Construction And Characterization Of Bioactive Hydrogel Nerve Grafts

Purpose:The effective repair and functional recovery after peripheral nerve injury is still a hot topic in clinical research,and it is also a major problem to be solved urgently.The purpose of this study was to construct a bioactive composite hydrogel nerve graft,explore its effects on the regeneration of peripheral nerves,and study its effective regulation of the growth behavior of Schwann cells.The effect of the composite hydrogel with bioactivity on the growth behavior of Schwann cells and its physicochemical characterization of the couduit as a 3D scaffold were discussed.This study may provide a powerful reference for the design and development of new artificial nerve grafts.Methods:The different components of sodium alginate,gelatin,polyacrylamide,graphene oxide were mixed in different volumes to optimize and screen out polyacrylamide/graphene oxide/gelatin/sodium alginate?PAM/GO/Gel/SA,PGGS?composite hydrogels with certain mechanical properties.Macroscopic observation,optical microscopy?OM?,scanning electron microscopy?SEM?were used to analyze the surface morphology of PAM/GO/Gel/SA multi-component composite hydrogel materials;Fourier transform infrared spectroscopy?FTIR?was used to analyze ingredient;The porosity of the hydrogel was tested to determine whether the composite hydrogel could exchange material to a certain extent,and the swelling properties of the hydrogel was tested under the corresponding conditions;Mechanical properties and stability of the hydrogel were evaluated by tensile and compression and in vitro degradation tests;The cytotoxicity was evaluated using L929 fibroblast;PAM/GO/Gel/SA four-component composite hydrogel was subjected to surface modifications of YIGSR and IKVAV peptides.The composite hydrogel co-culture with Schwann cells was optimized to evaluate the effects on the growth behavior of the cells.BCA protein concentration measurement was used to evaluate matrix protein adsorption;Real-time quantitative polymerase chain reaction was used to analyze the expression of cell genes on different composite hydrogels and the cell cycle was studied using flow cytometry.Based on the above studies,a biologically active hydrogel system that promoted cell growth was selected and a hydrogel composite couduit with biological activity was prepared,and the morphological structure of the couduit was characterized.Results:A PAM/GO/Gel/SA composite hydrogel was successfully prepared.The results of optical microscopy showed that with the change of hydrogel composition,the color of PAM/GO/Gel/SA composite hydrogel which added with sodium alginate and gelatin was lighter than PAM/GO_0.4 composite hydrogel,but the three PAM/GO/Gel/SA composite hydrogel had less color difference.Scanning electron microscopy?SEM?observation showed that the surface of the hydrogel had a certain pore structure.Fourier transform infrared?FTIR?spectroscopy results showed that different components could be successfully polymerized.The results of swelling ratio and porosity showed that the composite hydrogel had a certain water absorption performance.Mechanical testing had found that the composite hydrogel had good flexibility,which indicated that the addition of sodium alginate and gelatin changed the mechanical properties of the composite hydrogel in tension.Degradation experiments showed that the composite hydrogel had no obvious degradation after immersed in phosphate buffer solution?PBS?for 15 days to show good stability.The results of L929 fibroblast culture showed that the composite hydrogel was not cytotoxic and could support cell growth.Schwann cell co-culture results showed that the composite hydrogel after immobilization of YR polypeptide could well support the adhesion and proliferation of Schwann cells.Flow cytometric analysis showed that YR concentration was 0.5 mg/mL and 1 mg/mL(PGG_0.5SYR1 and PGG₁SYR0.5)were suitable for Schwann cell proliferation.The gene expression levels of GAP43,MBP,Sox10 on PGG_0.5SYR1 and PGG₁SYR0.5 were up-regulated and the DNA synthesis of Schwann cells on PGG_0.5SYR1 and PGG₁SYR0.5 was higher than that of other hydrogels,indicating that the composite hydrogel was suitable for Schwann cell growth.In addition,a PGG₁S hydrogel conduit was successfully fabricated using a self-made mold with an inner diameter of 2 mm and an outer diameter of 6 mm.The physicochemical properties of the conduit demonstrated that the hydrogel conduit had better elastic properties.The results of the internal and external diameters showed that the prepared hydrogel conduit tube wall was uniform.Conclusion:In this study,firstly,the PAM/GO/Gel/SA composite hydrogel was successfully prepared.It was found that the concentration of YR polypeptide at 0.5mg/mL and 1 mg/mL can effectively promote the growth of Schwann cells and its proliferation;Secondly,PGG₁S and PGG₁SYR0.5 hydrogel couduits were successfully prepared and expected to play an important role in nerve injury repair.Overall,this study can provide important theoretical and experimental evidence for the design,application and development of tissue engineering hydrogel scaffolds.