中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2018, Vol. 13 ›› Issue (8): 1455-1464.doi: 10.4103/1673-5374.235303

• 原著:脑损伤修复保护与再生 • 上一篇    下一篇

可用于神经组织修复的新型导电聚吡咯/丝素蛋白支架

  

  • 收稿日期:2018-05-28 出版日期:2018-08-15 发布日期:2018-08-15
  • 基金资助:

    中国国家自然科学基金项目（81671823, 81701835），中国重大研究发展项目（2016YFC1101603），南通市自然基金研究项目（MS12016056）

Novel conductive polypyrrole/silk fibroin scaffold for neural tissue repair

Ya-Hong Zhao1, 2, Chang-Mei Niu3, Jia-Qi Shi3, Ying-Yu Wang4, Yu-Min Yang1, 2, Hong-Bo Wang1   

  1. 1 Key Laboratory of Science and Technology of Eco-textiles, Ministry of Education, Jiangnan University, Wuxi, Jiangsu Province, China
    2 Key Laboratory of Neuroregeneration, Co-innovation Center of Neuroregeneration, Nantong University, Nantong, Jiangsu Province, China
    3 Medical School, Nantong University, Nantong, Jiangsu Province, China
    4 Wen Zheng College, Soochow University, Suzhou, Jiangsu Province, China
  • Received:2018-05-28 Online:2018-08-15 Published:2018-08-15
  • Contact: Yu-Min Yang, Ph.D. or Hong-Bo Wang, Ph.D., yangym@ntu.edu.cn or wxwanghb@163.com.
  • Supported by:

    This study was supported by the National Natural Science Foundation of China, No. 81671823, 81701835; a grant from the National Key Research and Development Program of China, No. 2016YFC1101603; a grant from the Natural Science Research Program of Nantong of China, No. MS12016056.

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摘要：

3D打印技术是一种理想的制造复杂三维生物结构的方法，并可通过计算机辅助逐层设计混合生物材料，但在制备精确模拟天然组织微纳米结构的生物材料领域仍有挑战性。制备静电纺技术的纳米级生物材料，有利于神经细胞的粘附，迁移，增殖，分化，以及修复损伤神经的再生。设计拟将3D生物打印联合静电纺丝制备新型导电聚吡咯/丝素蛋白支架。实验通过三维生物打印丝素蛋白支架，然后以聚吡咯包被支架，以自制静电纺丝装置将丝素蛋白纳米纤维沉积在聚吡咯/丝素蛋白支架，制成新型导电聚吡咯/丝素蛋白支架。以傅里叶变换红外光谱分析支架成分，以光镜和扫描电镜观察支架的表面形态，以万用表测量支架的电阻率，以光镜观察支架在37℃的水和DMEM浸泡30d后表面形态变化以评估其稳定性。见合成的导电聚吡咯/丝素蛋白支架存在聚吡咯及丝素蛋白的特征峰，表面存在静电纺丝纳米纤维层的组织结构，电导率为1×10−5–1×10−3 S/cm，稳定性为66.67%。此外，以MTT法测量支架的体外细胞毒性，以荧光显微镜观察EdU标记许旺细胞确定细胞增殖情况，以免疫组化染色检测细胞中S100β的免疫阳性反应，以扫描电镜观察粘附许旺细胞的形态。发现导电聚吡咯/丝素蛋白支架没有细胞毒性，不影响许旺细胞增殖，在支架上形成伪足，许旺细胞排列整齐，表明制备的导电聚吡咯/丝素蛋白支架具有良好的生物相容性，可作为神经组织工程中应用的适宜材料。

orcid:0000-0002-6211-8122(Ya-Hong Zhao)

关键词: 复合纳米纤维, 支架, 3D打印, 静电纺丝, 丝素蛋白, 聚吡咯, 导电性, L929细胞, 许旺细胞, 生物相容性, 神经修复, 神经再生

Abstract:

Three dimensional (3D) bioprinting, which involves depositing bioinks (mixed biomaterials) layer by layer to form computer-aided designs,is an ideal method for fabricating complex 3D biological structures. However, it remains challenging to prepare biomaterials with micro-nanostructures that accurately mimic the nanostructural features of natural tissues. A novel nanotechnological tool, electrospinning,permits the processing and modification of proper nanoscale biomaterials to enhance neural cell adhesion, migration, proliferation,differentiation, and subsequent nerve regeneration. The composite scaffold was prepared by combining 3D bioprinting with subsequent electrochemical deposition of polypyrrole and electrospinning of silk fibroin to form a composite polypyrrole/silk fibroin scaffold. Fourier transform infrared spectroscopy was used to analyze scaffold composition. The surface morphology of the scaffold was observed by light microscopy and scanning electron microscopy. A digital multimeter was used to measure the resistivity of prepared scaffolds. Light microscopy was applied to observe the surface morphology of scaffolds immersed in water or Dulbecco’s Modified Eagle’s Medium at 37°C for 30 days to assess stability. Results showed characteristic peaks of polypyrrole and silk fibroin in the synthesized conductive polypyrrole/silk fibroin scaffold, as well as the structure of the electrospun nanofiber layer on the surface. The electrical conductivity was 1 × 10−5–1 × 10−3 S/cm, while stability was 66.67%. A 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay was employed to measure scaffold cytotoxicity in vitro. Fluorescence microscopy was used to observe EdU-labeled Schwann cells to quantify cell  proliferation. Immunohistochemistry was utilized to detect S100β immunoreactivity, while scanning electron microscopy was applied to observe the morphology of adherent Schwann cells. Results demonstrated that the polypyrrole/silk fibroin scaffold was not cytotoxic and did not affect Schwann cell proliferation. Moreover, filopodia formed on the scaffold and Schwann cells were regularly arranged. Our findings verified that the composite polypyrrole/silk fibroin scaffold has good biocompatibility and may be a suitable material for neural tissue engineering.

Key words: nerve regeneration, composite nanofiber, scaffold, three dimensional bioprinting, electrospinning, silk fibroin, polypyrrole, L929 cells, conductivity, Schwann cells, biocompatibility, nerve repair, neural regeneration