基于脱细胞基质水凝胶与微流控仿生三维培养的运动/感觉神经选择性再生机制研究

The accurate entry of motor and sensory nerve is the key to improve clinical efficacy after peripheral nerve injury. It has been noticed that the nerve fiber growth relies on the topographical cues and biochemical signals within the regenerative sites and exhibits high selectivity in vivo. However, the role and mechanism for the preferential growth of motor/sensory nerves within microenvironment has not been clarified. Previously, we have prepared a decellularized nerve matrix hydrogel, which has shown its functional properties that mimic the extracellular matrix in vivo. Since the regenerative microenvironment cannot be ideally simulated by traditional 2D cell culturing, a 3D multi-channel cell culturing microfluidic system with precisely controlled architectures will be established to realize a highly biomimetic nerve regeneration process. In this approach, motoneurons and sensory neurons will be cultured in the decellularized nerve matrix hydrogel. Our preliminary experiments demonstrated that the preferential neurite growth relies on physical topography of the microchannels. In order to study the role of nerve reinnervation within the regenerative microenvironment, regarding to the sizes of microchannels, composition of the culture media, species and concentration of the growth factors, and Schwann cells distribution within the culturing environment. Overall, the implementation of this project will serve to understand the role of preferential motor/sensory nerve reinnervation and provide theoretical basis for detailed design and manufacture of functional tissue engineering nerve grafts for clinical applications.

运动和感觉神经的精准匹配再生是提高周围神经缺损修复临床疗效的关键。已知神经纤维的生长对再生区域物理构型和生化信号存在不同程度的依赖性和选择性，但微环境影响运动和感觉神经选择性再生的机制未明。课题组前期制备了脱细胞神经基质水凝胶，发现其作为培养介质可高度模拟周围神经细胞外基质。鉴于传统的二维细胞培养模型无法精准模拟体内微环境，我们构建了微流控三维细胞培养装置，初步证实了神经轴突的生长对微通道空间物理构型存在选择性。为深入探究运动/感觉神经的选择性再生规律，本项目拟利用尺寸与构型控制精准的多通道微流控装置，以高度仿生的脱细胞神经基质水凝胶模拟再生神经内环境，三维培养运动和感觉神经元，探讨通道孔径大小、介质成分、因子种类与浓度以及施万细胞分布等微环境因素对神经再生的影响。本项目的实施为探明运动/感觉神经的选择性再生机制、指导组织工程神经移植物的精确设计与生物制造提供了理论依据。

长段周围神经缺损的再生修复治疗是临床医学的重点、难点问题。决定神经能否成功再生的关键因素是运动和感觉神经轴突能否分别快速生长并准确进入远端相应的再生通道，从而分别重新支配肌肉和皮肤。前期工作提示，运动/感觉神经的这种选择性再生模式，可能与运动、感觉神经轴突所进入的再生通道具有特定物理构型和营养供给有关，但具体机制不明。运动和感觉神经的精准匹配再生是提高周围神经缺损修复临床疗效、促进机体功能恢复的关键。本项目利用精确的微纳加工技术设计并成功制作出一系列具有不同结构和尺寸的微通道细胞三维培养装置，以高度仿生的脱细胞神经基质水凝胶模拟再生神经内环境，三维培养运动和感觉神经，定时、定量地观测和分析了物理空间及细胞排布对神经轴突生长的影响和引导作用。通过神经元与施万细胞的三维共培养，掌握了神经轴突定向生长的基本规律，优化实验条件，促进神经轴突在微通道内的定向快速生长。项目通过对微流控三维培养装置的通道尺寸控制，研究并分析了神经轴突定向延伸、施万细胞迁移及排列、神经髓鞘化以及集束化等受环境物理因素影响的作用，探讨了二者在不同时空分布条件下协同生长的可能机制。本项目通过对神经纤维生长的体外三维培养，提出对长段缺损性周围神经修复材料的结构、功能化设计要求，为神经再生微环境的构建提供实验依据与理论指导。