| Objective: The regeneration and repair of peripher nerve inury has been one of the important directions of clinical research.Acellular neural matrix retains part of extracellular matrix and biomaterial for nerve repair,and it has become an ideal adjuvant material in the treatment of nerve injury.In this study,a novel double-layer nerve conduit of nerve extracellular matrix and polylactic acid-polycaprolactone(PLA-PCL)was prepared by electrospinning technique for nerve repair,and its effect on nerve regeneration was verified by various means.Methods: 1.Three kinds of conduits were perpared by electrospinning technique,which were extracellular matrix nerve conduit(ENC),bilayer nerve conduit(BNC)and PLAPCL conduit.2.The conduit was characterized and observed by means of type microscope and scanning electron microscope.3.The effects of fresh nerve decellularization were qualitatively and quantitatively evaluated by HE staining,DAPI staining,DNA content determination and DNA agarose gel electrophoresis of nerve and conduit samples.4.The effects of decellular treatment and electrospinning on nerve GAGs and collagen content were evaluated by glycosaminoglycan(GAGs),Masson staining and collagen content mesurement.5.Bioactive molecules(COL-Ⅰ,COL-Ⅳ,LN,and FN)and regenerationrelated factors(NGF and BDNF)were analyzed by immunohistochemistry,Western blot and Elisa.6.The safety and physical properties of the three kinds of conduits were evaluated by cytotoxicity,cytocompatibility,subcutaneous inflammatory reaction,mechanical property test,degradation rate test and water absorption test.7.A rat model of sciatic nerve injury at 10 mm was established.Twelve weeks after surgery,postoperative gross observation,footprint analysis,leg circumference measurement,electrophysiolocal test,toluidine blue staining were performed to evaluate the of nerve conduit on sciatic nerve regeneration in each group.Results:1.The results showed that the inner layer(extracellular matrix)and outer layer(PLA-PCL)of BNC were well bonded without obvious stratification.2.The results of HE staining and DAPI staining on cross sections of nerve and conduits showed that the presence of blue nucler was clearly visible in the fresh nerve group(FPN),while no nuclei and myelin sheath were visible in the acellular nerve group(DPN)and ENC group.DNA quantitative analysis and DNA agaross gel electrophoresis results showed that compared with FPN group,the DNA content in DPN group and ENC group were significantly decreased,both of which were less than 50ng/mg.The electrophoresis map clearly showed that there were more than 200 bp DNA bands in the FPN group,while there were no obvious DNA bands in the DPN group.3.Masson staining results showed that red myelin sheath and blue collagen fibers were evenly distributed in the FPN group,while most of the components in the DPN group and ENC group were blue collagen fibers.The quantitative detection results of collagen showed that compared with the FPN group,both the DPN group and the ENC group were significantly increased(P<0.05).Quantitative analysis of glycosaminoglycan showed that ENC group were decreased compared with FPN group(P<0.05),and glycosaminoglycan content of DPN group and ENC group were no significant difference(P>0.05).4.Immunohistochemical staining according to the results of four bioactive molecules in the nerve bundle of FPN group and DPN are expressed,but LN molecular was not expressed on the epineurium.According to results of Western Blot,COL-Ⅰ,LN,COL-Ⅳ and FN were expressed in DPN and ENC,and the intensity level of LN and COL-Ⅳ were significantly higher amount of DPN(P<0.05),and the intensity of FN and COL-Ⅰ molecular quantity was no significantly difference between ENC group and DPN group(P>0.05).ELISA test results showed that LN element in DPN group was significantly higher than ENC group(P<0.05),while LN molecular level in the FPN and DPN was no significantly differences(P>0.05),in addition,FN molecular content of DPN group was significantly higher than that of FPN group(P<0.05),FN content of DPN and FPN group had no significantly difference(P>0.05),COL-Ⅳ in DPN group was significantly higher than that of FPN group and ENCgroup,and COL-Ⅳ content of FPN and ENC had no statistical significance(P>0.05).ELISA results showed that NGF and BDNF levels in FPN group were slightly higher than those in DPN group,and the difference was no statistically significant(P>0.05).The content of NGF in DPN group was significantly higher than that in ENC group(P<0.05),while the contennt of BDNF factor in DPN group and ENC group was no significantly difference(P>0.05).5.The results of cytotoxicity test showed that the cell survival rate of ENC group and BNC group was significantly higher than that of PLA-PCL group and positive control group(p<0.05).When the concentration of extract was 100%,the cell survival rate of ENC group was significantly higher than that of negative control group(p<0.05).Scanning electron microscope observation of cell compatibility showed that the number and cell status of ENC and BNC group were better than that of PLA-PCL group.The resules of the subcutaneous implantation experment showed that the number of inflammatory cells in the BNC and ENC groups was less than that in the PLA-PCL group.The results of mechanical properties test showed that the maximum suture retention strength of BNC group and PLAPCL group was significantly higher than that of the epigneural membrane group and ENC group(P<0.05).The results of degradation rate test showed that by using PLA-PCL as the outer layer of BNC,the degradation rates were significantly reduced compared to ENC(p< 0.05).The water absorption test results showed that the water absorption of BNC and ENC groups was significantly higher than that of PLA-PCL group(P<0.05).6.After 12 weeks of nerve defect repair,the footprint test and ratio of calf circumference measurement of rats(affected side/healthy side)showed that the calf circumference ratio and sciatic nerve function index(SFI)of ENC and BNC groups were significantly higher than PLAPCL group,there were no significant differences in calf circumference ratio and SFI between ENC group and BNC group(P>0.05).Electrophysiological test results showed that nerve conduits velocity and amplitude ratio in ENC group and BNC group were significantly higher than in PLA-PCL group at 12 weeks postoperatively,and there was no statistical significance between the ENC and BNC group(P>0.05).The results of toluidine blue staining showed that the density of myelinated nerve fibers in ENC and BNC group were significantly higher than that in PLA-PCL group(P<0.05).and there was no significant difference in density of myelinated nerve fibers between BNC group and ENC group(P>0.05).Conclusion: 1.The BNC prepared by electrospinning technology has the characteristics of not easy to delamination,good mechanical properties,higher porosity,no hemolysis reaction and low inflammatory reaction,which completely meets the standard of nerve repair materials.In addition,the inner layer of BNC(extracellular matrix)retains various extracellular matrix components,bioactive molecules,and regeneration-related factors.2.BNC had good biocompatibility,it can more reffectively promote the adhesion,infiltration and migration of Schwann cells compared with conventional polymer conduits.Moreover,BNC was not only no cytotoxic,but also promotes cell proliferation.3.Compared with PLA-PCL conduit,BNC can guide regenerated nerve fibers through the defect site more effectively and promote the recovery of nerve function. |
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