Nanostructured Hydrogel Surfaces for Artificial Extracellular Matrix

项目摘要 支架干细胞移植有可能提供结构化的化学和机械线索， 引导细胞生长成再生医学的功能组织。然而，重大挑战限制了 这种方法的临床成功。植入大支架中的细胞通常具有有限的活力， 在宿主中的缺氧条件下，而单独或以小簇注射的细胞通常遭受差的缺氧。 保留在受伤部位。水凝胶通常用作干细胞支架材料，因为它们 在化学组成和配体整合方面赋予相当大的灵活性。然而，水凝胶是 通常也是无定形的，限制了对配体呈递的控制（对粘附和信号传导很重要），和 缺乏结构线索，如生物ECM中存在的纤维，这会影响机械强度。 我们最近已经证明，有可能产生稳定的，1 nm分辨率的功能模式， 无定形聚丙烯酰胺和聚二甲基硅氧烷表面，使用高度有序的亚纳米厚的膜， 聚二乙炔（PDA），其预组装并共价转移到水凝胶表面。我们 该方法可能解决与水凝胶干细胞相关的化学和机械挑战 支架，使得能够生成细胞指导性水凝胶带，其可以成形以创建3D支架。 然而，为了在临床环境中有用，这种策略需要通过以下方式进行验证：（1）常用 水凝胶干细胞支架材料，（2）水凝胶模量匹配通常与 组织，和（3）薄膜足够薄，以充分灌注，以防止缺氧，并使正常的分泌组 交互.在这里，我们开发了一种基于细胞指导性水凝胶胶带的平台技术， 它们的化学和机械性质，以及它们对人间充质干细胞（hMSC）的影响。 在目标1中，我们评估了PDA表面功能化可以改善对 再生医学中常见的水凝胶表面，配体呈现的定向和空间聚集， 以可预测的方式改变干细胞的生长。我们通过在设计为 维持干性或诱导特定的分化行为（血管生成，脂肪生成， 软骨形成），对常见的随机水凝胶修饰策略进行基准测试。 在目标2中，我们评估了我们的PDA表面功能化方法可以改善生物相容性的假设。 薄、软的水凝胶膜的机械和处理性能，使得能够产生细胞指导性水凝胶 胶粘带.我们生成并测试了成对胶带作为细胞三明治支架和3D结构的影响， 结构化的化学表面和机械环境，同时最大化细胞的灌注。 总的来说，该提议开发了一种模块化表面功能化策略，其可以容易地与 许多现有的用于组织支架的水凝胶，提供结构化配体呈现和机械强度 旨在提高临床细胞移植治疗的效用。

PROJECT SUMMARY Scaffolded stem cell transplantation has the potential to provide structured chemical and mechanical cues to guide cell growth into functional tissues for regenerative medicine. However, significant challenges have limited the clinical success of this approach. Cells implanted in large scaffolds often have limited viability due to hypoxic conditions in the host, while cells injected individually or in small clusters often suffer from poor retention at the site of injury. Hydrogels are commonly utilized as stem cell scaffold materials because they confer substantial flexibility in terms of chemical composition and ligand integration. However, hydrogels are also typically amorphous, limiting control over ligand presentation (important for adhesion and signaling), and lacking structural cues such as fibers that are present in biological ECM, which impacts mechanical strength. We have recently demonstrated that it is possible to generate stable, 1-nm-resolution functional patterns on amorphous polyacrylamide and polydimethylsiloxane surfaces, using sub-nm-thick films of highly ordered polydiacetylenes (PDAs) that are preassembled and covalently transferred to the hydrogel surface. Our approach potentially addresses both chemical and mechanical challenges associated with hydrogel stem cell scaffolds, enabling generation of cell-instructive hydrogel tapes that can be shaped to create 3D scaffolds. However, to be useful in clinical settings, this strategy will need to be validated with: (1) commonly used hydrogel stem cell scaffold materials, (2) hydrogel moduli matching the range commonly associated with tissues, and (3) films thin enough for adequate perfusion to prevent hypoxia and enable normal secretome interactions. Here, we develop a platform technology based on cell-instructive hydrogel tapes, benchmarking their chemical and mechanical properties, and their impacts on human mesenchymal stem cells (hMSCs). In Aim 1, we evaluate the hypothesis that PDA surface functionalization can improve chemical control over surfaces of hydrogels common in regenerative medicine, orienting and spatially clustering ligand presentation, to modify stem cell growth in a predictable fashion. We test this by culturing hMSCs on surfaces designed to maintain stemness or to induce specified differentiation behavior (angiogenesis, adipogenesis, chondrogenesis), benchmarking against common stochastic hydrogel modification strategies. In Aim 2, we evaluate the hypothesis that our PDA surface-functionalization approach can improve the mechanical and handling properties of thin, soft hydrogel films, enabling creation of cell-instructive hydrogel tapes. We generate and test impacts of paired tapes as cell sandwich scaffolds and 3D constructs that provide structured chemical surfaces and mechanical environments, while maximizing perfusion to and from cells. Overall, this proposal develops a modular surface functionalization strategy that can be easily integrated with many existing hydrogels for tissue scaffolds, providing structured ligand presentation and mechanical strength aimed at improving utility in clinical cell transplantation therapies.