Experiment Study Of Chitin-Polydopamine Conduit In Repairing Nerve Defect And Preventing Neuroma Formation

Peripheral nerve injury(PNI),a universal intractable clinical problem,is usually arised from traffic or industrial accidents.Nerve defect is one of the worst cases of PNI as it results in serious damage of motor function and sensory perception.Despite the certain extent of self-regenerative capability,repair of long-range peripheral nerve transection injury was still a formidable challenge in clinic and the therapeutic effect was normally unsatisfactory.Though the autograft transplantation remains the gold standard in large gap defects repair,neuroma formation,limited supplies,additional damages to the donors and size mismatch significantly impede its practical application.Besides,neuroma formation after PNI as a long-term deficiency leads to spontaneous neuropathic pain that impairs patients’ life quality.In order to prevent neuroma formation,utilizing nerve conduit to bridge or cap injured nerve has been considered as a promising approach.Numerous efforts have been devoted to fabricating the nerve guidance conduit as a reasonable alternative for autograft.The nerve guidance conduit is an affirmative clinical strategy for regenerating the critical-sized peripheral nerve injury and preventing neuroma formation.In our previous studies,the polysaccharide chitin was applied to fabricate the hydrogel film for inducing the impaired sciatic nerve regeneration through incorporating the conductive poly(3,4-ethylenedioxythiophene)nanoparticles(PEDOT NPs)and modifying with cell adhesive tetrapeptide Cys-Arg-Gly-Asp(CRGD)(ChT-PEDOT-p).The partial deacetylation process of chitin for exposing the amino groups was performed to(i)improve the electrostatic interaction between chitin and the negative charged PEDOT for enhancing the composite hydrogel strength;(ii)offer the active sites for peptide modification.The as prepared hydrogel remarkably promoted the in vitro RSC-96 adhesion and proliferation,as well as the Schwann cell activity related gene S100,NF-200 and MBP expression.Function of gastrocnemius muscle and thickness of myelinated axon in chitin/PEDOT groups were analogous to the autograft in 10 mm rat sciatic nerve defect.Immunofluorescence,immunohistochemistry,western blotting and toluidine blue staining analysis on the regenerated sciatic nerve were explained that the attachment and proliferation enhancement of RSC-96 and angiogenesis were the vital factors for the chitin/PEDOT composite to facilitate the nerve regeneration.In this study,the main contents of this work include the following aspects:(1)ChT-PDA NGC construction and characteristic evaluation: the crosslinked chitin solution dissolved in Na OH/uera at low temperature was molded by freeze-thaw method,and then immersed in Tri buffer containing dopamine monomer to obtain polydopamine-coated ChT-PDA NGC.The surface structural characteristics,stability and mechanical properties of nerve conduits were evaluated by scanning electron microscope,XPS,fourier infrared spectroscopy,mechanical testing machine,and thermogravimetric analysis.The composite NGCs exhibit controllable water uptake performance,uniform density porosity,ideal mechanical properties and stability.FT-IR analysis indicated the increased transformation amide group II and acetamide group I.(2)ChT-PDA NGC biocompatibility,cellular proliferation and neuronal differentiation evaluation: RSC 96 were applied to evaluate biocompatibility,cellular proliferation,neuronal differentiation of ChT-PDA NGC in vitro.Hemolysis testing,CCK-8,live/dead staining assay,scanning electron microscope and immunofluorescent were performed to evaluate the blood compatibility,cell proliferation,cell viability,cell morphology,cell attachment and neuronal differentiation of ChT-PDA NGC,respectively.The results indicated that both ChT-PDA and ChT NGC showed excellent blood compatibility and cytocompatibility.Meanwhile,ChT-PDA NGC demonstrated enhanced cell adhesion,migration,neurite extension,as well as upregulated expression of neurotrophic factors(Tuj1,S100 and GFAP)compared to ChT NGC.(3)ChT-PDA NGC promoted nerve regeneration: The designed NGCs were then used in 10 mm rat sciatic nerve defect model for assessing nerve regeneration in vivo.Functional index,H&E staining,immunofluorescence staining,toluidine blue staining,transmission electron microscopy and western blotting were performed to evaluate the effect of nerve regeneration.In vivo degradation and toxicity of ChT-PDA NGC were assessed by H&E staining.The results showed that ChT-PDA NGC could stimulate the secretion of neurotrophic factors(Tuj1,S100 and GFAP)in rats,evidently promote the improvement of functional restoration and increase the diameter,the density and thickness of the myelin sheath of the regenerated nerve,and attenuate muscle denervation.In addition,both of the NGCs were biodegradable in vivo and nontoxic on the main organs,such as heart,liver,spleen,lung and kidney.(4)ChT-PDA NGC prevented neuroma formation: In order to investigate the ability of the ChT-PDA NGC in preventing neuroma formation,rat sciatic nerve resection models were constructed by excising the distal ends of nerve and the proximal stumps were capped by ChT-PDA NGC in vivo.Autotomy behaviors,morphology,immunohistochemical,q PCR and transmission electron microscopy were performed to assess the therapeutic efficacy for preventing the formation of neuroma.The results indicated that the ChT-PDA NGC capping group obtained lower autotomy score with less inflammatory infiltrations and collagen deposition.Transmission electron microscopy of the regeneration of myelin sheath in both groups showed that abnormal myelination was only present in the uncapped groups.The ChT-PDA capping conduits not only acted as physical barriers to prevent the invasion of inflammatory infiltration in the nerve tissue but also provided a moderate microenvironment for promoting nerve repairing and inhibiting neuroma formation during nerve recovery.In summary,we fabricated ChT-PDA composite conduits with micropatterned inner structure which demonstrated considerable mechanical properties,porosity,and stability.Such conduits exhibited excellent biocompatibility and could facilitate RSC-96 proliferation,migration,and expression of versatile neurotrophic factors(Tuj1,S100 and GFAP).The ChT-PDA NGC could effectively promote axon growth and accelerate nerve regenerationin vivo.Furthermore,the ChT-PDA capping conduits also demonstrated a positive effect in preventing neuroma formation through coordinating the inflammation response,collagen deposition,and demyelination process.Collectively,these findings not only confirms that ChT-PDA NGC have great potential for peripheral nerve regeneration,but also provides promising evidence for inhibiting neuroma formation.This research may provide a new alternative application of chitin related materials in peripheral nerve tissue engineering field.