the improvement of the hospital chest pain center emergency system,the direct mortality rate after myocardial infarction has decreased,but the mortality rate is still high.Objective:To explore a simple method for preparing engineered myocardial patch by using skeletal muscle decellularized scaffolds obtained by cross-linking with EDC-NHS after mild decellularization of rat rectus abdominis,and to evaluate its biocompatibility,cardiomyocyte functionalization and performance in vitro.Vascularization of endothelial cells.Methods:The rectus abdominis muscle of rats were placed in-80℃ for repeated freezing and thawing three times,and then immersed in 10%alkyl glycoside solution at room temperature for 12 to 24 hours,decellularize with Mgcl2 solution containing DNANase and RNANase at 37℃ for 24 to 48 hours to obtain basic skeletal muscle decellularized scaffolds;carry out different concentrations of 1-(3-dimethylaminopropyl)-3-ethylcarbon Diimide hydrochloride(EDC)and N-hydroxysuccinimide(NHS)were cross-linked at 4℃ for 12 hours to obtain the desired engineered myocardial patch;skeletal muscle was observed by HE staining,MASSON staining and DAPI staining The decellularization of the decellularized scaffolds;the microstructure results of the skeletal muscle decellularized scaffolds were observed by scanning electron microscopy;the tensile force and strain of the skeletal muscle decellularized scaffolds under different EDC and NHS cross-linking were measured by a mechanical stretching machine.Mechanical properties;cultured H9C2 cells,primary cardiomyocytes and human umbilical vein endothelial cells and inoculated the cells on the scaffold material,detected their viable cell rate by Live&Dead kit,observed cytoskeleton,cell sarcomere,and cell blood vessels by immunohistochemical staining The biocompatibility,cardiomyocyte functionalization and endothelial cell vascularization of the skeletal muscle decellularized scaffolds were evaluated by the generation and RT-qPCR experiments to determine the VEGF protein and eNOS protein.RESULTS:After decellularization,no residual cells were remained in the decellularized matrix.After cross-linking with two chemical solutions of EDC and NHS,when EDC concentration was 0.5M,the calculated elastic modulus of skeletal muscle decellularized scaffold mechanics was the most moderate,in the range of elastic modulus of human myocardial tissue;The skeletal muscle decellularized scaffolds in the Lianhe 0.5M group contained more VEGF.In vitro experiments by planting H9C2 cells,it was found that the 0.5M group had the highest average live cell rate and live cell area,and the cytoskeleton was well stretched and some sarcomere structures were visible.The formation of intercellular sarcomere was observed after implantation of primary cardiomyocytes;angiogenesis was observed after implantation of human umbilical vein endothelial cells,and vascular-specific VEGF and eNOS proteins were verified by RT-qPCR experiments,which were significantly increased compared with the climbing film group.Conclusion:The skeletal muscle decellularized scaffolds with cross-linking agent EDC 0.5M have good mechanical properties and microscopic network structure,and can promote the maturation and functionalization of primary cardiomyocytes,and promote the vascularization of human umbilical vein endothelial cells. |
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