Repair Of Peripheral Nerve Defects Using Tissue Engineered Nerve And Molecular Mechanisms Underlying Functional Reconstruction

Tissue engineered nerve(TEN)constitute a promising alternative to autologous nerve grafts that have been recognized as the gold standard for surgical repair of peripheral nerve defects.Silk fibroin(SF)derived silkworms represent a type of highly biocompatible biomaterial for tissue engineering.We have previously investigated biocompatibility of silk fibroin with neural cells isolated from central nervous system or peripheral nerve system in vitro,and also developed a SF-based nerve graft conduit(NGC)or a TEN by introducing bone marrow mesenchymal stem cells(BMSCs),as support cells,into SF-based scaffold and evaluated the outcomes of peripheral nerve repair in rat model.As the extension of previous study,the electrospun technique was performed here to fabricate SF-based neural scaffold inserted with silk fibers for bridging a 30 mm-long sciatic nerve gap in dogs.An ensemble assessments as functional,histological and morphometrical analysis were applied 12 months after surgery.All the results indicated that SF-based neural scaffold group achieved satisfactory regenerative outcomes,which were close to those achieved by autologous nerve grafts as the golden standard for peripheral nerve repair.For achieving better axonal regeneration in the PNS,activating injured neural intrinsic growth status and enabling outgrowth of axons in a superior regenerative microenvironment are required.And a transcriptomic mechanism of TEN for peripheral nerve regeneration is increasingly demanded.In present work,the TEN constructed using skin-derived precursor induced Schwann cells(SKP-SCs)and chitosan/silk filament scaffold were approved to promoted peripheral nerve regeneration and functional restoration.Then,a combination of gene expression profiling and multi-facetted bioinformatic analysis further experimental validation were performed in the rat sciatic nerve grafts and the dorsal root ganglions(DRGs,L4-L6).The results showed the precise molecular mechanisms involved in biological process of cell apoptosis,axonal regeneration,myelination,vascularization and their transcriptional regulation,which revealed the unique gene-protein-expression patterns for the repair of TEN,autograft,scaffold groups and overlapping patterns among aforementioned groups following the sciatic nerve defect model in rat.For the regenerative microenvironment in the grafted segment,core biological process related to axonal regeneration shared the similar transitions(12 h and 4 d)of average expression trends.Three phases were naturally designated as "stress response phase","pre-regeneration phase",and "regeneration phase".Principally,there were two transitions(6 h and 4 d)of average expression trends in myelination.Thus,3 phases as "injury response phase","pre-myelination phase’",and"myelination phase" were naturally designated.And there were 3 transitions(6 h,4 d and 8 w)of average expression trends in vascularization.Four phases were designated as"injury response phase","pre-vascularization phase","vascularization phase" and "re-modulation phase".The functional transcriptomic information were also gathered in DRGs about the biological processes of cell apoptosis,axonal regeneration,myelination,vascularization,and their transcriptional regulations.Furthermore,the novel transcripts identified as candidate mediators of key transitions help define the mechanisms of peripheral nerve regeneration.Our work serves as a valuable resource to understand the molecular processes that define axonal injury-induced micro-environmental variation and to discover novel therapeutic targets for peripheral nerve injury(PNI).