Myocardial Infarction Repair Based On Silk Fibroin Protein/Alginate Natural Active Biomaterial

The high rate of morbidity and mortality of cardiovascular diseases has received considerable attention from researchers and seriously threatened human life and health.Cardiac tissue engineering research based on stem cells and biomaterials has important application prospects in the treatment of myocardial infarction（MI）,which includes two strategies:（1）Constructing of Engineered cardiac tissues（ECTs）in vitro and transplanting it in vivo.（2）Injectable strategy based on the injectable biomaterial.In the case of constructing ECTs for cardiac band-aid strategy,the silk fibroin porous scaffold for ECTs construction is lack of extracellular matrix component and microenvironment component.In terms of injectable strategy for MI repair,injectable biomaterials lack the ability to regulate the hostile microenvironment of MI,especially the microenvironment containing reactive oxygen species（ROS）in MI region.Therefore,it is of great significance to develop scaffold with myocardial micro-components and microstructures for ECTs construction,and injectable biomaterial with anti-oxidation,anti-inflammatory and regulating microenvironment for MI repair research.The key research work of this thesis is depicted as the follows:1.Developing cardiac fibroblasts-derived extracellular matrix（CFs-d ECM）coated natural active silk fibroin（SF）porous scaffold that mimic myocardial micro-component and microstructures,and melanin nanoparticles/Alginate（MNPs/Alg）natural active hydrogel scaffold with regulating microenvironment of MI.2.Clarifying the regular and molecular mechanisms of differentiation of brown adipose derived stem cells（BADSCs）into cardiomyocytes by CFs-d ECM coated SF porous scaffold.3.Elucidating the anti-oxidation,anti-inflammation of the MNPs/Alg natural active hydrogel scaffold and its influence on the differentiation of BADSCs into cardiomyocytes.4.Examining the MI repair effect of CFs-d ECM coated SF porous scaffold carrying BADSCs and MNPs/Alg hydrogel scaffold carrying BADSCs.In this study,CFs-d ECM,SF porous scaffold and CFs-d ECM-modified natural active SF porous scaffold were developed respectively.It was demonstrated that both CFs-d ECM and the CFs-d ECM modified SFs could promote the differentiation of BADSCs into cardiomyocytes by QPCR,western blotting,and immunofluorescence detecting.We further found that the CFs-d ECM also increased the expression of cardiomyocyte-specific proteins（e.g.,cardiac troponin T andα-actinin）of BADSCs.Notably,it was proved that CFs-d ECM was involved in the regulation of differentiation of BADSCs into cardiomyocytes through theβ1-integrin-dependent TGF-β1 signaling pathway during the construction of ECTs.The injectable MNPs/Alg natural active hydrogel scaffold for regulating the MI microenvironment was further developed and its antioxidant properties were systematically evaluated.The MNPs/Alg hydrogel scaffold showed good biocompatibility and antioxidant properties,and the anti-apoptotic and antioxidant properties of MNPs/Alg hydrogel on cardiomyocytes were examined by DHE staining,immunofluorescence,QPCR and western blotting.Then the proliferation and differentiation of BADSCs and the anti-inflammation and transformation of macrophages were examined by live/dead staining,QPCR and western blotting,the results showed that MNPs/Alg hydrogel scaffold could alleviate H₂O₂-induced apoptosis and promote antioxidation in cardiomyocytes.Besides,it also effectively promoted the proliferation of BADSCs and the expression of cardiomyocyte-specific proteins such as cardiac troponin T（c-Tn T）and connexin43（Cx-43）,inhibits the polarization of M0-type macrophages to M1-type macrophages under ROS environment and effectively facilitates their polarization to M2 repair type.The above experimental results laid the foundation for the subsequent in vivo MI repair by MNPs/Alg hydrogel scaffold.On this basis,a rat MI model was prepared.Researches of repairing MI by natural active SF porous scaffold carrying BADSCs and MNPs/Alg natural active hydrogel scaffold carrying BADSCs were carried out respectively.The effects of MNPs/Alg natural active hydrogel scaffold carrying BADSCs in the regulation of the MI microenvironment and repair of cardiac function were systematically evaluated by histological staining,immunofluorescence staining,QPCR,and heart ultrasound.The results showed that compared to the CFs-d ECM coated SF porous scaffold,MNPs/Alg natural active hydrogel scaffold effectively scavenged superoxide anions and hydroxyl radicals and promoted the transformation of M2-type macrophages.Regardless of whether MNPs/Alg natural active hydrogel scaffold carried BADSCs or not,the maintenance of cardiomyocyte structure,the formation of gap junctions and angiogenesis in the infarct region were promoted,which in turn contribute to the recovery of cardiac function.In summary,the natural active SF porous scaffold and MNPs/Alg natural active hydrogel scaffold were developed.The regulatory effects of the two types of biomaterials on the survival,proliferation and differentiation of BADSCs into cardiomyocytes were systematically demonstrated at the cellular,protein and gene levels.The possibility of treating MI with natural active SF porous scaffold and MNPs/Alg natural active hydrogel scaffold is further discussed,which provides a certain theoretical basis and experimental basis for future clinical applications based on cardiac tissue engineering.