Harnessing Neurotropism to Sort Sensory and Motor Axons

再生外周轴突的随机靶向损害神经损伤后的感觉和运动功能 修复，并限制了肢体截肢后再生假体界面的有用性。再生 运动神经轴突到皮肤的再生不提供功能;皮肤神经轴突到肌肉的再生降低感觉。 类似地，截肢后近端神经残端内的感觉/运动定位不良， 与特定功能相对应的孤立轴突。这些问题可以通过分选再生来克服 轴突形态。然而，以前的努力，分裂轴突人口享有有限的成功，为三个 原因：1）关于生长因子仅吸引感觉轴突或仅吸引运动轴突的比较数据很少; 2） 在迄今为止描述的结构中，很少有再生轴突被给予同等的机会来对两者做出反应。 生长因子; 3）我们最近已经表明感觉轴突粘附于运动轴突并限制它们的生长， 这是一个以前没有意识到的因素。该项目的目标是开发一种工程方法， 轴突从混合神经干再生到离散的感觉和运动通道。这些渠道可以 然后用于神经损伤后支配个体运动和感觉神经，或用于控制个体 肌肉通过再生假肢接口。该项目测试了两个核心假设：1）有可能 鉴定指导仅运动轴突或仅感觉轴突再生的亲神经生长因子（Aim I），和 2)这些因子的重叠梯度可用于分离再生的感觉和运动轴突（Aim II）。这些假设将在我们的混合神经再生的器官型模型中进行测试，其中感觉神经再生的器官型模型将在神经再生的器官型模型中进行测试。 表达番茄红的轴突和表达YFP的运动轴突在一个三维空间内结合， 周围神经节段。在切断神经后，颜色编码的轴突长出一个没有特征的 胶原蛋白/层粘连蛋白片，其中它们的寻路可由生长因子梯度引导。这些梯度将 使用微囊化生长因子和生物打印技术建立， 实验室在目标I中，我们将评估感觉或运动轴突对候选生长因子的反应， 改变生长因子浓度、梯度陡度和基质以使轴突转向最大化。In Aim II 我们将产生目标I中最有效因子的重叠梯度，同时阻止 感觉和运动轴突来最大化它们的转动。呈现再生轴突重叠的能力 在最小化轴突-轴突的环境中，真正的“仅运动”和“仅感觉”生长因子的梯度 因此，相互作用将使我们能够优化这些轴突群体的分离。如果成功的话， 然后，可以使用该平台来构建一个三维假体， 和体内运动轴突，可用于改善50，000例患者/年的结果， 神经修复和近300名最近上肢截肢的退伍军人。

Random targeting by regenerating peripheral axons compromises sensory and motor function after nerve repair, and limits the usefulness of regenerative prosthetic interfaces after extremity amputation. Regeneration of motor axons to skin provides no function; regeneration of cutaneous axons to muscle degrades sensation. Similarly, poor sensory/motor localization within the proximal nerve stump after amputation defeats attempts to isolate axons corresponding to specific functions. These problems can be overcome by sorting regenerating axons by modality. However, previous efforts to split axon populations have enjoyed limited success for three reasons: 1)There is little comparative data on which growth factors attract only sensory or only motor axons; 2) In the constructs described so far, few regenerating axons are given equal opportunity to respond to both growth factors; 3) We have recently shown that sensory axons adhere to motor axons and limit their outgrowth, a factor not previously appreciated. The goal of this project is to develop an engineering approach that directs axons regenerating from a mixed nerve trunk into discrete sensory and motor channels. These channels could then be used to innervate individual motor and sensory nerves after nerve injury, or to control individual muscles through a regenerative prosthetic interface. The project tests two core hypotheses: 1) It is possible to identify neurotropic growth factors that direct the regeneration of only motor or only sensory axons (Aim I), and 2) Overlapping gradients of these factors can be used to separate regenerating sensory and motor axons (Aim II). These hypotheses will be tested in our organotypic model of mixed nerve regeneration, in which sensory axons expressing tomato red and motor axons expressing YFP are combined within a three-dimensional segment of peripheral nerve. After this nerve is transected, the color-coded axons grow out onto a featureless collagen/laminin sheet, where their pathfinding can be directed by growth factor gradients. These gradients will be established using micro-encapsulated growth factors and bioprinting technology developed in our laboratory. In Aim I we will evaluate the response of sensory or motor axons to candidate growth factors, modifying growth factor concentrations, gradient steepness, and substrate to maximize axon turning. In Aim II we will generate overlapping gradients of the most effective factors from Aim I while blocking the interaction of sensory and motor axons to maximize their turning. The ability to present regenerating axons with overlapping gradients of truly "motor-only" and "sensory-only" growth factors in an environment that minimizes axon-axon interactions will thus allow us to optimize separation of these axon populations. If successful, this engineering platform could then be used to construct a three-dimensional prosthesis that separates regenerating sensory and motor axons in vivo, which can be used to improve outcomes in the 50,000 patients/year that undergo nerve repair and the nearly 300 veterans with recent upper extremity amputations.