三维微环境下神经干细胞定向分化的精细物理调控研究

For fundamentally alleviating or even healing diseases related to nerve damage and neurodegeneration, one probable approach is artificial mobilization of involved stem cells for participating in nerve regeneration. Physical stimulations have been proved to have definite significant effect on regulating stem cell behavior and promoting nerve regeneration. Cells are in three-dimensional (3D) micro-environment with complicated biophysical and biochemical factors, so that comprehensive, 3D and fine intervention in artificial bio-micro-environment may lead to more effective mobilization of related stem cells for promoting nerve regeneration. So far, 3D structured micro-distribution of biochemical composition in bio-micro-environments has been being utilized as an important and effective tool for manipulating stem cells; but the counterpart approach with 3D fine biophysical stimuli in bio-micro-environment are still far from being well exploited because of its special requirements of fabrication. In view of the above problems, this project intends to build artificial cell micro-niches integrated with physical-stimulation micro/nano-devices, flexibly and precisely manipulate physical stimuli like light, and electricity, etc., and simultaneously apply bio-chemical tools like 3D micro-distributed bio-factors. As a result, it should regulate nerve-regeneration-related stem cell behaviors like the directional differentiation of neural stem cells into nerve cells in the spinal cord, in order to provide new ideas and technical platforms for elucidating the effects of physical stimulations on neural stem cells and related mechanisms, and establishing new nerve regeneration strategies involving specific stem cells.

神经损伤和功能退行等疾病，从根本上得以缓解甚至治愈的可能方法之一为，人为动员相关干细胞参与神经再生修复。对于调控干细胞行为、促进神经再生，物理刺激已被证明具有明确的重要作用。而体内细胞处于物理、生化因素复杂的三维微环境中，故若在人工生物微环境下进行综合、三维、精细干预，可能更有效的动员干细胞参与再生修复等生理过程。目前，生化成分的微环境三维结构化散布已成为干预干细胞的重要有效手段；但与之对应的三维微环境中精细物理刺激“场”方案，因其特殊构建技术要求，仍难以较好实现。针对以上问题，本项目拟构建集成有物理刺激微纳器件的人工细胞微龛，在三维微环境中灵活、精确操纵光、电等物理刺激，兼顾生化成分三维结构化散布等生化因素，调控神经干细胞向脊髓神经细胞定向分化等神经再生相关干细胞行为，为阐明物理刺激对神经干细胞等的作用和机制、建立干细胞参与神经再生修复新策略，提供新的思路和技术支撑。

基于生物功能材料体系和“自上而下”微纳加工，模拟体内环境或者仿生驱动器，或者定制细胞刺激或者传感的生物界面材料和器件，实现精细构建多类型、多维度生物物理因子刺激和传感，是实现人工生物界面的关键之一。本项目面向细胞或者片上组织尺度的特殊跨尺度微纳器件化需求，在保证飞秒激光多光子光刻加工纳米分辨率的前提下（百纳米），优化了激光微纳加工系统的有效工作范围（约所用透镜平视场范围1.5倍，例如，用高数值孔径（1.42）60倍油镜实现~550-600微米的有效加工范围）和效率等；实现了利用飞秒激光直写等微纳加工技术，制备生物材料微纳光、电、机械驱动等器件，实现其较好的生物兼容和自身功能，可用于光、电、机械等微纳操控、物理介入刺激、生物传感等。取得的主要研究成果包括：（1）面向细胞或者片上组织的特殊跨尺度微纳器件化需求，即同时实现亚微米乃至纳米级空间分辨率和百微米、毫米级总尺寸，在保证飞秒激光多光子光刻加工纳米分辨率的前提下，优化激光微纳加工系统的有效工作范围和效率。（2）基于多光子聚合飞秒激光直写等微纳加工技术，制备生物材料基微纳光器件（如微纳波导），同时实现其较好的生物兼容和自身功能（导光、传感等），甚至在人工生物微环境中原位实时制备光等物理因子介入或者传感的微纳器件终端。（3）作为实现细胞微纳尺度诱导操控的关键因子之一，实现微纳机械驱动器，以及微纳尺度调控（例如浸润性、机械特性）的生物界面。（4）实现微纳高灵敏压力传感器，其可在三维微环境中精确构建和布置，以期用于细胞及亚细胞尺度的（干）细胞生长、运动实时原位检测、监控。在可穿戴生医传感器方面，取得了一些项目预期外的研究成果。