Fabrication And Biocompatibility Evaluation Of Chitosan/gelatin/hyaluronic Acid/MWCNTs Scaffold

Tissue engineering scaffolds have a porous structure that can provide enough space for the growth of cells,facilitate the transmission of nutrient and information between cells,so that can achieve in vitro expansion of cells.These properties make it be used as an important mean for nerve tissue regeneration and repair.Multi-walled carbon nanotubes(MWCNTs)have excellent properties such as mechanical strength,high conductivity and good thermal stability.They were combined with natural biological materials to prepare composite scaffolds,which can ensure that the scaffolds have well biocompatibility while improving its mechanical strength and conductivity.In this study,MWCNTs were introduced on the basis of chitosan/gelatin/hyaluronic acid(Cs/Gel/HA)scaffolds to prepare Cs/Gel/HA/MWCNTs conductive scaffolds by freeze-drying method.The physical and chemical properties,and the biocompatibility of the scaffolds which were affected by the content and modification of MWCNTs were investigated.And the scaffold with modified MWCNTs(mMWCNTs)content of 6% was screened for culturing NSCs.The effects of it on adhesion and proliferation of NSCs were investigated.Firstly,the surface chemical modification of MWCNTs was processed and the effects of reaction time and temperature on conductivity were investigated.According to the result of conductivity,the MWCNTs modified under the conditions of 2 h and 80 ℃ were chosen for the research.FTIR and Raman spectroscopy showed that the surfaces of(mMWCNTs)were grafted with-COOH.And the position of Raman spectra did not change,while the R value increased by 0.93 times.This result indicated that the chemical modification increased the chaotic degree of MWCNTs.The dispersity study showed that the chemical modification increased the dispersity of MWCNTs.The morphologic observation showed that PC12 cells could grow on MWCNTs,and the synapses on m MWCNTs were longer.Then the MWCNTs and mMWCNTs were used to prepared the Cs/Gel/HA/MWCNTs and Cs/Gel/HA/mMWCNTs conductive scaffolds,and the properties of them were researched.SEM and porosity,pore size and swelling ratio results showed that the scaffolds have a porous structure with a porosity of 83.74-96.94% and a pore size 93.93-131.77 μm,which can meet the cells growth.The addition of mMWCNTs can improve the electrical conductivity and mechanical strength of the scaffolds,the maximum can be respectively increased by 40.36% and 6.33 times.The biocompatibility results showed that Cs/Gel/HA/6%mMWCNTs conductive scaffolds have a higher cell viability,which can increased to 118.12±2.71% of control group.And there was no siginificant difference of cell adhesion rate compared to Cs/Gel/HA/10%mMWCNTs conductive scaffolds.Based on the conductivity and mechanical strength results,the Cs/Gel/HA/6%m MWCNTs conductive scaffolds were selected to culture NSCs.Finally,the effects of Cs/Gel/HA/6%mMWCNTs conductive scaffolds on the adhesion and proliferation of NSCs were investigated.NSCs immunocytochemical identification showed that the extracted NSCs had pluripotent differentiation potential and could be used as the seed cells for the experiment.NSCs cultured on scaffolds showed that the adhesion rate of NSCs on Cs/Gel/HA/6%mMWCNTs were increased to 110.27±4.41% of the control group and cell viability increased to 112.83±3.72% after culturing 7 days.The scaffolds can be used to investigate the impact of electrical stimulation on NSCs differentiation,and can be futher used to explore the application of them on the neural tissue engineering.