Preparation Of Chitosan/Carbon Nanotube-Based Composite Scaffold And Its Application In Neural Tissue Engineering

With the development of peripheral nerve tissue engineering,study on biodegradable materials have extensive applied prospect.Chitosan(Cs)is a candidate for neural tissue engineering due to its similar chemical structure to glycosaminoglycan,but it often needs to be used in combination with other materials due to its lack of elasticity and flexibility.Many natural materials can also be used to construct nerve scaffolds,such as hyaluronic acid(HA),gelatin(Gel)and collagen(Col).Generally speaking,these materials are not suitable for preparation of nerve scaffolds alone,because they are difficult to mold,and their water-solubility determines that they will not maintain a specific shape in tissue environment for a long time as a single material.Artificial synthetic materials are also commonly used in the development of nerve scaffolds,such as polyethylene glycol(PEG).However,a scaffold made with PEG alone cannot easily produce a very standardized shape.Therefore,the form of composite scaffolds is often used to improve various properties of these materials.Carbon nanotube(CNTs)composite materials are very attractive for neural tissue engineering and biosensor coatings.CNTs-scaffolds are excellent mimics of extracellular matrix due to their hydrophilicity,viscosity,and biocompatibility.On the other hand,CNTs can impart conductivity to other insulating materials,improve mechanical stability,guide neuronal cell behavior,and trigger axon regeneration.Therefore,the performance of composite scaffold can be optimized by introducing multi-wall carbon nanotubes(MWCNTs).In order to prepare a three-dimensional scaffold with good mechanical properties,degradable,non-toxic,and excellent biocompatibility,Cs was compounded with HA,Gel,Col and PEG respectively to prepare composite materials to complement their advantages.Finally,the composite scaffold with the best comprehensive performance was optimized by introducing CNTs.In this regard,this research uses different materials to construct two three-dimensional neuronal cell culture models.The specific research contents are as follows:(1)Cs/HA/Gel,Cs/Col and Cs/PEG composite scaffolds were prepared by freeze-drying method,and characterized by SEM,mechanical properties,swelling behaviour,porosity and conductivity.Compared with pure Cs,the addition of other materials reduced the average pore size,but improved the mechanical properties of the composite scaffold.Furthermore,MTT,live/dead staining,cell morphology,and immunofluorescence staining were performed to evaluate the cytotoxicity,biocompatibility,and differentiation of rat pheochromocytoma(PC12)cells on the composite scaffolds.Our results indicated that three chitosan-based composite scaffolds had good biocompatibility,while Cs/PEG scaffolds possessed higher cell survival rate and could promote the adhesion,proliferation,and differentiation of PC12 cells.To sum up,the chitosan-based composite scaffolds developed in this study can serve as promising substitutes for neural tissue regeneration.(2)The performance of the Cs/PEG composite scaffold can be optimized by introducing MWCNTs.Cs/PEG/CNTs composite scaffolds with CNTs content of 1%,3%,and 5% were prepared by freeze-drying,then their physical and chemical properties and biocompatibility were evaluated.SEM and Young's modulus measurement showed that MWCNTs increased the roughness of the pore wall,improved the hydrophobicity and enhanced the elastic modulus of the scaffold.The porosity of the scaffold ranges from 83% to 96%.It reaches a stable water swelling state within 24 h and decreases with the increase of the MWCNTs concentration.The electrical conductivity and the cell adhesion rate of the scaffold increases with the increase of MWCNTs content.Immunofluorescence showed that PC12 cells grown in scaffolds have similar characteristics to nerve cells.We measured changes in the expression of nerve cell markers by quantitative real-time polymerase chain reaction(q RT-PCR),and found that PC12 cells cultured in scaffolds expressed GAP43,NGFR and Tubb3 proteins.Preliminary research shows that the prepared Cs/PEG/CNTs scaffold has good biocompatibility and can be further applied to neural tissue engineering research.