Biocompatible chitosan/polyethylene glycol/multi-walled carbon nanotube composite scaffolds for neural tissue engineering

Abstract

Carbon nanotube (CNT) composite materials are very attractive for use in neural tissue engineering and biosensor coatings. CNT scaffolds are excellent mimics of extracellular matrix due to their hydrophilicity, viscosity, and biocompatibility. CNTs can also impart conductivity to other insulating materials improve mechanical stability guide neuronal cell behavior and trigger axon regeneration. The performance of chitosan (CS)/polyethylene glycol (PEG) composite scaffolds could be optimized by introducing multi-walled CNTs (MWCNTs). CS/PEG/CNT composite scaffolds with CNT content of 1%, 3%, and 5% (1%=0.01 g/mL) were prepared by freeze-drying. Their physical and chemical properties and biocompatibility were evaluated. Scanning electron microscopy (SEM) showed that the composite scaffolds had a highly connected porous structure. Transmission electron microscope (TEM) and Raman spectroscopy proved that the CNTs were well dispersed in the CS/PEG matrix and combined with the CS/PEG nanofiber bundles. MWCNTs enhanced the elastic modulus of the scaffold. The porosity of the scaffolds ranged from 83% to 96%. They reached a stable water swelling state within 24 h, and swelling decreased with increasing MWCNT concentration. The electrical conductivity and cell adhesion rate of the scaffolds increased with increasing MWCNT content. Immunofluorescence showed that rat pheochromocytoma (PC12) cells grown in the scaffolds had characteristics similar to nerve cells. We measured changes in the expression of nerve cell markers by quantitative real-time polymerase chain reaction (qRT-PCR), and found that PC12 cells cultured in the scaffolds expressed growth-associated protein 43 (GAP43), nerve growth factor receptor (NGFR), and class III β-tubulin (TUBB3) proteins. Preliminary research showed that the prepared CS/PEG/CNT scaffold has good biocompatibility and can be further applied to neural tissue engineering research.

概述

目的

对壳聚糖/聚乙二醇/多壁碳纳米管(CS/PEG/MWCNT)复合支架的理化性质及对神经细胞的生物相容性进行研究, 探究碳纳米管(CNT)的最佳浓度, 同时判断其是否可以进一步应用于神经组织工程研究。

创新点

通过引入MWCNT来优化CS/PEG复合支架的综合性能。

方法

通过冷冻干燥制备CNTs的质量体积分数(g/mL)分别为0%、1%、3%和5%的CS/PEG/CNT神经复合支架, 对其进行一系列的物理化学性质的表征, 包括扫描电镜(SEM)、透射电镜(TEM)、红外光谱、拉曼光谱、杨氏模量、孔隙率、水溶胀以及导电率等。进而将神经细胞接种到支架上进行体外培养, 通过对支架上的细胞增殖及粘附、活/死细胞染色来观察所制备神经支架的细胞毒性, 通过免疫荧光及实时定量聚合酶链式反应(qRT-PCR)检测了神经细胞标志物表达的变化, 进一步探究不同支架对神经细胞分化的影响。

结论

CS/PEG/CNTs支架表现出紧密而整洁的中空网络结构, 且CNTs很好地分散在CS/PEG基质中。CNTs的添加增加了支架孔壁的粗糙度、导电性和疏水性, 提高了支架的力学性能, 降低了溶胀率和生物降解率。PC12细胞体外生物学评价结果表明:CS/PEG/CNTs支架具有良好的生物相容性, 无细胞毒性, 增强了体外神经元细胞的增殖和分化能力。总体而言, CS/PEG/3% CNTs支架具有优异的综合性能, 有望用作周围神经再生的神经元生长基质。