Study On The Repair And Application Of Succinylated Chitosan(NSC)in HUVECs Cells Damaged By Inflammatory Hyperglycemia

The repair of chronic wounds has been a difficult clinical problem.The persistent inflammatory response brings huge physical and psychological burdens to patients.To slow down or even eliminate the local inflammatory response is of great significance for the repair of chronic wounds.Chitosan is natural polymer materials,and they had been widely used and studied in the field of biomaterials because of their good biocompatibility.Succinate can inactivate gastrin D(GSDMD)and prevent cell pyroptosis.Therefore,we used succinic anhydride to modify chitosan and got N-succinylated chitosan.We established an endothelial cell injury model that induced endothelial cell dysfunction and inflammatory stress by inflammatory hyperglycemia(H+T),and to explore the repair effect on the normal function of damaged endothelial cells and the inhibitory effect on inflammation.We prepared a composite hydrogel based on the characteristics of NSC materials,and evaluated its mechanical properties,swelling properties,water vapor permeability,in vitro stability,microscopic morphology,cytocompatibility,etc.The main experimental content and results of this study are as follows:1.Firestly,we prepared N-succinylated chitosan with substitution degrees of9.6%,22.4%,31.1%,38.4%,and 46.7% by the reaction of succinic anhydride and chitosan.Through a simple cell proliferation experiment,we confirmed that the best material concentration and substitution degree to promote cells proliferationy was 0.5wt% and 38.4%.We use(H+T)to damage endothelial cell and induce endothelial cell dysfunction and inflammatory stress,and through observating cell morphology and the expression of endothelial cell marker proteins and inflammatory factors to confirme the successful establishment of the model.Use NSC to treat Injured cells and evaluat index of correlation.The test results showed that: NSC increased the proliferation rate and cell migration rate of damaged cells,increased the expression of VEGF and CD31 in damaged cells to varying degrees,and improved the cell state,which proved that NSC had a certain repair effect on damaged cells.The expression of inflammation-related proteins and the results of q PCR experiments showed that the expression of inflammatory factor TNF-α and anti-inflammatory factor IL-10 in injured cells were down-regulated after NSC treatment,which proved that the material has a certain ability to regulate inflammation.2.We further evaluated the ability of NSC to repair damaged cells in the co-culture system by co-culture of macrophages and endothelial cells.We used transwell plates to carry out Co-culture experiments and evaluated related indicators.The results showed that the proliferation rate of injured endothelial cells was further increased in the co-culture system,and at the same time,the upper macrophages migrated toward endothelial cells through the pores.However,the migration of macrophages in the NSC-treated group was less than that in the untreated group.In addition,in the co-culture system,the measurement results of angiogenesis-related factors and inflammatory factors were optimized and expanded to varying degrees,indicating that the repairing effect of NSC was enhanced by the co-culture system.Finally,we evaluated the polarization of macrophages in the system,and the results showed that M2-type macrophages increased in the NSC-treated group,which proved that the NSC material exerts a good anti-inflammatory ability even in the presence of macrophages.3.Using κ-carrageenan as the base material,and KCl and introduced glycerol to adjust its properties,and compounded different proportions of NSC.Evaluated The swelling properties,thermal stability,mechanical properties,stability,water vapor permeability,cross-sectional micromorphology and cell compatibility of the composite hydrogels.The results show that those composite hydrogels have good cytocompatibility,thermal stability,mechanical properties and swelling properties.The introduction of glycerol component enhances the toughness and swelling ability of the composite hydrogels.The introduction of NSC endows the composite hydrogel with certain wound repairing ability and anti-inflammatory properties,but the water vapor permeability is decreased.The composite hydrogel can be used for the repair of deep wounds and should have good application prospects in wound repair materials.