Construction And Conductive Property Analysis Of Engineered Cardiac Conduction Tract In Vitro And The Research For Seed Cell Candidate

Background and Objective: Complete atrioventricular block(AVB)is one type of severe arrhythmia that suffer many children or adults,for which the main strategy is placement of electric cardiac pacemaker.However,pacemaker implantation is not the perfect choice for children,as there are many limitations in pacemakers such as the durable time,electrode life,electrode length,pacing site etc.Engineered cardiac conduction tract(ECCT)is theoretically a perfect alternative for pacemaker therapy,but the mechanical properties of constructs that have ever been used are poor,or the scaffold component was not completely biodegradable,which may limit their long term researches in vivo.The uniform gelatin/polycaprolactone(PCL)fibrous scaffolds newly developed by our team have good mechanical properties and cell biocompatibility,and the conduction tract constructed by neonatal rat ventricular myocytes(NRVCMs)as the seed cells could repair heart conduction block [1],thus the cardiac conduction tract constructed by NRVCMs and uniform gelatin/PCL fiber scaffold was supposed to be able to repair conduction block,and the scaffolds we would use possess mechanical properties and cell biocompatibility,thus could better meet the needs of cardiac tissue engineering research.Our prior research indicated that the cardiac conduction tract constructed by uniform fibrous scaffolds and NRVCMs could effectively repair heart block model.However,the source of immature cardiomyocytes is limited,and the conduction velocity is comparably slow than native conduction system,thus the exploration for potential seed cell is impending.Transplantation of differentiated adipose-derived stem cells containing cardiomyocytes(CMs)and conduction system cells(CCSs)could improve the atrioventricular block in mice [66],and the conduction velocity of the engineered tract constructed by the above cells is much higher than ventricular slices [25].Therefore,the cell population containing CMs and CCSs might be better seed cells for cardiac conduction tract.Based on the above analysis,construction and conductive property of tissue engineered cardiac conduction tracts(ECCT)were studied in this research.On the one hand,NRVCMs were seeded on the uniform gelatin/PCL fiber scaffolds to construct ECCT,and electrical conductive characteristics and the repair potential for cardiac conduction block of the ECCT were measured.On the other hand,the differentiation potential of rat brown adipose stem cells into CMS and CCS were explored,and then an efficient differentiation induction protocol were developed.Furthermore,the conduction characteristics of cardiac conduction tracts constructed by induced cardiomyocytes derived from r ADSCs were analyzed.Therefore,the research was divided into the following two parts.Part 1 Construction and conductive property analysis of engineered cardiac conduction tract from gelatin/PCL fibrous scaffolds and neonatal rat ventricular cardiomyocytesThe gelatin and PCL composite were dissolved in the modified solvent to prepare electrospun fibre scaffold.The diameters of the fibers and arrangement of cells on the fibers were measured by scanning electron microscopy,then the mechanical properties and hydrophilicity of different fibrous scaffolds were measured.Next,NRVCMs were seeded on the tissue culture polystyrene,random and uniform gelatin/PCL fibrous scaffold to test the adhesion ability,cell viability and cell proliferation.And the morphology and alignment of NRVCMs on different fibrous scaffolds were visualized at different time points by immunofluorescence staining.Furthermore,the engineered cardiac conduction tract was constructed by seeding NRVCMs on fibrous scaffolds,and the electrical conduction characteristics and repair ability of ECCT were measured through microelectrodes assay in vitro.Moreover,the ECCTs were transplanted on the atrioventricular groove in rats to test their conductive ability.Results: The uniform fibrous scaffolds constructed from modified method displayed anisotropic arrangement,well mechanical properties and perfect hydrophilicity.Meanwhile,the anisotropic scaffolds had good cell adhesion and viability with NRVCMs,and promoted NRVCMs proliferation in vitro.Furthermore,uniformly aligned fibrous scaffolds guided NRVCMs parallel to the longitudinal axis and affected cell morphology.Further research indicated that the organization and conductive characteristics of ECCT constructed from uniform Gt/PCL fibrous scaffolds and NRVCMs were similar to that of natural cardiac tissue,and the ECCT could repair cardiac conduction block model in vitro.The ECCT transplanted on the atrioventricular groove of rats could adhere well to the recipient heart,and the ECG after 2 weeks’ transplantation suggested that there might be an atrioventricular bypass exist.Part 2 Research of potential seed cell for cardiac conduction tractRat brown adipose stem cells(r ADSCs)were administered to 5-azacytidine(5-Aza),transforming growth factor-β(TGFβ1)and bone morphogenetic protein 4(BMP4),and the expression of cardiomyocyte-specific markers and cardiac conduction system cell-specific markers in r ADSCs were analysed through RT-PCR and immunofluorescence staining.Then we try to find a new cardiomyogenic differentiation protocol with higher efficiency and little negative effect on cell viability based on the above results.Then the differentiated cardiomyocytes,derived from r ADSCs using the new protocol,were seeded on uniform fibrous scaffolds to construct cardiac conduction tract,the morphology and electrical conduction characteristics were measured.Results : The results indicated that r ADSCs could be induced into cardiomyocytes(CMs)and cardiac conduction system cells(CCSs)with stimulators,and the differentiated cardiomyocytes were a mixture of CMs and CCSs.The cardiomyogenic differentiation efficiency of several stimulators was as following :5-Aza > BMP4 > Tgf β1.Moreover,low dose combination of 5-Aza and BMP4 turned to be more efficient and associated with higher cell viability than 5-Aza alone.Furthermore,the cardiac conduction tract constructed by differentiated CMs and uniform fibrous scaffolds displayed longitudinal cell arrangement and high conduction velocity.Conclusions: In summary,on the one hand,our research indicated that the uniformly aligned gelatin/PCL fibrous scaffolds using new method have good physical properties and biocompatibility,and the ECCT constructed of the uniform fibrous scaffolds and NRVCMs could simulate cell arrangement and the electrical conduction characteristics of the natural myocardial tissue.Furthermore,the ECCT could repair electrical conduction block in vitro,and when the ECCTs were transplanted on the rat heart,they could adhere well to the recipient heart and might act as atrioventricular bypass.Thus,the constructed ECCT provide a potential choice for the treatment of atrioventricular block.On the other hand,the study showed that differentiated CD29+CD31-r ADSCs are mixture of CMs and CCSs,and the combination of 5-Aza and BMP4 at low dose turned out to be an effective and safe protocol to induce r ADSCs into cardiomyocytes.The cardiac conduction tract constructed by differentiated cardiomyocytes and uniform fibrous scaffolds,have similar cell arrangement with atrioventricular bundle and higher conduction velocity.Therefore,the differentiated CMs and CCSs derived from r ADSCs are better seed cell for cardiac conduction tract.This conduction tract has the potential to repair cardiac conduction block,thus may meet the needs of individualized treatment and open application prospect in future.