Cell Responsive Hydrogels to Improve Functional Recovery after Spinal Cord Injury

 描述(申请人提供)：在整个组织发育和动态平衡过程中，细胞与细胞外基质(ECM)动态相互作用。然而，大多数用于调节干细胞命运和促进组织再生的材料主要是细胞指导材料，为细胞提供机械和生化信号，而不是细胞响应材料，即它们不会对交付细胞中引发的变化做出反应。大多数对细胞有反应的物质只是对细胞分泌的蛋白酶做出反应而降解。一种对干细胞分化引起的特定表型变化做出反应的材料，如发育过程中发生的变化，仍有待开发。这种材料的潜在治疗靶点是脊髓损伤(SCI)。脊髓损伤往往导致患者的严重虚弱，临床上可用的治疗选择有限。神经再生受以下因素限制 身体的自然炎症反应，迅速将受伤的脊髓组织替换为疤痕组织。此外，这种炎症过程会对存活的神经元造成严重的氧化损伤，从而进一步阻碍再生。这个项目的目标是通过将神经干细胞(NSCs)输送到损伤部位的材料中来修复这种炎症造成的损害，这种材料既是细胞指导性的，促进被传递细胞的植入和分化，又是细胞反应性的，释放出一种神经保护肽。 对神经元分化的反应。在具体目标1中，我将合成一种材料，通过释放一种神经保护肽来动态响应NSC的分化。该多肽将通过使用叠氮-炔“点击”化学的可蛋白分解的连接体连接到弹性蛋白样蛋白(ELP)上。尿激酶型纤溶酶原激活剂(UPA)是一种丝氨酸蛋白酶，由轴突生长锥体分泌，在神经元发育中发挥作用。我推测，在ELP水凝胶中培养的神经干细胞的神经元分化会导致uPA活性增加，进而在神经元分化时选择性地释放神经保护肽。在具体目标2中，我将开发一个计算模型来改进CELL响应性材料设计。采用Michaelis-Menten反应扩散模型模拟神经保护性多肽的释放过程，并通过实验确定相关参数。该模型将通过培养和分化细胞响应性ELP水凝胶中的神经干细胞，并使细胞受到氧化应激来验证。在具体目标3中，我将在细胞反应性水凝胶中将神经干细胞输送到啮齿动物脊髓挫伤模型的损伤部位，并评估功能恢复。材料保留将通过活体动物成像进行评估，神经干细胞的存活、植入和分化将通过组织学进行评估。恢复情况将通过追踪再生神经和通过行为测试进行评估。我推测，细胞响应性材料将提高移植的神经干细胞的活性，从而改善使用细胞响应性材料治疗的动物的功能恢复。

 DESCRIPTION (provided by applicant): Throughout tissue development and homeostasis, cells dynamically interact with the extracellular matrix (ECM). However, most materials developed to regulate stem cell fate and facilitate tissue regeneration are primarily cell-instructive, providing mechanical and biochemical signals to cells, and are not cell-responsive, that is they do not respond to the changes elicited in the delivered cells. The majority of materials that are cell-responsive simply degrade in response to cell-secreted proteases. A material that reacts to specific phenotypic changes elicited upon stem cell differentiation, such as those that occur during development, remains to be developed. A potential therapeutic target for such a material is spinal cord injury (SCI). SCI often results in severely debilitating conditins for patients, with limited clinically available treatment options. Nerve regeneration is limited by the body's natural inflammatory response that rapidly replaces injured spinal cord tissue with scar tissue. Furthermore, this inflammatory process results in significant oxidative damage to the surviving neurons, which further hampers regeneration. The goal of this project is to remediate the damage caused by this inflammation by delivering neural stem cells (NSCs) at the injury site within a material that is both cell-instructive, facilitating engraftment and differentiation of the delivered cells, and cell-responsive, releasing a neuroprotective peptide in response to neuronal differentiation. In Specific Aim 1, I will synthesize a material that dynamically responds to NSC differentiation by releasing a neuroprotective peptide. The peptide will be conjugated to an elastin-like protein (ELP) via a proteolytically cleavable linker using azide-alkyne "click" chemistry. Urokinase plasminogen activator (uPA) is a serine protease known to play a role in neuronal development, as it is secreted from the growth cones of axons. I hypothesize that neuronal differentiation of NSCs cultured in ELP hydrogels will result in increased uPA activity, which in turn will selectively release the neuroprotective peptide upon neuronal differentiation. In Specific Aim 2, I will develop a computational model to refine the cel-responsive material design. A reaction-diffusion model with Michaelis-Menten kinetics will be used to simulate the release of the neuroprotective peptide, and the relevant parameters will be experimentally determined. The model will be validated by culturing and differentiating NSCs in the cell-responsive ELP hydrogels and subjecting the cells to oxidative stress. In Specific Aim 3, I will deliver NSCs in the cell-responsive hydrogels to injury sites in rodent SCI contusion models and evaluate functional recovery. Material retention will be assessed with live-animal imaging, and NSC survival, engraftment, and differentiation will be assessed by histology. Recovery will be evaluated by tracing the regenerating nerves and through behavioral testing. I hypothesize that the cell-responsive material will improve the viability of the transplanted NSCs, resulting in improved functional recovery in animals treated with the cell-responsive materials.