Biological Evaluation And Therapy Ofmyocardial Infarction Of Nanomaterials Based Reconstituted Cardiac Tissues

Cardiac tissue engineering with the rapid development has been one of the promising noval therapy. Researchers have obtained a lot of exciting redults in the field of construction and transplantation of engineered cardiac tissues(ECTs) in vivo, recently various ECTs based on different scaffolds have been constructed in vitro and transplanted into host myocardium to repair the infarcted myocardium in vivo successfully. However, concsidering the conductivity of natural myocardium, tranditional biomaterials for ECTs could not mimic the conductive microenvironment of native myocardium, and the ECTs based on the tranditional biomaterials did not have developed contraction-excitation coupling. Graphene with the strongest mechanical property and the best conductivity of nano biomaterials as far has been proved to be good biocompatibile to cells, and the application of graphene showed to be promising in biomedical medicine especially tissue engineering. Besides, cell tracking in vivo is very important to evaluate the repair of cardiac function of infarcted heart with tranplantation of ECTs into injured myocardium, iron oxide magnetic nanoparticles(IRONs) has been one of the promising cell labeling due to its good biocompatibility and strong magnetic response, but the influence of IRONs on the microstructures and cell activity of cardiomyocytes, especially on the intercalated discs of cardiac cells in the three dimensional ECTs has not been elucidated.Therefore, this projects was focused on the construction of collagen/Matrigel scaffolds and the influence of IRONs on microstructure and cell activity of cardiomyocytes. Meanwhile, after the evaluation of effects of graphene oxide(GO) on the cardiac differentiation of brown adipose derived stem cells(BADSCs), the distribution and repair of infarcted heart with transplantation of GO based hydrogel delivering BADSCs labeled with IRONs was studied. The following is the mian contents:(1) The effects of iron oxide magnetic nanoparticles on microstructure and biological activity: The DMSA-IRONs were prepared through co-precipitation and the surface modification of dimercaptosuccinic acid(DMSA). And the effects of DMSA-IRONs on microstructures of cardiomyocytes including cytoskeleton, gap junctions and adherens junctions were confirmed by immunofluorescence staining, histological staining and western blotting assay. The results indicated that DMSA-IRONs have rare influence on the microstructure of cardiomyocytes, which guaranteed the safety of its application in vivo. Meanwhile, in the effects of cell activity of cardiomyocytes, di-azoaminobenzene(DAB) color reaction revealed that DMSA-IRONs scavenged the intracellular reactive oxygen species(ROS), and also DMSA-IRONs performed the similar activity of antioxidant enzyme in normal and H2O2 treated condition. Therefore, DMSA-IRONs protected cardiomyocytes through diminishing the intracellular ROS contents.(2) The effects of iron oxide magnetic nanoparticles on intercalated discs in engineered cardiact tissue: We firstly constructed ECTs using collagen/Matrigel hydrogel scaffold and cardiac cells, and treated ECTs with DMSA-IRONs for 24 h. With the observation of transmission electronic microscope(TEM) and prussian blue staining, we confirmed that the internalization of DMSA-IRONs by cardiac cells was closely associated with the location of cells in ECTs. Cells in the border of ECTs absorbed DMSA-IRONs extensively, while the internal cells in ECTs internalized only a little. Furthermore, the internalized DMSA-IRONs distributed in the cytoplasm and visicles. In the effects of DMSA-IRONs on intercalated discs, the expression of connexin 43, N-cadherin, desmoplakins and plakoglobin through western blotting and immunofluorescence staining showed that DMSA-IRONs promoted the formation of gap structure, reducing the formation of adherens junctions and desmosomes.(3) The effects of graphene oxide(GO)-gelatin with methyl acrylate(GO-Gel MA) hydrogel on the cardiac differentiation of brown adipose derived stem cells(BADSCs): GO-Gel MA hydrogel was prepared. The determination of scanning electron microscope(SEM), Raman spectroscopy and cyclic voltammetry convinced that the supplement of GO did not affect the porous structure of GO-Gel MA hydrogel, but enhanced the electric capacitance. We screened GO-Gel MA hydrogel with proper GO concentration for BADSCs using crystal violet staining and MTT assay. And the rare effects of GO on proliferation of BADSCs were proved by the Brd U incorporation assay. Moreover, immunofluorescence staining and western blotting were performed to determine the effects of GO-Gel MA hydrogel on the differentiation and expression of conductivity protein of BADSCs. The results showed that GO-Gel MA hydrogel promoted the cardiac differentiation and polerization of connexin43 in BADSCs. And also, the ILK-Akt-Erk1/2 signalling pathway was induced in the modulation of cardiac differentiation and promotion of conductivity of BADSCs by GO-Gel MA hydrogel.(4) The repair of myocardial infarction with GO-Gel MA hydrogel delivering BADSCs labeled with magnetic nanoparticles: The zinc doped iron oxide nanoparticles(Zn-MNPs) with diameter of 4.8±0.6nm were prepared by the hythermal decomposition, and further modified with DMSA to increase the hydrophilic dispersion. BADSCs were labeled with Zn-MNPs and were delivered by GO-Gel MA hydrogel into the infarcted myocardium area of rats with acute myocardial infarction. Through determination of echocardiography, MRI technology, Prussian blue staining, histological staining and Masson staining, the results showed that GO-Gel MA hydrogel significantly promoted the cell retention of BADSCs in the infarcted myocardium area, increased the cardiac ventricular wall thickness and improved the contractile function of heart. Furthermore, GO-Gel MA hydrogel promoted the migration of BADSCs in the infarcted myocardium area and the differentiation of BADSCs into cardiomyocytes.In summary, we firstly confirmed the effects of DMSA-IRONs on microstructure and cell activity showing that the internalied DMSA-IRONs acted as antioxidant enzyme to diminish the intracellular ROS and protected cardiomyocytes; while in 3D ECTs, the incubation of DMSA-IRONs promoted the formation of gap junctions, reduced adherens junctions and desmosome of cardiac cells. Meanwhile, the research of the effects of graphene oxide on the cardiac differentiation and the transplantation of stem cells in vivo indicated that GO-Gel MA hydrogel enhanced the spread and the cardiac differentiation of BADSCs. Furthermore, the incorporation of GO induced the expression and polarization of conduction related protein-connexin 43, which was related with the ILK-Akt-Erk1/2 pathway. And GO-Gel MA hydrogel delivering BADSCs promoted cardiac function significantly. This study about magnetic nanoparticles and graphene oxide in cardiac tissue engineering, cell labeling and transplantation provide helpful information for the further application in vivo.