Study On The Preparation Of Carbon Fiber Reinforced Polyvinyl Alcohol Conductive Hydrogel And The Construction Of Engineered Myocardial Tissue

Objective:Myocardial injury caused by myocardial infarction,chronic cardiovascular disease can lead to the formation of fibrous scar tissue,affecting the hearth contraction and pumping function.Engineered cardiac tissue based on seed cells and biomaterials may offer an alternative promising strategy for myocardium repair and drug screening in vitro.Scaffold materials play a key role in the construction of engineered cardiac tissue,which provide cells with appropriate extracellular matrix,mechanics and electrical microenvironment to promote cell differentiation and maturation.’The natural myocardium is an electroactive tissue in which Purkinje fibers with electrical conduction are distributed;when the electrical impulses originating from the sinoatrial node,transmitted throughout the myocardium,induced synchronous contraction of the cardiac cells by "excitation-contraction" coupling.At the same time,the natural myocardium is of strength to withstand the continuous stretching/relaxing motion of the myocardium that occurs during each heartbeat.Therefore,the scaffold used for the construction of engineered cardiac tissue should have both excellent electrical and mechanical properties.Hydrogels hold advantageous features for cardiac tissue constructions,including high water content,excellent biocompatibility,structural similarity to native ECM,and controllable biochemical and biophysical properties.It has been widely used in the research of cardiac tissue engineering.However,hydrogels are usually electrically insulating materials,which hinder the efficient transmission of electrical signals between cells.In addition,the hydrogels generally have a low modulus and are not ideal for supporting cardiomyocyte contraction.In the study,we simulated the distribution and electrical conduction of Purkinje fibers in natural myocardial tissue,combining carbon fiber(CF)that is both highly conductive and of high modulus with biocompatible polyvinyl alcohol(PVA)to construct a carbon fiber reinforced polyvinyl alcohol conductive hydrogel(PVA/CFs),in which CFs were expected to bring both conductivity and stiffness to the hydrogels.The effects of PVA/CFs on cardiomyocyte proliferation,maturation and electrophysiological function were investigated,and the responses of cardiomyocyte-hydrogel construct(NRCM@PVA)/CFs)to drugs were evaluated as well.In addition,interactions of PVA/CFs to vascular endothelial cells and macrophages were examined on the endothelial junction integrity and inflammatory reactions respectively.This research was aimed to provide a new kind of scaffold for myocardial defect repair and for quick screening of cardiovascular system drugs.Methods:Carbon fiber reinforced polyvinyl alcohol conductive hydrogels(PVA/CFs)with different content of carbon fiber were prepared by cyclic freeze-thawing method;microstructure was observed by scanning electron microscope,electrical conductivity was measured by four-probe test method,the elastic modulus was measured by electronic universal testing machine,the swelling ratio and degradation degree were determined by gravimetric method.The growth and proliferation of cardiomyocytes,human umbilical vein endothelial cells(HUVEC)and macrophages(RAW264.7)on PVA/CFs were detected by CCK-8 kit(Cell Counting Kit-8);The viability of NRCMs was determined using LIVE/DEAD Cell Imaging Kit.The cell morphology of cardiomyocytes and HUVEC on different hydrogels,the expression of cardiac-specific contractile proteins a-actinin,Cx43 and cTnT,and expression of the protein VE-Cadherin of HUVEC were observed by immunofluorescence staining and laser scanning confocal microscopy.Western Blot experiments were selected to further semi-quantitatively analyze the expression of a-actinin,cTnT,Cx43 and VE-cadherin7and the effect on mechanotransduction pathway of a5pl-integrin/ILBC/P-AKT.The transient intracellular Ca2+concentration in cardiomyocytes during spontaneous contraction and drug treatment were measured using a fluorescent calcium indicator(Fluo 4-AM)and Real-Time live confocal fluorescence microscopy.The expression of TNF-α,IL-1β and IL-6 in inflammatory response genes of macrophages(RAW264.7)was detected by Real Time PCR.The secretion of cytokins such as TNF-α,IL-1β and IL-6 were tested by ELISA kits.Results:(1)Carbon fiber reinforced polyvinyl alcohol conductive hydrogels(PVA/CFs)were prepared by cyclic freeze-thawing methods.The carbon fibers homogeneously distributed in the PVA hydrogel,and there were numerous interconnecting pores on the surface of PVA/CFs,similar to the microstructure of the natural extracellular matrix;PVA/CFs with high swelling ratio,which is beneficial to the diffusion of nutrients and oxygen in cardiomyocytes;the conductivity of PVA/CFs was between 0.1-0.4 S/m,which was significantly higher than that of PVA,and close to the electrical conductivity of myocardial tissue(0.03-0.6 S/m);in addition,elastic modulus of PVA/CFs was close to the modulus of active myocardium during systole.(2)PVA/CFs supported the growth and proliferation of neonatal rat cardiomyocytes(NRCMs),rat cardiomyocyte cell line(H9c2),human umbilical vein endothelial cells(HUVEC),and mouse macrophage(RAW 264.7);promoted the distribution and orientation of the cytoskeleton proteins,and enhanced the maturation of cardiomyocytes;HUVECs cultured on PVA/CFs exhibited a characteristic "cobblestone" morphology,and the expression of VE-cadherin was more intact.CFs addition did not affect expression of TNF-α,IL-1β and IL-6 in RAW 264.7 both in genes and cytokines levels.(3)PVA/CFs remarkably enhanced the maturation of neonatal rat cardiomyocytes(NRCMs)in vitro by upregulating the expression of α-actinin,troponin T,and connexin-43,and enhanced the electrophysiological function of neonatal rat cardiomyocytes(NRCMs).The spontaneous beating rate of NRCMs on PVA/CFs hydrogel was significantly faster than that of PVA,showing much more synchronous,stable,and robust beating behavior.When exposed to a cardiotoxic molecule doxorubicin or isoprenaline speeding up heart contraction,the engineered cell-hydrogel constructs(NRCM@PVA/CFs)could retain effective beating for much longer time or change the contractile rate much faster than NRCM@PVA.(4)The mechanistic study indicated that CFs activated a5pl integrin-mediated a5pl-integrin/ILK/p-AKT mechanotransduction pathway and up-regulated the level of Rho family small GTPase RhoA and hypoxia-inducible factor(HIF-la).Conclusion:Carbon fiber reinforced polyvinyl alcohol conductive hydrogel(PVA/CFs)remarkably enhanced the maturation and electrophysiological performance of neonatal rat cardiomyocytes in vitro by activating α5β1 integrin-mediated mechanotransduction pathway,and increasing the level of RhoA and hypoxia-inducible factor-1α.The resulting cardiomyocytes-conductive hydrogel constructs(NRCM@PVA/CFs)had a good drug response function to cardiotoxic or accelerated heart contraction drugs.Meanwhile,the scaffold supported the growth of vascular endothelial cells and macrophages,and benefited the adherens junction to the endothelial cells as well as inhibited the inflammatory reactions to the macrophages.