Nanostructured Hydrogel Surfaces for Artificial Extracellular Matrix

项目总结 支架干细胞移植有可能提供结构化的化学和机械线索 引导细胞向功能组织生长，用于再生医学。然而，重大挑战有限。 这种方法在临床上的成功。移植到大型支架中的细胞通常由于 宿主的低氧条件，而单独注射或以小簇形式注射的细胞往往表现不佳 在受伤部位滞留。水凝胶通常被用作干细胞支架材料，因为它们 在化学组成和配体集成方面赋予了相当大的灵活性。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(对于粘合和信号传递很重要)，以及 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，有可能在上产生稳定的1 nm分辨率的功能图案 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薄膜 预组装并共价转移到水凝胶表面的聚二乙炔(PDA)。我们的 该方法潜在地解决了与水凝胶干细胞相关的化学和机械挑战 支架，能够产生细胞指导性水凝胶带，可以成形以创建3D支架。 然而，为了在临床环境中有用，这一策略将需要通过：(1)常用的 水凝胶干细胞支架材料，(2)水凝胶模数与通常相关的范围相匹配 组织，以及(3)足够薄的薄膜，足以防止缺氧和正常分泌 互动。在这里，我们开发了一种基于细胞的平台技术-水凝胶胶带，标杆 它们的化学和机械特性，以及它们对人骨髓间充质干细胞(HMSCs)的影响。 在目标1中，我们评估了pda表面功能化可以改善化学控制的假设。 再生医学中常见的水凝胶表面，定向和空间聚集配体呈现， 以可预测的方式改变干细胞的生长。我们通过培养hMSCs来测试这一点 维持茎或诱导特定的分化行为(血管生成、脂肪生成、 软骨形成)，对照常见的随机水凝胶修饰策略。 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 薄薄、柔软的水凝胶膜的机械和操作性能，使创建细胞指导型水凝胶成为可能 录像带。我们生成并测试成对胶带作为细胞夹层支架和3D结构的影响，这些结构提供了 结构化的化学表面和机械环境，同时最大限度地向细胞和从细胞灌流。 总体而言，该提案开发了一种模块化表面功能化策略，可以很容易地与 许多现有的用于组织支架的水凝胶，提供结构化的配体呈现和机械强度 旨在提高临床细胞移植治疗的实用性。

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.