Construction Of New Tissue Engieered Nerve And Repair Of Rat Sciatic Nerve Defects

ObjectiveThrough the systematic study of the relationship between immune response and nerve regeneration after peripheral nerve transection,we explored the potential target molecules for peripheral nerve regeneration at the molecular level.In order to obtain better repair effect,tissue engineering technique was used to construct a new tissue engineered nerve for repairing rat sciatic nerve defects,and the effect of nerve repair was evaluated comprehensively to guide the clinical treatment of nerve injury in the future.MethodsGene microarray analysis was used to identify the differentially expressed genes which are related to immune response and nerve regeneration.The interaction between differentially expressed genes and proteins was analyzed comprehensively.Real-time PCR and immunohistochemically staining were used to verify the differential genes.SKPs were isolated from the skin of juvenile SD rats,and were induced differentiation of SKP-Neurons and SKP-SCs.SKP-Neurons and SKP-SCs were co-cultured with chitosan / silk fibroin scaffold to construct novel tissue engineered nerve.Constructed of new tissue engineered nerve was used to repair rat sciatic nerve 10 mm defect.At last,many indicators were used to evaluate the effect of repair,which were contain rat gait study,neurophysiology research,retrogradetracing of Fluoro – Gold research,morphological evaluation of sciatic nerve and the target muscles that were dominated by sciatic nerve.Results1.Gene analysis and validation: there are more than 600 differentiated genes Differential gene screening results showed that there were more than 600 genes were screened out which involved more than 10 classical pathways in the immune response,and approximately 60 genes involved in nerve regeneration of the five classical pathways.A total of six involved in multi-channel gene in those were used for real-time PCR and immunohistochemistry.The result of validation demonstrate the accuracy of gene analysis,and the differential genes that were screened out may provide experimental basis for targeted therapy of nerve injury in clinic in the future.2.Construction of new tissue engineered nerve: SKP-Neurons and SKP-SCs were co-cultured with chitosan / silk fibrosis scaffold to build a new tissue engineered nerve,and that was stimulated to secrete extracellular matrix.Cells in scaffold were in good condition,and extracellular matrix was secreted rich.New tissue engineered nerve was constructed success,and the cellular components are consistent with normal nerve,contains axonal,Schwann cells and extracellular matrix components.3.Sciatic nerve function index(SFI)analysis: There was significant difference(p< 0.05)in 8 w after surgery,and no significant difference(p > 0.05)in 4 w and 12 w after surgery compared between the experimental group and the autograft group.There was significant difference(p < 0.01)between the experimental group and the scaffold group in 4 w,8 w and 12 w after surgery.4.Nerve electrophysiology: Nerve electrophysiology was tested in 12 w after surgery which contained the compound muscle amplitude potential(CMAP)and the motor nerve conduction velocity(MCV).There were no significant difference(p >0.05)in both the near and distal CMAP between the experimental group and the autograft group.However,the autograft group was faster than the experimental group(p < 0.05)on MCV.There was a certain gap compared between all operated groups and normal nerve which is harvested on non-operative side(p < 0.01).5.Morphological analysis of regeneration nerve: The full length sciatic nerve was harvest at 4 d,1 w,2 w and 3 w.Some individuals in the experimental group of sciatic nerve bridge proximal axons have been going across tissue engineered nerve to the distal nerve.The distal regeneration of sciatic nerve was harvest at 12 w after surgery.Immunohistochemistry and transmission electron microscopy showed the distal nerve axon density,myelinated fiber diameter,myelin sheath thickness and myelin sheath number,the autograft group was superior to experimental group,and the experimental group was better than scaffold group.But all operative groups had significant difference compared with non-operative side(p < 0.05).6.Fluorescence gold retrograde tracer: The distal of regenerative nerve was injected fluorescent gold tracer after 12 weeks surgery.Spinal cord and dorsal root ganglion were harvest 2 weeks later.The statistical results of the number of spinal motor neurons and the percentage of dorsal root ganglion neurons showed that there was no significant difference between the experimental group and the autograft group(p > 0.05),but both of them were better than the scaffold group(p < 0.01).There were no significant difference both the experimental group and autograft group compared with the non-operative side respectively(p > 0.05).7.Morphological evaluation of the target muscles: The wet weight of gastrocnemius muscle: There was no significant difference between the experimental group and the autologous group(p > 0.05),but both of them were better than scaffold group and nongrafted group(p < 0.05).Masson trichrome staining showed gastrocnemius muscle atrophy in nongrafted group.The cross-sectional area of the gastrocnemius muscle fibers showed there had a progressive decrease arranged by normal group,autograft group,experimental group,scaffold group and nongrafted group(p < 0.05).The percentage of collagen fibers in the gastrocnemius muscleshowed there was no different between the experimental group and the autologous group(p > 0.05).Motor end plates were atrophy seriously in nongrafted group.Conclusions1.Through gene chip technology,we got information of differential expression of the genes both in immune response and nerve regeneration after the sciatic nerve transection.The interaction between proteins which were coded by differential expression of genes that we got before was obtained.Real-time PCR and immunohistochemistry were used to verify the reliability of the gene analysis results.It provides not only new clues for the process of nerve regeneration,but also the basis of micro-environment regulation and targeted drug therapy in peripheral nerve injury in the future.2.A new tissue-engineered nerve had been constructed by SKP – SCs,SKP-Neurons and the degradable chitosan / silk fibroin scaffold successfully.The cell components were similar to normal nerves which contains SCs,Neurons and abundant extracellular matrix.3.The tissue engineering nerve which contained SKP-SCs and SKP-Neurons and chitosan / silk fibroin scaffold repaired 10 mm of sciatic nerve in rats.The most of morphological and physiological function had recovered both in sciatic nerves and the innervated muscles after 12 weeks of operation,and were basically the same with autologous group(p > 0.05).This study provides new clues and new experimental evidence both in construction of tissue engineering nerve and the clinical application of autologous skin in peripheral nerve injury.