Effects Of Composite Electrostatic Nano-spinning Materials On The Growth And Maturation Of Ventricular Myocytes

Objective:Cardiac tissue engineering(CTE)is a promising strategy for the development of functional extracellular matrix(ECM)-like scaffolds for the treatment of cardiovascular diseases such as myocardial infarction.The aim of this study was to develop a novel therapeutic hybrid stent that combines electrical,mechanical and biological properties and is an ideal material for promoting the development and maturation of heart tissue.Different concentrations of go/gold nanoparticles composite electrostatic spinning nanofiber materials were prepared.The effects of in vitro culture on the adhesion and proliferation of neonatal rat ventricular myocytes were observed.Then the effects of GO /Au NPs composite electrospun nanofibers on the growth and maturation of NRVM were investigated,providing a basis for further application in cardiac tissue engineering.Methods:1)GO/Au NPs composite electrostatic nanofibers with different GO concentrations were constructed.The microstructure of the materials was characterized at different levels using SEM and TEM,and the conductivity of the materials was measured using a conductivity meter;2)Live/dead and cck-8 experiments were used to evaluate the biocompatibility and toxicity of GO/Au NPs composite electrostatic nano-spinning fibers to NRVM,so as to select the optimal concentration suitable for cell adhesion growth and development.F-actin staining was used to observe the different cytoskeleton morphology of each group,and the cell morphology and extension of each group were observed under the environment scanning electron microscope.3)the expression of disk-related proteins in each group and the functional maturity of NRVM were detected by laser confocal immunofluorescence and Western blotting at the level of morphology and molecular biology.Results :(1)Environmental scanning electron microscopy(ESEM)showed that there were no significant differences in the size and pore size of the electrostatic nanofibers prepared from different concentrations of graphene oxide polycaprolactam polymers.Transmission electron microscopy(TEM)showed that go could be dispersed well in nanofibers at lower concentration.The conductivity test results showed that the conductivity of the composites increased with the increase of the go concentration.The results of Live/dead and cck-8experiments showed that the 0.05 mg /ml GO/Au NPs composite electrospun nano-fiber materials had good biocompatibility,low cytotoxicity,significantly enhanced cell metabolic activity.F-actin staining showed 0.05 mg/ml GO/Au NPs group cell adhesion number more than the control way of collagen,the spreading area of the collagen in the control group,the ratio of the cells and the length of the shaft and the col group increased,the GO/Au NPs electrostatic nanometer fiber composite materials stretch cells showed similar natural myocardial cell of long spindle,stretching,and col group NRVM configuration spherical,and there are gathered phenomenon.The morphology and elongation of the cells in the 0.05mg/ml GO/Au NP group were better than those in the control group.The cells in the material group showed long fusiform extension similar to that of cardiomyocytes,while the cells in the control group were mostly spherical with poor polarity.B)laser confocal immunofluorescence staining showed that the range of cx-43 and NC proteins at each time point in the 0.05mg/ml GO/Au NPs composite electrostatic nanospinning fiber group was larger than that in the collagen group.With the increase of culture time,cx-43 and NC proteins showed linear distribution between cells.Western blotting showed that NC protein was less expressed in the GO/Au NPs group and Col group on day 3,and no significant difference was observed.On the 7th day,the NC protein expression in the GO/Au NPs group was higher than that in the collagen group,with P value less than 0.05,which was statistically significant.The expression of cx-43 protein was higher in the GO/Au NPs group than in the collagen group at all time points,and the difference was statistically significant(p<0.05).Conclusion :1)The conductivity of the composites was enhanced by go and the surface mechanical properties of Au NPs-Col were changed by a simple synthesis method.Moreover,the combination of electrospinning fibers and collagen can better simulate the structure of the extracellular matrix of cardiac myocytes in vivo,so as to promote the expression of bundle like extension of cardiac myocytes in vivo.2)GO/Au NPs electrospun nanofibers can promote the expression of NRVM leap disc related proteins(cx-43,NC).Thus promoting the assembly of intermyocardial leap disc(IDs)and the maturation of cell function.