Preparation Of Fullerene Based Scaffold And Its’ Application In Cardiac Tissue Enineering

In China, more and more young people are suffering from myocardial infarction with a high morbidity and mortality. Traditional clinical strategies, such as drug therapy and coronary intervetion, can only alleviate the symptoms and improve the life quality of patients in a certain extent and can not fundamentally repair and reconstruct the damaged myocardial tissues. The strategy of injectable myocardial tissue engineering provides new methods for the effective repair of damaged myocardial tissues. However, the current design of injectable materials had not considered the harsh microenvironment of redundant oxygen radicals(ROS) in myocardial infarction area and the electrical conductivity of the myocardium. Therefore, it is significant to develop hydrogels that contain both antioxidation property and conductivity. Fullerene has good conductivity, and it is more important that its derivative fullerenol has excellent antioxidation property. Therefore, this paper will focus on elucidating the regulation and related molecular mechanism of fullerene and its derivative on the survival, proliferationand myocardial differentiationof rat brown adipose stem cells(BADSCs). On this basis, fullerenol/alginate hydrogel with both antioxidation capacity and co nductive was prepared. The regulation and related molecular mechanism of the survival, proliferation and myocardial differentiationof BADSCs in the ROS microenvironment were studied and its repair of damaged myocardium in vivo was explored.The regulation and molecular mechanism of different concentrations of fullerene on the toxicity, proliferation and myocardial differentiation of BADSCs were systematically investigated by the measurements of microscopy, flow cytometry, immunofluorescence staining, transmission electron microscopy, western blotting and real time PCR, etc. The results showed that fullerene has good cell compatibility and up to 100μg/m L of fullerene nanomaterials still had no cytotoxicity to BADSCs. It could enter the BADSCs to regulate the expression of MAPK signal pathway(ERK, p38) protein and promote the expression of cardiac specific proteins and genes of BADSCs, so as to improve the ability of BADSCs proliferation and myocardial differentiation. Simultaneously, it could improve the expression of gap junction protein Connexin 43 and promote the intercellular communication.The water-soluble fullerene derivatives-fullerenol was prepared through chemical modification technology. And injectable fullerenol/alginate hydrogel was also prepared with biocompatible alginate as substrate and calcium gluconate as crosslinking agent. The control and regulation of the hydrogel composition on its antioxidation capacity and conductivity was systematically studied. In this study, it is suggested that the hydrogel has good conductivity and it gradually increased with the content of fullerenol. When the concentration of fullerenol was 600μg/m L, its conductivity could reach(0.41+0.09)×10-4 S?cm-1. At the same time, the hydrogel was able to effectively remove hydroxyl radicals and organic oxygen radicals, and the scavenging efficiency was directly proportional to the concentration of fullerenol. On this basis, BADSCs were encapsulated into the fullerenol/alginate hydrogel and the regulation and related molecular mechanism of the material properties on the oxidative stress injury, proliferation and myocardial differentiation of BADSCs in ROS microenvironment were systematically studied by the measurements of histological staining, immunofluorescence staining, tr ansmission electron micoscopy, western blotting and real time PCR, etc. The results showed that the fullerenol/alginate hydrogel has good biocompatibility. It is more important that the introduction of fullerenol could significantly reduce the oxidative st ress injury to BADSCs of ROS, increase the expression of survival and proliferation related signal pathway of p-ERK and p-p38 and reduce the expression of apoptosis related signal pathway of p-JNK. And it can also promote the expression of myocardial specific protein c Tn T, α-Actinin and Connexin 43 and then regulate their ability to differentiate into cardiomyocytes. After the comprehensive analysis, when the concentration of fullerenol was 100μg/m L, the antioxidation effects of fullerenol/alginate hydrogel, the promotion of BADSCs proliferation and myocardial differentiation effect is more ideal.The rat model of myocardial infarction was prepared and the developed fullerenol/alginate hydrogel with BADSCs was injected to rat myocardial infarction area. By histochemical staining, immunofluorescence staining, cardiac ultrasound and laser Doppler techniques, its regulation on myocardial infarction microenvironment in vivo and its repair of damaged myocardium were studied in detail. The study found that the introduction of fullerenol could significantly reduce the number of active oxygen radicals in myocardial infarction area. And compared to the control group, the hydrogel with a fullerenol concentration of 100 μg/m L with BADSCs had the best effectto repair the myocardial infarction. It could significantly reduce the infarction area, increase ventricular wall thickness, increasethe vascular density of myocardial infarction, improve the cardiac function and delay the postremodeling process of infarction ventricula r.In summary, this study successfully developed a cardiac tissue engineering hydrogel material with both antioxidation capacity and conductivity. And it also systematically elucidated the regulation and related molecular mechanism of material property on the survival, proliferation and myocardial differentiation from the material level, cellular level, protein level, gene level and animal level. It explored the possibility of the material in treating myocardial infarction and provided theoretical and experimental basis for the development of myocardial tissue engineering biomaterials.