Smithmyer Megan E, Spohn Joseph B, Kloxin April M
Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware 19716, United States.
ACS Biomater Sci Eng. 2018 Sep 10;4(9):3304-3316. doi: 10.1021/acsbiomaterials.8b00491. Epub 2018 Jul 27.
Synthetic hydrogels with well-defined mechanical properties have become invaluable tools for probing cell response to extracellular cues including matrix stiffness and integrin binding. These synthetic matrices are often decorated with either proteins or integrin-binding peptides to promote cell adhesion and to direct or probe cell behavior. For example, both collagen I-functionalized polyacrylamide and peptide-functionalized poly(ethylene glycol) hydrogels have been instrumental in elucidating the role of the elasticity or 'stiffness' of the matrix in promoting fibroblast activation in wound healing and fibrosis. However, the two methods of promoting integrin binding are not often directly compared in the same system, partly owing to differences in material designs, despite the potential differences in the way cells interact with whole proteins and protein mimetic peptides. We hypothesized that such a comparison could provide insight into the ways integrin binding affects fibroblast activation within commonly utilized cell culture models, and more broadly, to inform the design of materials to modulate fibroblast activation in studies of wound healing and disease. To enable this comparison, we developed a method to conjugate whole proteins to step-growth poly(ethylene glycol) (PEG) hydrogels and investigated fibroblast response to protein-peptide pairs: fibronectin and PHSRN(G)RGDS or collagen I and (POG)POGFOGER(POG), which are important in matrix remodeling and relevant to fibroblast activation. With this approach, we observed that human pulmonary fibroblasts adopted a similar morphology on fibronectin and PHSRN(G)RGDS, although with a slight increase in the percentage of alpha smooth muscle actin (αSMA) expressing cells on PHSRN(G)RGDS. Interestingly, we observed that fibroblasts formed activated clusters on the collagen mimic (POG)POGFOGER(POG) while exhibiting less activation on collagen I. This cell activation and clustering is reminiscent of fibroblast foci that are observed in lung fibrosis, suggesting the relevance of these well-defined polymer-peptide hydrogels for investigating fibrosis and decoupling biochemical and biophysical cues.
具有明确机械性能的合成水凝胶已成为探究细胞对细胞外信号（包括基质硬度和整合素结合）反应的重要工具。这些合成基质通常用蛋白质或整合素结合肽进行修饰，以促进细胞黏附并指导或探测细胞行为。例如，I型胶原蛋白功能化的聚丙烯酰胺和肽功能化的聚乙二醇水凝胶在阐明基质弹性或“硬度”在促进伤口愈合和纤维化过程中成纤维细胞活化方面发挥了重要作用。然而，尽管细胞与完整蛋白质和蛋白质模拟肽相互作用的方式可能存在差异，但促进整合素结合的这两种方法在同一系统中并不常被直接比较，部分原因是材料设计的差异。我们推测，这样的比较可以深入了解整合素结合在常用细胞培养模型中影响成纤维细胞活化的方式，更广泛地说，为在伤口愈合和疾病研究中调节成纤维细胞活化的材料设计提供信息。为了进行这种比较，我们开发了一种将完整蛋白质与逐步生长的聚乙二醇（PEG）水凝胶偶联的方法，并研究了成纤维细胞对蛋白质 - 肽对的反应：纤连蛋白和PHSRN(G)RGDS，或I型胶原蛋白和(POG)POGFOGER(POG)，它们在基质重塑中很重要且与成纤维细胞活化相关。通过这种方法，我们观察到人类肺成纤维细胞在纤连蛋白和PHSRN(G)RGDS上呈现出相似的形态，尽管在PHSRN(G)RGDS上表达α平滑肌肌动蛋白（αSMA）的细胞百分比略有增加。有趣的是，我们观察到成纤维细胞在胶原蛋白模拟物(POG)POGFOGER(POG)上形成活化簇，而在I型胶原蛋白上的活化程度较低。这种细胞活化和聚集让人联想到在肺纤维化中观察到的成纤维细胞灶，表明这些明确的聚合物 - 肽水凝胶对于研究纤维化以及分离生化和生物物理信号具有相关性。
