| BackgroundCoronary heart disease is the leading cause of the rising incidence of heart failure worldwide[1].Following MI,the limited regenerative potential of the heart causes scar formation in and around the infarcted region,leading to abnormal electric signal propagation and desynchronized cardiac contraction[2].The lack of electric connection between healthy myocardium and the scar sites results in most of progressive functional decompensation[2,3].The engineered electrical cardiac patches(ECPs)have proven to be a promising alternative biological treatment after MI to create functional cardiac syncytium,replace dysfunctional parts of damaged myocardial tissue,reduce adverse remodeling and preserve cardiac function[4-6].The prerequisites for successful myocardial repair of the implanted ECPs include electrically coupling with host tissue,triggering propagation of electrical impulses throughout the heart,participating in the synchronous contraction of the whole heart,and inducing cardiomyocytes into a functional syncytium[6-10]The electrically active scaffold is beneficial to improve cardiomyocyte function by increasing connexin 43(CX43)expression[11],which help to regulate cell-cell communication,to increase electrical coupling,and to promote contractile behavior[12].Conducting polymers such as polypyrrole(pPy)[13],carbon nano tube(CNT)[14],polyaniline(PANi)[15]and poly(3,4-ethylenedioxythiophene)(PEDOT)[16],exhibit many excellent properties such as biocompatibility,conductivity and redox stability.Among these,pPy is one of the most extensively studied conducting polymer in the application of tissue engineering scaffolds due to its easy preparation,better biocompatibility,inherent electrical conductivity,controllability of surface biochemical properties and suitable hydrophobicity for cell adhesion[17,18].It has been demonstrated that pPy could sustain the electrophysiological maturation and functionality of cardiomyocytes with less cytotoxicity and its conductivity could also guide the regular beating of heart,which are hardly achieved by utilizing the conventional nonconductive polymeric biomaterials[18,19].These promising results indicate the potential application of pPy for cardiac regeneration due to its possibility of electrical signal transmission.However,the weak mechanical property of pPy limits its application in cardiac tissue engineering(CTE).To overcome this shortcoming,conductive pPy used to be hybridized with other biomaterials to construct ECPs with stronger contractile and electrical properties[20].In view of biomimetics,the main purpose is to create cell-compatible or tissue-favored environments for various damaged tissues,and an optimal choice is to utilize the underlying properties of natural biological systems[21].The architectures at submicrometer scale are abundant in native ECMs and play a key role in regulating cellular behavior.Decellularization of the native omentum[22]or the whole heart could be taken as the native tissues scaffolds for CTE[22,23],but source of these native tissues is rare.Inspired by marine creatures like mussels,a series of ideal materials have been developed,such as extracellular matrix substrates[24],strong adhesives[25,26],and other bioengineering materials[27].We previously developed a mussel shell-derived scaffold with multilayer,interconnected porous structure for wound repair[28].The main component of this scaffold is chitosan,which exhibits a minimal immune reaction[29,30].It is possible that the chitosan-based shell material could be served as a 3D matrix scaffold to efficiently deliver host cells to the damaged cardiac tissues.To test the possibility,we crosslinked the pPy with the shell-derived chitosan membrane to develop a novel scaffold(shell-pPy)for restoring MI.Dopamine(DA),a small molecule containing catechol and amine groups,is similar to adhesive proteins of marine mussels[27].Under a weak alkaline pH condition,it can self-polymerize to form an adherent polydopamine(PDA),which displays striking adhesion property to render materials with secondary modification[31].As a biocompatible adhesive,the polydopamine could also attenuate toxicity caused by intrinsic pPy in scaffolds[32].Objective:To study the effects of four kinds scaffolds,the shell-pPy,the shell-pPy-PDA,the shell-PDA-pPy and the pure shell,which hybridized among pPy,PDA with chitosan-based scaffold(shell)derived from mussel shells after hybrid crosslinking on the repair of myocardial infarction.1.High elastic,regular prism porous,homogeneous,chitosan scaffolds were obtained,and then modified in situ with polypyrrole,or coated with dopamine,to construct shell conductive film with different porosity.2.Different 3D myocar-dial engineering patches were constructed and the tissue,maturity and function of cardiomyocytes were investigated in vitro.3,in vivo to explore the repair effect of the patch on myocardial infarction.Methods1.Constructed the conductive film material of the mussel shell,SEM for structural observation,and Fluorescent staining to testing the biocompatibility and adhesion of the materials.2.The neonatal exogenous cardiomyocytes were co-cultured on different shell membrane materials to construct myocardial engineering patches.Scanning electron microscopy(SEM)was used to observe the ultrastructure of myocapial membrane material,which was inoculated with cardiomyocytes,and to analyze the calcium transient of different myocardial patches by Fluo-4 fluorescence.In vitro immunofluorescence and Western blot were used to detect the expression of connexin 43,α-actinin and calcium in different patches.3.The model of acute myocardial infarction was constructed by ligating the left anterior descending branch of SD rats.After 14 days,the surviving rats were examined by ultrasonic electrocardiogram.The FS<30%rats were randomly divided into groups,and each group was implanted with different patches(Shell Group,shell-pPy group,shell-pPy-DA group,shell-DA-pPy group),respectively.One month later,echocardiography was used to detect cardiac parameters in each group,and cardiac slices were taken.Masson staining was used to measure cell content in infarcted area.Fluorescence detection of alpha-actinin and CX43 and CM-Dil staining were used to trace the migration and distribution of exogenous myocardial cells in the infarcted area.4.Statistical analysis.The results of statistical analysis using SPSS.20 statistical software were expressed with mean standard deviation(Mean±SD).A single factor analysis of variance(One-way ANOVA)was used in the comparison between the groups.The Tukey method was used to compare the difference between groups.When the variance was not equal,the Dunnett’s T3 method was used between the groups.P<0.05 thought there was a significant difference.Results:1.The shell membrane materials with different biocompatibility,adhesion,conductivity and porosity were successfully prepared,and the ductility of myocardial cells on the modified membrane materials was better.2.The successful construction of different mussel shell membrane material engineering myocardial patch,myocardial cell organization degree of patch and different in different flow,calcium ions are significantly different.3.In different animal models,the migration and distribution trend of exogenous cardiomyocytes to cardiac stem area is different,and the number of new blood vessels is different from that of cardiac function recovery.ConclusionsElectro-active scaffolds have attracted much attention due to their conductive properties in cardiac tissue engineering.The pPy,an extensively used conductive material,provided an inductive microenvironment for enhancing the electrical communications among CMs as well as promoting the maturity of CMs and cardiac tissues regeneration.Here,using a facile acid-base treatment,a flexible matrix scaffold was fabricated in situ shell with multilayer,interconnected porous structure.The pPy was then grafted onto the flexible shell material to develop a pristine electrically conductive shell-derived scaffold(shell-pPy).We found that the shell-pPy scaffold could retain the subtle porous structure of the chitosan shell.The pPy incorporation could even increase the roughness on the inner wall of the pores in the shell and endow the pure shell with a suitable conductivity.Polydopamine(PDA),one of the most versatile moleculars for functionalizing material surfaces,was previously applied to neutralize the toxicity of pPy in the functional cardiac patches.To verify the roles of PDA,we fabricated two different pPy/PDA modified hybrids,including the shell-pPy-PDA and the shell-PDA-pPy scaffolds.Compared with pPy/PDA modified shell scaffold,merely modification of pure pPy(shell-pPy)provide a more biocompatible 3D electrophysiological microenvironment for exogenous CMs growth,cell-cell crosstalk and maturation,along with synchronous contraction of CMs.We found that PDA-coating could decrease the porous size and porosity of the shell scaffolds.The fine spatial structure,kept in shell-pPy ECP,is very important for the engineered ECP tissues,while the pPy/PDA-modified scaffolds(shell-pPy-PDA and shell-PDA-pPy)exhibited a weak synchronous beating and an inorganized sarcomere structure.After being treated with the shell-pPy ECP,the fibrosis processing was drastically attenuated,abundant angiogenesis was triggered,and heart function was significantly improved in the MI rats.We summarized that well porosity and subtle spatial structures of the electro-active shell-pPy scaffold,assisting to provide a suitable 3D microenvironment,could facilitate the maturation and functionalization of recruited CMs and reach a promising cardiac repair efficacy of MI rats. |
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