Repair Of Sciatic Nerve Defect In Rats By Transplantation Of Plant-derived Nerve Conduit With SHH-EMSCs

Objective:There are many reports on the repair of peripheral nerve injury by nerve conduit bridging nerve gap instead of autogenous nerve graft.The Mesenchymal stem cell is an important seed cell for the transplantation of peripheral nerve injury.Stem Cell/tissue engineering scaffold transplantation has attracted much attention of researchers.The aim of this study was to investigate:(1)The effects of plant-derived nerve conduits and in vitro cultured nasal mucosa-derived ectodermal Mesenchymal stem cell(NSCS)Biocompatibility on the proliferation and differentiation of NSCs;(2)To study whether EMSCs can express the sonic hedgehog factor stably.(3)To establish a rat model of Sciatic nerve injury and to evaluate the repair effect of SHH-EMSCs on rat Sciatic nerve defect.Method:(1)The natural plant tubular culms with an internal diameter of 0.2 cm were used to physically and chemically remove the flesh of the culms,retain their fibrous tubular shape,and the surface of the plant conduits was crosslinked with Laminin and modified with extracellular matrix;(2)EMSCs were isolated and cultured in vitro,and their surface markers were identified by immunofluorescence assay.And seeded generations of EMSCs on the surface of plant conduits.(3)To evaluate the Biocompatibility of plant-derived vascular scaffolds and the proliferation and differentiation of EMSCs: MTT assay was used to test the effect of the scaffolds on the proliferation and differentiation of EMSCs;the morphology and growth of the cells on the surface of the scaffolds were observed under the Fluorescence microscope;The expression of MBP and S100 protein was detected by immunofluorescence and Western blot.(4)EMSCs were transfected with recombinant adenovirus carrying SHH gene,and the expression of Shh was detected by Western blotting.SHH-EMSCs modified by Shh Gene were planted on the surface of Laminin-modified plant vessel,and the morphology and growth of the cells were observed under Fluorescence microscope(5)Combined use of Laminin-modified plant conduit and SHH-EMSCs transplantation to repair Sciatic nerve defect in SD rats: twenty-five healthy SD rats were selected to establish Sciatic nerve injury model.The experimental animals were randomly divided into five groups: simple Sciatic nerve injury group(Group A),single Sciatic nerve injury group(Group B),combined Sciatic nerve injury group(Group C),and single graft group(Group B),sciatic nerve injury was treated with Laminin to modify the vessel and SHH-EMSCs Group(Group D)and Sham Operation Group(Group E);(6)To evaluate the effect of the treatment: after the operation,the hindlimb motor function was dynamically tested and the Sciatic nerve index(SFIRRB-was calculated.After 19 weeks of survival,the injured nerve was injected with fluorescein,and the dorsal root ganglion(DRG)was traced.After 1 week,the bilateral DRG was fixed and sectioned,the number of positive neurons in the dorsal root ganglion of each group was compared by Fluorescence microscope.The Sciatic nerve tissue sections were stained with NF-200 antibody and the regeneration of nerve fibers was observed.The relative expression levels of NF-200 and MBP protein were detected by Western-blotting Quantitative analysis after the tissue proteins were extracted from some fresh Sciatic nerve samples.After weighing the fresh gastrocnemius muscle,HE staining was used to observe and compare the cross-sectional area of the gastrocnemius muscle fibers in each group.Result:(1)Under the microscope: after the treatment of physical and chemical treatment,the surface of the stent is arranged vertically and parallel stripes.After lamin modification,the spontaneous red fluorescence can be observed in the Fluorescence microscope,the stripes are more clear.(2)The high expression of Nestin and Vimentin was observed by immunofluorescence staining in the subcultured EMSCs,which indicated that the EMSCs isolated and cultured in this experiment had high purity.(3)EMSCs grew well and oriented on the surface of plant duct fiber.The growth curve of EMSCs was s-shaped and similar to that of control group by MTT assay.The results of immunofluorescence and western blotting showed that the expression of MBP and S100 in EMSCs implanted on the surface of the catheter were higher than that in other groups(p < 0.05).(4)EMSCs could stably express SHH after transfection with recombinant Shh Adenovirus.(5)The recovery of SFI in SHH-EMSCs Group(D group)was faster than that in other injury groups(p < 0.05);(6)At 5 months postoperatively,the grafts were in good bridging state with the injured nerve,and there was no obvious local inflammatory reaction in all groups In Group D,there were more positive nerve cells in dorsal root ganglion than in other experimental groups,the cross-sectional area of gastrocnemius fibers was larger than that in other injury groups,and The density of NF-200 positive nerve fibers in the catheter in group D was significantly higher than that in other injury groups,and the relative expression levels of NF-200 and MBP were higher in group D than in other injury groups(P<0.05).Conclusion:(1)The plant conduit has good Biocompatibility with EMSCs,and the Lamininmodified plant-derived nerve conduit can promote the differentiation of EMSCs into Schwann cells,the results may provide a new theoretical basis for the transplantation and repair of Sciatic nerve with natural plant-derived nerve conduits loaded with seed cells.(2)The morphological and functional recovery of SD rat Sciatic nerve defects can be improved by the combination of Laminin modified plant derived nerve conduit and SHH-EMSCs transplantation...