The Effect Of Functional Electrospun Nanofibers On Nerve Cells And Their Application In Nerve Tissue Engineering

The nervous system is a functional regulation system which plays a leading role in the body.It has complex structures and extensive distribution.The injury of peripheral nerve is a common clinical problem,which can be caused by trauma,neurodegeneration,ischemic anoxia,demyelination and so on.Once the nerve is damaged,the normal function of the organ,which is controlled by it,will also be affected.How to promote the regeneration of the injured peripheral nerve and restore the nerve function has been the focus of attention in the medical field.At present,the main methods to repair the nerve fracture and defect include surgical suture,autologous or allogenic nerve transplantation,but these methods have some limitations.The appearance of tissue engineering provides a new way of thinking for the repair and regeneration of nerve damage.Construction of tissue-engineered nerve conduits using tissue engineering technology,directional guide axon growth,improve nerve fibers on the accuracy,thus replacing autologous or allogeneic nerve graft repair of peripheral nerve injury,has become a hot research topic in the field of tissue engineering.The core of the peripheral nerve tissue engineering is to simulate the natural structure of the peripheral nerve and to construct an ideal tissue engineered nerve conduit scaffold.Scaffolds are required to mimic the structure and biological properties of natural extracellular matrix(ECM)as much as possible and to provide the most suitable living environment for cells and tissues.The electrospun nanofibers which can mimetic ECM in structure have been widely applied to the preparation of various tissue engineering scaffolds,which are also widely used in nerve tissue engineering.It is of great significance to further prepare the electrospun nanofiber scaffolds which are more suitable for the repair and regeneration of the injured nerve tissue.Based on the regeneration mechanism and regeneration process after peripheral nerve injury,we can see that the ideal nerve repair should maximize the three functions of nerve chemotaxis,neurotrophic and contact guidance.Therefore,the ideal neural tissue engineering conduit scaffold should first mimic the natural neural structure,enhance the migration of Schwann cells and guide the growth of the regenerated axon.Secondly,it also has inducible activity,inducing neural differentiation of seed cells and synaptic regeneration and extension of neurons in situ,such as loading nerve growth factor in scaffolds.It also should be conductive and can transmit electrical signals in the process of nerve regeneration so as to accelerate nerve regeneration.In addition to this,it should have appropriate degradation properties,matching mechanical properties,and so on.The factors that affect the application of electrospun nanofiber as scaffold materials to neural tissue engineering are mainly the physical and chemical properties of the fibers.The physical properties mainly include the surface morphology,diameter,orientation and electrical activity of the fibers.The chemical properties mainly include the drugs and the active factors carried by the fibers and the corresponding degradation products.Therefore,the purpose of this study is to improve the physical and chemical properties of electrospun nanofibers through functionalization,hoping to enhance the application of electrospun nanofiber scaffolds in neural tissue engineering.Based on the requirements of ideal nerve conduit scaffolds and bionics of peripheral nerve regeneration microenvironment,we have prepared functional nanofibers and evaluate the feasibility used in peripheral nerve tissue repair and regeneration.The main contents of the research are as follows:(1)In order to improve the biological activity of the fibrous scaffold,it is one of the effective means to load the bioactive molecules in the scaffold.Studies have shown that plant polysaccharides have a wide range of pharmacological activities,such as enhancing immune function,lowering blood sugar,regulating blood lipids,anti-tumor,anti-oxidation,neuroprotection and so on.More and more evidences show that Lycium barbarum polysaccharide(LBP)has protective function on nerve,which increases the feasibility of using LBP as a therapeutic drug after nerve injury.It is the first attempt to study the effects of different concentrations of LBP on the proliferation and differentiation of nerve cells.It is found that LBP can effectively promote the proliferation of PC12 and Schwann cells,and the neuronal differentiation of NGF induced PC12 cells.At the same time,the optimum concentration was determined at 50-100 ?g/m L.The LBP was loaded into nanofibers by coaxial electrospinning to make the scaffold with LBP sustained release properties.It also characterized the morphology,structure and properties of scaffolds,the controlled release properties of fiber scaffolds on LBP and the effects on nerve cell behavior,so as to evaluate the feasibility of the active scaffolds for peripheral nerve tissue engineering.The results of in vitro release show that LBP can be released slowly for more than 2 months,indicating that the fibrous scaffold has the function of load and controlled-release on LBP.Further cell experiments confirmed that the LBP sustained-release fibrous scaffold exhibited good biocompatibility for PC12 cells and Schwann cells,especially PLGA-LBP30 fiber scaffolds,which could significantly promote the proliferation of PC12 cells and Schwann cells.The results of the differentiation of PC12 on the scaffold showed that the LBP released in the scaffold had a significant effect on the neuro-differentiation of NGF induced PC12 cells.Moreover,the expression of myelin basic protein(MBP)in Schwann cells and the synapse extension of dorsal root ganglion(DRG)neurons also showed significant enhancement.The experimental results reveal the positive role of LBP in the regeneration of peripheral nerve tissue,while the sustained-release functional nanofiber scaffold loaded with LBP has potential application in the repair and regeneration of peripheral nerve injury.(2)Above studies have found that LBP can promote the induction activity of NGF,while LBP alone could not achieve good induction effect.This also confirmed that NGF played an irreplaceable role in neuronal differentiation and synaptic elongation.Therefore,the introduction of NGF in the scaffold is an effective way to further improve the function of the scaffold.The NGF sustained-release microspheres were further introduced on the basis of the preparation of LBP sustained-release fibrous scaffold to enhance the induced activity of the scaffold.First,by exploring the influence of solvents,polymer solution concentration and load voltage on the morphology and particle size of the electronic spray microspheres,we determined the best conditions for preparing NGF loaded microspheres.Microspheres / fiber composite scaffolds with simultaneous loading of NGF and LBP were prepared by combined electrospray and electrospinning.In vitro release behavior showed that LBP and NGF could release slowly for more than 40 days from the composite scaffold,indicating that the composite scaffold as a drug delivery system has good load and control ability for LBP and NGF,and meets the requirement of peripheral nerve repair cycle.The cell proliferation test showed that the load of NGF sustained-release microspheres had no significant effect on the biocompatibility of the scaffolds.The results of differentiation of PC12 cells on scaffolds showed that NGF released from the microspheres of the scaffolds maintained good bioactivity,and had a good induction effect on neuronal differentiation of PC12 cells,and had higher induction efficiency than exogenous NGF.Therefore,the microspheres / fiber composite scaffold can be used not only for nerve tissue engineering scaffolds to support the growth of nerve cells,but also can be used as a delivery system sustained release of LBP and NGF.The synergistic effect of LBP and NGF provides a better microenvironment for the proliferation and differentiation of nerve cells,and has the potential to be used in neural engineering.(3)In order to further improve the function of the fiber scaffold,a multi wall carbon nanotube(MWCNTs)with conductive activity was introduced into the fiber.First,the influence of the load of MWCNTs on the morphology and performance of the fiber was studied.The results of SEM observation showed that when the load of MWCNTs increased to 12%,a large number of reunions were formed on the surface of the scaffold.Therefore,the fiber of 2%,4% and 8% of the MWCNTs was selected for further study.From the mechanical properties test,we found that the introduction of MWCNTs makes the mechanical properties of the fiber scaffold including breaking strength and elongation decreased.This may be attributed to the addition of Span 80 in order to increase the dispersity of MWCNTs in the preparation of the scaffold,and was also related to the distribution of MWCNTs in the scaffold.In order to improve the surface hydrophilicity of fiber scaffolds,air plasma treatment was carried out on the scaffold.The results showed that the surface morphology and mechanical properties of the scaffolds did not change significantly after the plasma treatment,but the hydrophilicity increased significantly.The cell adhesion experiment further confirmed the improvement of the hydrophilicity and biocompatibility of the scaffolds.Further cell experiments showed that under 8% MWCNTs loading,scaffolds did not produce cytotoxicity on PC12 cells and Schwann cells,and the load of MWCNTs promoted the proliferation of two kinds of cells on scaffolds.SEM and immunofluorescence staining results showed that the scaffolds loaded with MWCNTs significantly promoted the differentiation and synaptic growth of PC12 cells,the axonal extension of DRG neurons,and the growth of Schwann cells,especially when the content of MWCNTs was 8%.All the results indicated that nanofibrous scaffolds containing MWCNTs can provide good microenvironment for the proliferation,differentiation and synaptic extension of neural cells,and can serve as effective neural conduit scaffolds for peripheral nerve regeneration.(4)On the basis of above,in order to further improve the guiding role of scaffolds for nerve cell growth,a highly aligned MWCNTs containing nanofiber was prepared by using high-speed rotating roller as receiving device.In addition,a large number of experiments show that appropriate electrical stimulation can effectively activate damaged neurons,and guide growth cone directional regeneration,that is,electrical signals can promote damaged nerve regeneration.Therefore,the response of neural cells to MWCNTs-based highly oriented nanofibrous scaffolds was studied by further combined external electrical stimulation.After the Poly-L-lysine(PLL)was adsorbed on the surface of the scaffold,the surface hydrophilicity and cell adhesion were significantly improved.Further cellular experiments showed that the proliferation of PC12 and Schwann cells was enhanced in the oriented fibrous scaffolds,and the orientation of scaffold fibers significantly guided the differentiation and extension of PC12 cells and the migration and growth of Schwann cells.After additional electrical stimulation,it was found that the synergistic effect of the fiber aligned topology and exogenous electrical signals could further guide the neuronal differentiation of PC12 cells and the axonal extension of DRG neurons in rats.In addition,the proliferation and migration of Schwann cells and the expression of myelinated proteins were also jointly induced by the orientation fiber and the exogenous electrical stimulation.Therefore,the results showed that the conductive fiber scaffold combined with appropriate electrical stimulation had the great potential to promote the repair and regeneration of the injured peripheral nerve.(5)Based on the previous research results,we try to load NGF and LBP into PLGA/MWCNTs nanofiber scaffolds at the same time,and prepare a conductive fiber composite scaffold with the orientation of sustained release LBP and NGF.So that the scaffolds are more active and functional,and get the composite scaffolds that integrate LBP and NGF's biochemical signals,fiber orientation topology,conductivity and so on.The synergistic effect of conductive scaffold combined with electrical signal on nerve cell differentiation and axon extension was explored.Through the study of the release behavior of LBP and NGF in vitro,it was found that both LBP and NGF could be released slowly from the scaffold.The proliferation of PC12 cells and Schwann cells on scaffolds were effectively enhanced,and the differentiation of PC12 cells and the growth and migration of Schwann cells were also further improved.In addition,the release of LBP and NGF from the scaffolds increased significantly after being exposed to electrical stimulation.After additional exogenous stimulation,the neural differentiation of PC12 cells and the growth of synapse elongation of primary neurons were further enhanced.The above results suggested that the scaffold could serve as a scaffold to provide effective support and contact guidance for cell proliferation,differentiation and migration.On the other hand,it could be used as a drug delivery system to release LBP and NGF at the damaged part,so as to further improve the rate of nerve regeneration,and had potential application in peripheral nerve tissue repair and regeneration.Combined with external electrical stimulation,it could further exert its potential.(6)After nerve injury,inflammatory reaction often occurs.Moreover,many scaffolds made of synthetic materials often cause inflammation and oxidative stress.Scaffolds with antioxidant properties can effectively overcome this problem.PCL was grafted onto natural antioxidant lignin by solvent free ring opening polymerization,and two lignin/PCL copolymers(LP2 and LP4)with antioxidant properties were obtained.The nanofiber scaffolds containing the copolymer were prepared by the electrospinning technology.Compared with the commonly used antioxidants,vitamin E,lignin/PCL copolymers and nanofiber scaffolds containing the copolymers showed better antioxidant properties.Cell proliferation assay further showed that the antioxidant nanofiber scaffold had good biocompatibility and showed a good protective effect on oxidative damage of Schwann cells caused by H2O2.By immunofluorescence staining and laser scanning confocal microscopy observation,it was found that fibrous scaffolds containing LP2 and LP4 could promote the expression of myelin basic protein(MBP)in Schwann cells and the synaptic growth and elongation rate of primary DRG neurons.And the effect of LP4 is more obvious than that of LP2.These results showed that the antioxidant fibrous scaffold has the potential to be used as a nerve conduit material for peripheral nerve tissue engineering.