导电丝素蛋白仿生组织工程支架中的微环境动态调控及神经再生

Microenvironment of cells is very significant for nerve repair and regeneration. Due to the simple structures and poor biocompatibility, it is hard for most of the artificial nerve tissue engineering scaffolds to precisely regulate the growth, proliferation of neural cells. Some applications, such as long-segment nerve repair, were limited accordingly. Recently, the applicant successfully prepared regenerated silk fibroin (RSF) tissue engineering scaffolds with good mechanical properties, biocompatibility and biodegradability. However, the microenvironment of cells in the electrospun scaffolds cannot be regulated well. This project constructs biomimetic RSF based scaffolds with micro-channel structures by using microfluidic technology. To fabricate a novel conductive RSF channels, derivatives of poly (3,4-ethylenedioxythiophene) (PEDOT) are synthesized with / or deposited on RSF in the channels via in situ reaction of microfluids. Dynamic cell culture systems are constructed by perfusing cell culture medium and applying electric fields in the microfluidic channels. The purpose of this project is to boost the neural cell growth, morphology, distribution and orientation in the scaffolds by precisely regulating the microenvironment of cells, such as pressure driven mechanical stimulation of fluids, electric stimulation and nutrition feeding. It may also provide new ideas and methods for the design of conductive tissue engineering scaffolds and the construction of flexible bio-electronic devices.

细胞微环境对神经修复和再生非常重要。人工神经组织工程支架通常结构简单、生物相容性欠佳，难以高效引导和精确调控神经细胞的生长、增殖等活动，限制了其在长段神经修复等领域的应用。近期申请者成功制备出力学性能优异、生物相容性良好且可降解的再生丝素蛋白（RSF）静电纺组织工程支架，但尚无法精确调控细胞生长的微环境。本项目拟采用RSF作为支架构筑材料，基于微流体技术构建支架微通道结构，通过微通道内流体原位反应，实现聚（3,4-乙烯二氧噻吩）衍生物在RSF微通道表面的导电复合修饰，制备导电RSF新材料。通过压力和电渗驱动并施加电场，仿生构建神经细胞的体外动态灌注培养系统，精确调控神经细胞生长微环境，促进其在支架中的有序生长和增殖；揭示微通道中流体力学刺激、电信号刺激及营养供给对神经细胞的生长形态、分布取向的单一及协同作用机制，为导电神经组织工程支架的设计及柔性生物电子器件的构建提供新思路与方法。

细胞微环境对神经修复和再生非常重要，开发具有优异力学性能、生物相容性且可仿生调控细胞生长微环境的导电神经组织工程支架具有重要意义。本项目从再生丝素蛋白（RSF）薄膜的导电改性出发，设计制备了多种RSF导电薄膜，构建了RSF导电微流体支架，并用其构筑动态培养神经细胞的微环境，揭示了流体力学刺激和电刺激对神经细胞的诱导分化作用。设计了基于十二烷基硫酸钠（SDS）的胶束水相体系，实现了羟甲基-3,4乙烯二氧噻吩（EDOT-OH）在RSF薄膜表面有效牢固地氧化聚合沉积，制备了电导率为0.006 S/cm的RSF导电薄膜。发展了过硫酸铵（APS）/ FeCl3双氧化剂引发体系，进一步提升RSF薄膜导电性（电导率为0.089 S/cm）的同时，改善了其光学透明性，在可见光区的透光率超过70%，其有利于PC12细胞的黏附、分化，并实现了对PC12细胞的电刺激培养及该过程中细胞的实时原位观察。进一步地构筑了微通道导电的透明RSF微流体支架，其两端电阻在培养环境中低至100000 Ω，采用压力驱动的方式动态灌流培养细胞，并对细胞施加电刺激，仿生构建了神经细胞的体外生长微环境，验证了电刺激可诱导PC12细胞的生长和分化。同时，通过构建石墨烯网络镶嵌修饰的RSF导电纤维支架，证实了电刺激对神经细胞生长和分化的调控作用及RSF导电支架在神经修复领域应用的潜力。为实时监测RSF支架微通道内的作用力，基于RSF开发了平均断裂强度为710.2 MPa、光损耗低至1.0 dB/cm、声速可达3.0 km/s的可编织生物光纤，有望为后续在RSF微通道中集成可降解的光学传感器提供构筑材料。为提高导电RSF微流体体系的生物相容性、生物降解性，并进一步实现体系自供电，基于可控剥离技术制备了单分子层厚的丝素纳米带，以其为构筑基元，制备了全降解型摩擦纳米发电机，为后续与RSF微流体支架集成、实现体系的全降解及自供电奠定了基础。本项目的研究为构筑新型RSF基功能材料提供了新的思路和方法，并对扩展RSF材料在神经修复、组织再生、生物电子领域的应用具有重要启示。