Neural Tissue Engineering Based on Combinatorial Effect of Multiple Guidance Cues

产品说明：本申请的目的是提供一个机制的理解梯度的物理和化学的指导线索（GC），单独和组合，指导和调制轴突生长。这项研究将具体回答以下问题：（1）生长锥的立即转向取决于生长锥左侧和右侧的浓度梯度之间的差异;（2）立即转向和偏置转向以及生长速率调节共同作用，以引导轴突朝向其目标;（3）多种线索梯度的整合可以为轴突导向提供精确的调节机制。轴突被细胞外环境中的吸引和排斥线索的梯度引导沿着特定的通路。为了理解引导线索的梯度单独或组合对生长锥转向和生长速率调节的影响，开发能够产生精确控制的引导线索的形状梯度的平台是必不可少的。我建议开发一种廉价和高通量的技术，能够提供精确的，可重复的，和任意形状的梯度的物理和生物化学线索，以指导和调节轴突生长。在这些研究中，我们将首先在微加工电极阵列上制造载有神经生长因子的导电聚合物纳米管。为了释放截留的神经生长因子，我们将通过施加电压来驱动这些纳米管。通过改变电极阵列上的致动电压，我们将在这些微电极上产生精确控制的神经生长因子释放梯度。接下来，我们将在电极阵列上的导电聚合物纳米管上产生与基底结合的分子梯度，在这种情况下是层粘连蛋白。在导电聚合物的电聚合过程中，通过使用这种蛋白质作为掺杂剂，将层粘连蛋白包含在纳米管上。我们将在各个电极部位使用不同浓度的层粘连蛋白以获得所需的梯度分布。为了产生表面形貌的梯度，我们将在微加工电极阵列上对齐的导电聚合物纳米管的直径和表面粗糙度中产生梯度。我们将通过改变导电聚合物的电化学聚合时间来调节（a）导电聚合物纳米管的直径，以及通过改变电沉积期间施加的电流密度来调节（B）导电聚合物纳米管的表面粗糙度。最后，我们将开发一个3D导管组成的PDMS引导通道，包含纳米结构的导电聚合物，提供（i）物理和生化生长线索，和（ii）低阻抗电极监测轴突生长的电生理记录沿着再生途径。该多功能导管将在体外和体内进行测试，以确定多种引导线索的梯度对轴突生长方向和速率的影响。这些研究的结果可能会对社会产生重大影响，为解决轴突再生和指导的主要临床问题铺平道路。

DESCRIPTION: This application aims to provide a mechanistic understanding of the effect of gradients of physical and chemical guidance cues (GCs), individually and combinatory, on guidance and modulation of axonal growth. The proposed study will specifically answer to questions whether: (1) immediate turning of growth cone depends on the difference between the concentration gradients on the left- and right-hand sides of the growth cone; (2) immediate and biased turning and growth-rate modulation work together to guide axons towards their targets; (3) integration of gradients of multiple cues can provide a precise regulation mechanism for axonal guidance. Axons are guided along specific pathways by gradients of attractive and repulsive cues in their extracellular environment. To understand the effect of gradients of guidance cues individually or in combination on growth cone turning and growth rate modulation, the development of platforms that are capable of producing precisely controlled shape gradients of guidance cues is essential. I propose to develop an inexpensive and high- throughput technology that is capable of providing precise, reproducible, and arbitrarily shaped gradients of physical and biochemical cues to direct and modulate axonal growth. For these studies, we will first fabricate aligned nanotubes of conducting polymer loaded with nerve growth factor on micro-fabricated electrode arrays. To release the entrapped nerve growth factor, we will actuate these nanotubes by applying electrical voltages. By varying the actuating voltage across the electrode array, we will create precisely controlled gradients of released nerve growth factor on these microelectrodes. Next, we will generate gradients of substrate-bound molecules, in this case laminin, on conducting polymer nanotubes across the electrode array. Inclusion of laminin on the nanotubes will be achieved by using this protein as a dopant during electropolymerization of conducting polymer. We will employ different concentrations of laminin on individual electrode sites to achieve the desired gradient profile. To generate gradients of surface topography, we will create gradients in diameter and surface roughness of aligned conducting polymer nanotubes on the micro-fabricated electrode arrays. We will modulate (a) the diameter of conducting polymer nanotubes by varying the time of electrochemical polymerization of conducting polymer, and (b) the surface roughness of conducting polymer nanotubes by varying the current density applied during electrodeposition. Finally, we will develop a 3D conduit consisting of a PDMS guidance channel that contains nanostructured conducting polymers that provide (i) physical and biochemical growth cues, and (ii) low impedance electrodes to monitor axonal growth by electrophysiological recording along the regeneration pathway. This multifunctional conduit will be tested in vitro and vivo to determine the effect of gradient of multiple guidance cues on axonal growth direction and rate. The results of these studies may significantly impact society by paving the way for a solution to the major clinical problem of axon regeneration and guidance.