Remyelination During The Repair Of Peripheral Nerve Defects Using Tissue Engineered Nerves

Objective:The present study was aimed to investigate the basic biological process of remyelination and the potential gene network underlying the complex biological process after the repair of rat sciatic nerve defect with different types of nerve grafts.We sought to provide new content for the remyelination study during peripheral nerve regeneration.Methods:1.Construction of tissue engineered nerve graft(TENG)in vitro.The artificial tissue engineered nerve was constructed in a perfused rotatory cell culture system(RCCS),and finally,to form a trinity structure composed of chitosan conduit,silk fibroin fibers and rat skin-derived precursor Schwann cells(SKP-SCs).2.Preparation of rat sciatic nerve defect/repair model.The rats sciatic nerve defect,measuring 10 mm in length,were bridged by chitosan/silk fibroin-based scaffold(Sca group),TENG(TENG group)and autologous nerve graft(Auto group),respectively.The experiment was divided into 8 time points,including 1d,4d,1w,2w,3w,4w,8w,12 w,in addition,a sham operation group was set as control at each time point(Sham group).3.Morphological observation of remyelination.The bridge segments in 3 groups(Sca,TENG and Auto)were harvested at 1d,4d,1w,2w and 3w after operation.The biological process of remyelination was respectively observed by optical microscope and transmission electron microscope.4.Microarry detection and bioinformatics analysis.The graft segments together with both nerve stumps(2mm)were collected at 1d,4d,7d,1w,2w,3w,4w,8w,12 w post-surgery and nerve segment of sham group in the corresponding position,respectively.Microarray data analysis was performed after RNA extraction and microarray detection,with the aid of several bioinformatics analysis software,i.e.MeV(MultiExperiment Viewer),PCA(Principal Components Analysis),IPA(Ingenuity Pathway Analysis)and Venn diagram.The expression of the target genes were verified by immunohistochemistry and quantitative real-time PCR.Results:1.One day post-surgery,there were few Schwann cells showing a proliferating state.Nerve degeneration was limited at the broken edge of the proximal and distal nerve stumps in all 3 groups.In the Auto group,the morphology of myelin sheath of graft segment was close to that of the normal,but the structure of myelin sheath was slightly loose,and the intra-axonal granules disappeared.2.Four days post-surgery,a large number of Schwann cells in the proximal and distal nerve stumps showed active proliferation condition in all 3 groups,and a few of SKP-SCs in the TENG group as well.A small amount of Schwann cells migrated from the proximal nerve stumps in the TENG and Auto groups.Furthermore,nerve degeneration of graft segment in the Auto group was remarkable at this stage.3.One week post-surgery,more Schwann cells migrated from the proximal and distal nerve stumps,besides,the regenerating axons extended from the proximal nerve stump along Schwann cells toward the distal stump in all 3 groups.Schwann cells did not yet wrap around the regenerating axons in the Sca and TENG groups.Degeneration peaked at this time in the Auto group,while a few axons were wrapped by Schwann cells.Supplementary experiment of the TENG group showed that 10 days post-surgery,there was well co-located between the SKP-SCs and regenerating axons in the proximal stump.4.Two weeks post-surgery,a poorly characterized tissue bridge forms,connecting the proximal and distal stumps in the Sca and TENG groups.The number of migrating Schwann cells increased significantly in both groups,and axons from proximal stump continued to regenerate along the cell bridge.The distal Schwann cells in TENG group contained suspected cell debris of SKP-SCs.Compared with the Sca and TENG groups,regenerating axons in Auto group had already grown to a long distance through its original structure.A small amount of axons were wrapped by Schwann cells,without lamellar structure formation in the Sca and TENG groups.Degeneration came to a close in the Auto group,and a large number of axons were wrapped by Schwann cells arranging in fascicles,in which some had formed a thin myelin sheath.5.Three weeks post-surgery,Schwann cells migrated from the proximal and distal nerve stumps gathered together in the middle of the lumen.A small amount of regenerating axons passed through the bridge into the distal nerve stump in the TENG group while a large number of regenerating axons crossed the bridge into the distal stump in the Auto group.In the Sca and TENG groups,there was a large number of loosely structured myelin sheath,by contrast,myelin sheath possessed dense structure in the Auto group.6.Microarray data clustering analysis fitted perfectly the biological process of remyelination.Gene network analysis combined with the Venn diagram showed that there was little difference about molecular involved in the network among the three groups.7.The immunohistochemical staining showed that JNK translocated into the Schwann cell nucleus 1 day post-surgery in the proximal nerve stumps in all 3 groups,RAC1 and BRG1 showed well co-located with Schwann cells 2 weeks post-surgery in all 3 groups,BRG1 also showed well co-located with SKP-SCs 10 days post-surgery in the TENG group,CAMK2 B displayed well co-located with Schwann cells 3 weeks post-surgery in the Auto group.8.The quantitative real-time PCR detection of the related genes involved in remyelination showed that the expression pattern of genes was similar to the microarray results.Conclusions:1.The biological process of remyelination during peripheral nerve regeneration was similar among different groups,i.e.dedifferentiation of Schwann cells,proliferation and migration of Schwann cells,differentiation and myelination of Schwann cells and so on,while the regenerating myelin sheath needed to undergo the process of lamellar compaction when repaired by the artificial grafts.2.The result of remyelination in Auto group was much better than that in Sca or TENG group within 3weeks post-surgery.SKP-SCs as the supporting cells could promote Schwann cells migration and guide axon regeneration.3.Genes involved in the network displayed little difference among the three groups,but the action phase of the specific gene i.e.initiation and termination was significantly different due to the different regeneration rate of remyelination.