Research On Functionalized Polymer-coated Nerve Tissue Grafts Combined With Exogenous Electrical Stimulation For Peripheral Nerve Defect Repair

Background:Peripheral nerve defects have always been one of the greatest challenges for neurosurgeons.When the distance of the nerve defect is long and direct suturing of the severed end is not possible,autologous nerve transplantation(ANT)has become the preferred treatment for this disease at this stage.However,the problems of limited number of donors,donor area damage,nerve diameter mismatch,and local neuroma formation have not been solved.To overcome these problems,tissue-engineered nerve guide conduits(NGCs)are expected to be an effective method for repairing longdistance peripheral nerve defects.There are many techniques to fabricate NGCs,such as freeze-drying,solvent casting,phase separation,three-dimensional(3D)printing,and electrospinning.Electrospun NGCs have been shown to be effective in promoting nerve regeneration in the field of peripheral nerve regeneration(PNR)by connecting defects between nerve dissections and assisting in the recovery of nerve function.Biocompatible conducting polymers(CP)have been investigated and used in various fields,such as tissue engineering,biosensors,drug delivery,and neural probes.CP can effectively enhance the adhesion and proliferation of cells.Poly(3,4-ethylenedioxythiophene)(PEDOT)is a biocompatible CP that is used in several fields due to the fine electrical conductivity,thermal and oxidative stability.In order to obtain a water-soluble polyelectrolyte system with good film-forming properties,PEDOT is doped with polystyrene sulfonate sodium(PSS).Due to the electrochemical,thermal and oxidative stability,PEDOT:PSS has received a lot of attention from researchers.These properties allow PEDOT:PSS to be used in a wide range of applications,such as nanocomposites,flexible electrodes,electrochromic displays,and transistors.In addition,there is an increasing interest in PEDOT:PSS for biomedical applications due to its good oxidative stability.CP scaffolds made of PEDOT:PSS have been used in bone repair,skin repair and PNR with good results.The nanotopography on the surface of electrospun fibers can modulate cellular functions for various tissue engineering applications.It can improve the proliferation and migration of human mesenchymal stem cells and modestly regulates the cytokine expression of macrophages.Porous poly(lactic-co-glycolic acid)(PLGA)electrospun fibers have been shown to improve the adhesion and proliferation of neural cells,and such porous nanofibers are also effective in reducing microbial colonization of their surfaces,which is also critical in the field of tissue engineering neur al repair.The pore-forming conditions of porous PLGA electrospun fibers have not been systematically explored,and the effectiveness of porous PLGA electrospun fibers NGC for in vivo experiments is unclear,and few studies have been reported on the use of PEDOT:PSS as a coating to improve the effectiveness of PNR.Purpose:This study focuses on the use of polymer-coated neural tissue grafts with exogenous electrical stimulation(ES)for the repair of peripheral nerve defects.PLGA was used to prepare porous electrospun fibers and to explore the optimal alignment and porogenic conditions.PEDOT:PSS was chosen as the polymer coating and its optimal coating concentration was explored.The physicochemical properties of the coated PLGA electrospun fiber films and their effects on nerve cell behavior were fully characterized,and then they were made into nerve tissue grafts and implanted into animals to explore the effects on promoting PNR in conjunction with exogenous ES and to investigate the mechanisms of promoting PNR.Materials and Methods:To address these main challenges,we designed and prepared a functionalized polymer-coated nerve tissue graft,characterized its physicochemical properties,and investigated its effects on nerve cell behavior through in vitro experiments,and finally implanted it into a rat sciatic nerve defect model with exogenous ES to study its role in promoting PNR and functional recovery in the rat lower extremity.The specific research protocol included the following 3 aspects:(1)We prepared PLGA electrospun fiber films with different alignments using different rotational speeds of the electrospun machine rollers and characterized them by scanning electron microscope(SEM)and water contact angle.After determining the optimal alignment,we used a phase separation method for the porogenesis of PLGA electrospun fibers and explored the porogenic effect at different dichloromethane(DCM)and dimethyl formamide(DMF)ratios.The porous PLGA electrospun films were characterized in terms of physical properties,such as density,porosity,and water absorption.We also tested the effect of different concentrations of PEDOT:PSS aqueous solutions on neuronal cell proliferation to determine the optimal coating concentration.After we coated the porous PLGA electrospun films with the optimal concentration of PEDOT:PSS solution,we performed detailed characterization of the properties of the coated electrospun films in terms of electrical conductivity,surface morphology,surface elements,mechanical properties,specific functional groups,hydrophilicity,in vitro degradation and in vivo degradation.(2)The effects of the coated electrospun films on the behavior of pheochromocytoma(PC12)cells and schwann cells(SCs)were verified in vitro by characterizing cell morphology,cytoskeleton staining and cell viability staining.(3)Then NGC made from coated electrospun films was implanted into rats and combined with exogenous ES for the repair of 10 mm sciatic nerve defect model,and the nerve regeneration and functional recovery of the lower limbs of rats were evaluated at 2 and 3 months after surgery based on the motor function,neurophysiology,bilateral gastrocnemius muscle changes,neurohistology,gastrocnemius motor end plate(MEP)and expression of neurospecific proteins.Results and Conclusions:In this study,we explored the optimal alignment and optimal surface porosity conditions of PLGA electrospun fibers and successfully prepared porous PLGA electrospun films with optimal alignment,high porosity,and water absorption.We determined the optimal coating concentration of PEDOT:PSS solution by proliferation experiments with SCs and PC12 cells.After coating the aligned porous PLGA electrospun films with this concentration of PEDOT:PSS solution,the electrospun films exhibited good electrical conductivity,surface morphology,surface element distribution,mechanical properties,hydrophilicity,and suitable degradation rates in vitro and in vivo.In vivo degradation experiments of coated and uncoated electrospun films at 1 month,2 months,and 3 months showed no significant inflammatory reactions.In vitro cellular experiments showed that the porous PLGA electrospun films coated with appropriate concentrations of PEDOT:PSS solution could effectively promote the adhesion and bipolar growth of neural cells and showed no significant cytotoxicity.Animal experiments demonstrated that the PEDOT:PSS-coated aligned porous PLGA electrospun NGC in concert with exogenous ES could effectively promote the regeneration of sciatic nerve defects in rats.The nanotopography provided by the porous PLGA electrospun films could further promote the adhesion and proliferation of nerve cells.The appropriate concentration of PEDOT:PSS coating not only increased the hydrophilicity of the electrospun films and further enhanced the bioaffinity,but also effectively improved the electrical conductivity of them.They provided a good basis for the synergistic use of functionalized polymer-coated neural tissue grafts with exogenous ES to promote nerve regeneration.In vivo experiments of SFI scores,electrophysiological tests,transmission electron microscope(TEM)characterization of axon and myelin thickness,comparison of Masson staining and MEP staining of bilateral gastrocnemius,and Neurofilament-200(NF200),myelin basic protein(MB Myelin basic protein(MBP),glial fibrillary acidic portein(GFAP),and ?3-Tubulin(Tuj-1)were shown to indicate the effect of axonal regeneration and myelination.In conclusion,functionalized polymer-coated neural tissue grafts with exogenous ES can improve the PNR effect and are expected to increase the precise repair length of neural defects,thus yielding positive economic and social benefits.