Data_Sheet_1_Porous Scaffold-Hydrogel Composites Spatially Regulate 3D Cellular Mechanosensing.docx

Cells encapsulated in 3D hydrogels exhibit differences in cellular mechanosensing based on their ability to remodel their surrounding hydrogel environment. Although cells in tissue interfaces feature a range of mechanosensitive states, it is challenging to recreate this in 3D biomaterials. Human mesenchymal stem cells (MSCs) encapsulated in methacrylated gelatin (GelMe) hydrogels remodel their local hydrogel environment in a time-dependent manner, with a significant increase in cell volume and nuclear Yes-associated protein (YAP) localization between 3 and 5 days in culture. A finite element analysis model of compression showed spatial differences in hydrogel stress of compressed GelMe hydrogels, and MSC-laden GelMe hydrogels were compressed (0–50%) for 3 days to evaluate the role of spatial differences in hydrogel stress on 3D cellular mechanosensing. MSCs in the edge (high stress) were significantly larger, less round, and had increased nuclear YAP in comparison to MSCs in the center (low stress) of 25% compressed GelMe hydrogels. At 50% compression, GelMe hydrogels were under high stress throughout, and this resulted in a consistent increase in MSC volume and nuclear YAP across the entire hydrogel. To recreate heterogeneous mechanical signals present in tissue interfaces, porous polycaprolactone (PCL) scaffolds were perfused with an MSC-laden GelMe hydrogel solution. MSCs in different pore diameter (~280–430 μm) constructs showed an increased range in morphology and nuclear YAP with increasing pore size. Hydrogel stress influences MSC mechanosensing, and porous scaffold-hydrogel composites that expose MSCs to diverse mechanical signals are a unique biomaterial for studying and designing tissue interfaces.

包封于三维水凝胶（3D hydrogels）中的细胞，其细胞机械感知（cellular mechanosensing）能力存在差异，该差异取决于它们重塑周围水凝胶微环境的能力。尽管组织界面（tissue interfaces）处的细胞具有多种机械敏感状态，但在三维生物材料中复现这一现象仍颇具挑战。包封于甲基丙烯酸酯化明胶（methacrylated gelatin, GelMe）水凝胶中的人骨髓间充质干细胞（human mesenchymal stem cells, MSCs），会以时间依赖性方式重塑局部水凝胶微环境：培养3至5天时，细胞体积与细胞核内Yes相关蛋白（Yes-associated protein, YAP）的定位水平均显著升高。通过压缩有限元分析（finite element analysis）模型，可观察到压缩GelMe水凝胶的应力存在空间差异；为此，我们将负载MSCs的GelMe水凝胶进行0%至50%的压缩处理并培养3天，以探究水凝胶应力的空间差异对三维细胞机械感知的调控作用。在压缩率为25%的GelMe水凝胶中，处于边缘（高应力区域）的MSCs体积显著更大、形态更偏离圆形，且细胞核内YAP水平相较于中心（低应力区域）的MSCs显著升高。当压缩率提升至50%时，GelMe水凝胶整体均处于高应力状态，此时整个水凝胶内的MSCs体积与细胞核YAP水平均呈现一致性升高。为复现组织界面处存在的异质性机械信号，我们将负载MSCs的GelMe水凝胶溶液灌注至聚己内酯（polycaprolactone, PCL）多孔支架中。不同孔径（约280–430 μm）的支架构建体中，MSCs的形态分布范围与细胞核YAP水平均随孔径增大而升高。综上，水凝胶应力可调控MSCs的机械感知；而为MSCs提供多样化机械信号的多孔支架-水凝胶复合生物材料，是研究与设计组织界面的独特仿生材料体系。