Design And Preparation Of Graphene Microfibers And Its Guidance To Neurons

Neuron is the most basic functional unit of the nervous system.It is composed of soma and neurite(neurite,also known as nerve fiber,which is divided into axon and dendrite).It has the function of sensing and transmitting information.Peripheral nerve is a bridge connecting the nerve center and peripheral target organs.If deficiency,the cripple rate is very high,which is part of a common clinical sickness.When the defect distance is less than 5 mm,the axis cylinder branches and buds from the proximal side of the broken end can directly pass through the distal side of the broken end under appropriate conditions such as current stimulation,and accurately bridge the fibers at ends,so as to repair nerve repair.However,when the defect is more than 10 mm,the body cannot rebuild itself and repair.As an implant to assist the repair and regeneration of damaged nerves,autologous nerve transplantation(ANT)and synthetic nerve guide conduits(NGC)still have many disadvantages,such as poor bridge matching and difficult function recovery.After a long period of development,neural tissue engineering has developed a large number of tissue-engineered nerve grafts(TENGs).However,so far,ANT is still the gold standard for the repair of human long-distance(>30 mm)nerve defects.According to the characteristics of nerve tissue and the special orientation arrangement structure of peripheral nervous system,many scientists are committed to developing the third generation of artificial synthetic nerve conduit,conductive nerve tissue engineering graft with orientation structure.In recent years,conductive polymers and inorganic materials,such as graphene based materials(GBMs),namely graphene and its derivatives graphene oxide(GO),reduced graphene oxide(rGO)and their composites,have attracted extensive research interest in the field of tissue engineering.With excellent biocompatibility,mechanical strength,stiffness and conductivity,GBMs have increasingly become new biomaterials in the field of tissue engineering.At present,studies have shown that GBMs scaffolds with topological patterned two-dimensional substrate or three-dimensional orientation structure have the function of stimulating axon germination and guiding axon directional growth.GBMs have the potential to become implants for repairing peripheral nerve defects.However,the research is mainly carried out by coating GO or rGO on the surface of polymer catheter or doping in polymer to form nerve scaffold or implant.As a modified material,GBMs have limited conductivity enhancement ability(almost all within 100 Sm^-1)and narrow regulation range of conductivity.Furthermore,the large specific surface area and structural advantages of GBMs are difficult to fulfill.Therefore,in this thesis,the natural flake graphite was used as the precursor materials to prepare the required GO nanosheets through the improved Hummers method.On this basis,the surface functional groups were modified,and the graphene microfibers were prepared by simple and effective one-step dimensionally confined hydrothermal(DCH).The toxicity of graphene fibers to nerve cells was evaluated,and the mechanism of directional induction to nerve cells was preliminarily discussed.This thesis has been carried out as the work from the following four aspects:(1)Preparation and characterization of graphene microfibersGO nanosheets with lateral size in three different ranges were prepared by changing oxidation time and centrifugal speed:>5?m,1.2-3?m and<1?m;Three kinds of graphene microfibers were prepared by DCH method,which correspond to rGOF_L,rGOF_M and rGOF_S,respectively.The structure,morphology and properties of the samples were analyzed by SEM,AFM,FTIR,XPS,Raman,electrochemical workstation and universal tensile testing machine.(2)Preparation and characterization of graphene microfibers modified by surface functional groupsCOOH-GO and NH₂-GO nanosheets were prepared by adding citric acid,ethylene glycol and ammonia into the initial GO suspension,respectively;Similarly,COOH-rGOF and NH₂-rGOF were prepared by DCH method,and the samples were characterized by SEM,XPS,FTIR,Raman and electrochemical workstation.(3)Graphene microfibers regulate the behavior of rat trigeminal neurons(TGNs)The primary TGNs of young rats were used to evaluate the guiding effect of rGOF surface topology and conductivity on the axon growth and nerve filament extension of primary neurons;CLSM and SEM were used to observe the morphology and count the cells of the scaffolds stained by immunofluorescence;The directional extension of TGNs cells on the scaffold was judged by observing the axon angle of TGNs cells on rGOF.(4)Study on the behavior of PC12 cells seeded on graphene microfibersPC12 cells and rGOF_M,COOH-rGOF_M and NH₂-rGOF_M with concentrations of 8mg ml^-1 and 10 mg ml^-1 were selected to form composite scaffolds,respectively.The cells were stained with Calcein-AM and PI,and the survival number of cells on the composite scaffolds was observed by fluorescence microscope to judge the cytotoxicity of the scaffolds.The results indicated that rGOF,COOH-rGOF and NH₂-rGOF prepared by DCH method have the adjustable physicochemical propertis,excellent biocompatibility,and the ability to promote nerve cell adhesion and directional extension,which ensure their widely use in the fields of biosensor,electrochemistry and peripheral or central nerve tissue engineering.