Neural Tissue Engineering Based on Combinatorial Effect of Multiple Guidance Cues

描述：本应用旨在提供对单独和组合的物理和化学引导线索(GC)的梯度对轴突生长的引导和调节的影响的机械性理解。这项拟议的研究将具体回答以下问题：(1)生长锥的即时转向取决于生长锥左右两侧浓度梯度的差异；(2)即时转向和偏向转向以及生长速率调节共同作用，引导轴突朝向其目标；(3)多个线索的梯度的整合可以为轴突引导提供精确的调节机制。轴突在其细胞外环境中由吸引和排斥的线索的梯度引导沿着特定的路径。为了了解引导线索的梯度单独或组合对生长锥体转动和生长速度调节的影响，开发能够产生精确控制的引导线索形状梯度的平台是至关重要的。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物理和生化线索的梯度，以指导和调节轴突生长。对于这些研究，我们将首先在微制造电极阵列上制备负载神经生长因子的定向导电聚合物纳米管。为了释放包裹的神经生长因子，我们将通过施加电压来启动这些纳米管。通过改变电极阵列上的驱动电压，我们将在这些微电极上创建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底物结合分子的梯度，在本例中为层粘连蛋白。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，可以实现层粘连蛋白在纳米管上的包合。我们将在每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，我们将在微制造电极阵列上产生取向导电聚合物纳米管的直径和表面粗糙度的梯度。我们将通过改变导电聚合物的电化学聚合时间来调节导电聚合物纳米管的直径，以及(B)通过改变电沉积过程中施加的电流密度来调节导电聚合物纳米管的表面粗糙度。最后，我们将开发一种由PDMS引导通道组成的3D管道，其中包含纳米结构导电聚合物，这些聚合物提供(I)物理和生化生长线索，以及(Ii)低阻抗电极，通过沿再生路径的电生理记录来监测轴突生长。这种多功能管道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度的影响。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这一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.