Construction Of Graphene Composite Nanofiber Neural Scaffolds For Peripheral Nerve Regeneration

Peripheral nerve injury caused by trauma,neurodegeneration,ischemia and hypoxia has become one of the common clinical diseases.Once the nerve is broken or damaged,it will lead to the interruption of nerve signal transmission pathway,which will affect the function of the corresponding target organs,and then the function of the body will be obstructed.How to promote the regeneration and function recovery of the damaged peripheral nerve has been the focus of attention in the medical field.Up to now,autologous or allogeneic transplantation is still the common clinical treatment for peripheral nerve injury,but there are some limitations to restrict their wide application.The development of tissue engineering provides an alternative strategy for the construction of nerve grafts.Neural tissue engineering scaffold,as a temporary micro-environment for the growth of nerve cells,is the key to ensure the success of nerve regeneration.The ideal neural scaffold should have(1)simulate the natural neural structure,providing the most suitable microenvironment for the nerve cells/tissues growth;(2)mechanical properties to provide mechanical support for nerve regeneration;(3)certain inducible activity to promote the migration,proliferation,differentiation of seed cells and growth of regenerative axons;(4)suitable electrical conductivity,which can transmit bioelectrical signals to promote nerve regeneration and functional recovery;(5)appropriate degradation performance,providing enough space for the nerve tissue growth,and avoiding squeezing the new nerve.Electrospinning is a simple yet versatile method to fabricate multiscale nanofibers with great similarity to the native extracellular matrix(ECM),which has been widely used in nerve tissue engineering.Graphene and graphene oxide,a novel carbon nanomaterial,have attracted great attention in the biomedicine field owing to their unique 2D honeycomb nanostructure and excellent physicochemical properties including high specific surface area,excellent mechanical properties and electrical conductivity.Studies have clearly demonstrated that graphene and its derivatives can promote the nerve cells proliferation and differentiation.Therefore,based on the biomimetic peripheral nerve regeneration micro-environment and the basic requirements of ideal neural scaffold,this project aims to modify the physical and chemical properties of nanofibers with the unique biological activity or electrical conductivity of graphene to further enhance their application in nerve tissue engineering.The study mainly consists of following contents:(1)Ap F/PLCL nanofiber scaffolds with different weight ratios were prepared via electrospinning.Through characterization of the morphology,structure,surface wettability and mechanical properties,the performance differences of Ap F/PLCL nanofibers with different weight ratios were systematically compared.The Schwann cell(SCs)proliferation and morphology were studied on different scaffolds via MTT and SEM.The results showed that compared with other nanofiber scaffolds,when the weight ratio of Ap F to PLCL was 25:75,its mechanical properties and biocompatibility were better.The feasibility of the scaffold as a tissue engineering nerve scaffold was clarified,which provided the basis for further studies.(2)Based on the research in chapter 2,bioactive graphene oxide(GO)was modified on the surface of Ap F/PLCL nanofibers to prepare bioactive composite nanofiber scaffolds.SEM,FTIR,Raman,XPS and other basic physicochemical properties showed that GO was successfully modified on the surface of the nanofibers without destroying the unique bionic structure of the nanofibers.Meanwhile,the presence of GO improved the mechanical and hydrophilic properties of the scaffolds,and this effect is enhanced with the increase of GO content.In vitro,GO functionalized Ap F/PLCL nanofibers significantly promoted SC migration and proliferation.Furthermore,GO modified nanofiber scaffolds promoted the PC12 cells differentiation by up-regulating the focal adhesion kinase(FAK)expression.In vivo,GO modified NGC scaffolds could significantly promote nerve regeneration and function recovery.The above results showed that GO modified scaffolds could provide a good bioactive microenvironment for nerve cells and tissues.(3)Based on the research in chapter 3,GO modified nanofiber scaffolds were reduced to obtain conductive reduced oxide graphene(RGO)composite nanofiber scaffolds.Light microscopy,Raman,XPS and contact angle confirmed that GO on the fiber scaffold was successfully reduced to RGO.The RGO coating on the surface of nanofibrous scaffolds significantly increased the conductivity and this effect is enhanced with the increase of RGO content.In vitro,the biological response of SCs and PC12 cells on conductive RGO modified scaffolds were systematically studied by further combined external electrical stimulation(ES).The results showed that the conductive RGO modified scaffolds significantly enhanced SC migration,proliferation,and myelination including myelin-specific gene expression and neurotrophic factor secretion via ES.In addition,these conductive scaffolds could induce PC12 cells differentiation under ES without the use of additional chemical inducer.Furthermore,the conductive NGC was fabricated and implanted into the rat sciatic nerve defect.After 4 and 12 weeks,through the evaluation of the sciatic nerve function index(SFI),Masson staining of the resected triceps surae muscle(TSM),TSM weight analysis,histology and morphology,immunohistochemistry,and immunofluorescence,it was consistently found that the existence of the conductive RGO can significantly promote nerve regeneration and function recovery.These results indicated that conductive RGO scaffolds combined with appropriate ES have great potential in peripheral nerve tissue engineering.(4)To simulate the fascicle-like architecture of nerve tissues,based on the research in chapter 2 and 3,a multiscale strategy was adopted to fabricate 3D biomimetic multichannel spongecontaining nerve guidance conduit(MCS-NGC)from Ap F/PLCL/GO nanofibers via nanofiber dispersion,template-molding,freeze-drying and crosslinking.The physicochemical properties of MCS such as morphology,mechanical properties,and degradation were characterized.The resultant MCS exhibited parallel multichannels(? = 125 ?m)surrounded by biomimetic fibrous fragments and the porosity is 92.7%.The degradation of MCS rate can be regulated by crosslinking time and MCS scaffold showed elastic property.In vitro studies showed that MCS supported the attachment and growth of SCs mainly along the luminal surface of microchannels.Meanwhile,MCS had the ability to enhance the SCs myelination,and the NGF from SCs secretion has biological activity which could significantly promote PC12 cells differentiation.Furthermore,the MCS was used as filler to prepared MCS-NGC and implanted rat sciatic nerve defects.After 12 weeks,based on a series of analysis(walking track,triceps weight,morphogenesis,vascularization,axonal regrowth and myelination),The MCS could provide more adhesion sites for the SCs growth and promote axonal growth for peripheral nerve regeneration by promoting SCs myelination.In addition,MCS as a filler for NGC almost completely degraded within 12 weeks.These findings demonstrate that the 3D hierarchical nerve guidance conduit with fascicle-like structure and bioactivity have great potential for peripheral nerve repair.In conclusion,based on the current hot spots,three kinds of nerve tissue engineering scaffolds were designed by combining the unique physicochemical properties of graphene with the bionic structure of nanofibers.The potential of these scaffolds in peripheral nerve regeneration was investigated in vitro and in vivo.This study provides a new method to construct functional neural graft and an alternative strategy to facilitate in situ nerve regeneration.