Localized immobilization of ephrin-B2 for neurite guidance in 3D culture

描述（由申请人提供）：中枢神经系统疾病通常与很少或没有恢复相关，部分原因是神经再生能力差、在初始神经元损伤后持续存在的损伤机制和髓磷脂固有的抑制特性。利用合成或天然材料作为允许环境以最大化轴突延伸的再生医学策略可能不足以恢复视力，因为从每个视网膜延伸的轴突在视交叉处交叉并选择性地投射到大脑的两个半球。蛋白质配体，如ephrin-B2，激活视网膜神经节细胞上的受体以诱导定向轴突生长，从而在胚胎发育期间形成发散的神经投射。发育配体，如这些可能能够指导再生轴突时，工程到生物材料的组织工程。然而，有一个关键的需要，生理和预防相关的文化模式，支持系统的调查，可能会影响组织生长的结构和分子参数。该项目的总体目标是开发一个3D组织培养模型，以研究视网膜神经突对工程线索的反应，这些线索模拟了发育过程中视交叉处发现的配体的空间分布。具体来说，我们假设肝配蛋白-B2，固定在一个合成的三维矩阵内的空间特异性的方式，将选择性地直接从胚胎视网膜外植体的神经突生长在一个结构配置，模仿视交叉。为了验证这一假设，我们提出了以下具体目标：目标1：合成一种光不稳定的肽水凝胶，用于蛋白质配体的局部固定。目的2：开发用于3D视网膜神经突生长和固定化蛋白配体掺入的双重水凝胶平台。目标3：评价肝配蛋白-B2，局部固定在3D肽水凝胶，选择性地指导神经突从胚胎视网膜外植体生长的功效。从这项工作中开发的技术将允许系统操纵的空间排列的结构和分子的线索，指导神经元的生长。因此，预计这项工作将建立一个新的实验平台，研究神经生长和指导，并提出潜在的治疗策略，以探索在未来的研究。中枢神经系统疾病，包括视神经病，由于神经再生能力差而难以治疗。在视神经中，即使最大化神经轴突再生也可能不会导致视力恢复，因为来自每只眼睛的神经在视交叉处交叉并投射到大脑两侧的特定区域。使用能够选择性引导轴突生长的功能性生物材料可能为视神经和其他中枢神经系统疾病提出新的治疗策略。

DESCRIPTION (provided by applicant): Disorders of the central nervous system are often associated with little or no recovery due to in part to poor neural regenerative capacity, damaging mechanisms that persist after initial neuronal injury, and inhibitory properties intrinsic to myelin. Regenerative medicine strategies that utilize synthetic or natural materials as permissive environments for maximizing axonal extension may not be sufficient for restoration of sight because axons that extend from each retina cross at the optic chiasm and selectively project to both hemispheres of the brain. Protein ligands, such as ephrin-B2, activate receptors on retinal ganglion cells to induce directional axon growth in order to form the divergent neural projections during embryogenesis. Development ligands such as these may be able to guide regenerating axons when engineered into biomaterials for tissue engineering. However, there is a critical need for physiologically- and translationally-relevant culture models that support the systematic investigation of structural and molecular parameters that may influence tissue growth. The overall goal of this project is to develop a 3D tissue culture model to study the guidance of retinal neurites in response to engineered cues that mimic the spatial distribution of ligands found at the optic chiasm during development. Specifically, we hypothesize that ephrin-B2, immobilized in a spatially-specific manner within a synthetic three-dimensional matrix, will selectively direct neurite outgrowth from embryonic retinal explants in a structural configuration that mimics the optic chiasm. In order to evaluate this hypothesis, we propose the following specific aims: Aim 1: Synthesize a photo-labile peptide hydrogel for localized immobilization of protein ligands. Aim 2: Develop a dual hydrogel platform for 3D retinal neurite outgrowth and incorporation of immobilized protein ligands. Aim 3: Evaluate the efficacy of ephrin-B2, locally immobilized in a 3D peptide hydrogel, to selectively direct neurite outgrowth from embryonic retinal explants. The techniques developed from this work will allow for the systematic manipulation of the spatial arrangement of structural and molecular cues for directing neuronal growth. Thus, it is anticipated that this work will establish a new experimental platform to study neural growth and guidance and also suggest potential treatment strategies to be explored in future studies. Disorders of the central nervous system, including optic neuropathies, are difficult to treat due to a poor capacity for nerves there to regenerate. In the optic nerve, even maximizing nerve axon regeneration may not lead to restoration of sight because nerves from each eye cross at the optic chiasm and project to specific regions at either side of the brain. The use of functional biomaterials that are able to selectively guide growing axons may suggest new treatment strategies for optic nerve and other central nervous system disorders.