Study On Promoting Microvascularization Of Il-4 Multicellular Gelma/Sodium Alginate-chitosan Microsphere Scaffold

Object: To explore the effect and mechanism of the immune factor interleukin-4(IL-4)through immune regulation to promote macrophage M2 polarization on the vascularization of vascular endothelial progenitor cells,and to provide evidence for it to cooperate with macrophage polarization to promote tissue engineering vascularization.Construction of IL-4loaded multicellular methacrylic acid anhydride gelatin/sodium alginate-chitosan microspheres(Gel MA/ACS)scaffold composite system to evaluate and explore the role of IL-4 in promoting macrophage M2 polarization and alginic acid the supporting role of sodium-chitosan microspheres in tissue engineering vascularization and related mechanisms provide new ideas and experimental basis for tissue engineering to promote vascularization.Method: The construction of vascularized tissue is a major challenge for tissue engineering.The formation of the microvascular network that provides oxygen and nutrients to cells is closely related to the success or failure of tissue engineering regeneration.Almost all tissues in the human body depend on the branch vascular system,and the optimal diffusion distance is less than 200 μm.Due to the insufficient distance between capillaries,the transplantation of tissue engineering grafts fails.Therefore,there is a special need for a method that can quickly and efficiently promote tissue engineering vascularization.A number of studies have shown that inflammation and immune regulation can promote the vascularization of tissue engineering implants.IL-4 is a common immunoregulatory factor that can promote M2 polarization of macrophages,and M2 macrophages can secrete angiogenic factors(VEGF,etc.)to promote angiogenesis.Preliminary studies have confirmed that the sodium alginate-chitosan(AC)microsphere scaffold can physically and electrostatically adsorb and support cells,and can well promote cell migration and differentiation,as well as angiogenesis and growth.Therefore,we prepared a kind of sodium alginate-chitosan microsphere loaded with IL-4.The microspheres have a double-layer structure of "core-shell";and then combined them with endothelial progenitor cells(EPCs),RAW264.7macrophages are mixed,and a 3D Gel MA/ACS gel scaffold containing IL-4 mixed with multi-cells is constructed using the methacrylic anhydride gelatin(Gel MA)hydrogel system.Based on the electrostatic effect on the surface of AC microspheres,the bionic “loofah vine”adsorbs and guides cell growth,and can support the structure of blood vessels.The IL-4factor released by the microspheres promotes the polarization of macrophages to the M2 type,and secretes factors that promote angiogenesis(VEGF,etc.),which can further promote tissue vascularization and microvascular stability.In this article,we evaluated the cytotoxicity of the gel scaffold by the CCK-8 method,and measured the IL-4 factor release and the secretion level of related vascular growth factors from the gel scaffold by the ELISA method,and performed in vitro EPCs cell formation experiments and macrophages.Cell polarization experiment and endothelial cell marker CD31 expression immunofluorescence experiment evaluate the angiogenesis performance.In order to further confirm the effect of IL-4 mixed multi-cell 3D Gel MA/ACS gel scaffold on tissue vascularization and angiogenesis,in vivo experiments under the skin of C57BL/6J mice were used to verify the in vivo vascularization effect.The vascularization of the gel stent was evaluated by observing,H&E staining,Masson staining and immunofluorescence techniques.Result: The results show that the 3D Gel MA/ACS gel scaffold loaded with IL-4 mixed multi-cells is not cytotoxic to EPCs and macrophages and can release the expression of IL-4factors and vascular growth factors.In vitro endothelial cell EPCs tube formation experiments,macrophage polarization experiments,and endothelial cell marker CD31 expression experiments confirmed that the gel scaffold can promote the tubularization of EPCs,the polarization of macrophages to M2 type,and the high expression of CD31.ELISA detected the secretion level of pro-vascularization factors in the gel stent,and the levels of VEGF,MCP-1 and PDGF-BB increased significantly within two days of culture.Observing,H&E staining,Masson staining and immunofluorescence staining were used to evaluate the in vivo vascularization experiments in mice.Conclusion: The 3D Gel MA/ACS gel scaffold with IL-4 and multi-cells had the best vascularization effect.In this study,in vivo and in vitro experiments confirmed that the IL-4loaded multicellular Gel MA/ACS gel scaffold has good biocompatibility,the electrostatic effect of AC microspheres can guide the adhesion and growth of endothelial progenitor cells,and the released IL-4 can induce macrophages Cells are polarized toward M2,which promotes the expression of angiogenic factors.The synergistic effect of the two can stably and rapidly promote the formation of microvascularization,providing new methods and ideas for tissue engineering vascularization.