Cell Responsive Hydrogels to Improve Functional Recovery after Spinal Cord Injury

 描述（由申请人提供）：在整个组织发育和体内平衡过程中，细胞与细胞外基质（ECM）动态相互作用。然而，开发用于调节干细胞命运和促进组织再生的大多数材料主要是细胞指导性的，向细胞提供机械和生物化学信号，并且不是细胞响应性的，即它们不响应递送细胞中引起的变化。大多数细胞响应性材料仅响应于细胞分泌的蛋白酶而降解。一种对干细胞分化引起的特定表型变化（如发育过程中发生的表型变化）起反应的材料仍有待开发。这种材料的潜在治疗靶点是脊髓损伤（SCI）。SCI常常导致患者严重衰弱，临床上可用治疗选择有限。神经再生受限于 身体的自然炎症反应，迅速取代受伤的脊髓组织与疤痕组织。此外，这种炎症过程导致对存活神经元的显著氧化损伤，这进一步阻碍了再生。该项目的目标是通过将神经干细胞（NSC）递送到损伤部位的材料中来修复由这种炎症引起的损伤，所述材料既具有细胞指导性，促进递送细胞的植入和分化，又具有细胞响应性，释放神经保护肽。 对神经元分化的反应。在具体目标1中，我将合成一种通过释放神经保护肽动态响应NSC分化的材料。使用叠氮化物-炔“点击”化学，通过蛋白水解可切割接头将肽缀合至弹性蛋白样蛋白（ELP）。尿激酶纤溶酶原激活物（uPA）是已知在神经元发育中起作用的丝氨酸蛋白酶，因为其从轴突的生长锥分泌。我推测ELP水凝胶中培养的神经干细胞的神经元分化将导致uPA活性增加，这反过来将选择性地释放神经元分化后的神经保护肽。在具体目标2中，我将开发一个计算模型来改进细胞响应材料的设计。将使用具有Michaelis-Menten动力学的反应扩散模型来模拟神经保护肽的释放，并通过实验确定相关参数。该模型将通过在细胞响应性ELP水凝胶中培养和分化NSC并使细胞经受氧化应激来验证。在具体目标3中，我将在细胞响应性水凝胶中将神经干细胞递送到啮齿动物SCI挫伤模型的损伤部位，并评估功能恢复。将通过活体动物成像评估材料保留，并通过组织学评估NSC存活、植入和分化。通过追踪再生神经和行为测试评估恢复情况。我假设细胞反应性材料将提高移植的神经干细胞的活力，从而改善用细胞反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.