Department of Ophthalmology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9057, USA.
Exp Eye Res. 2012 Jun;99:36-44. doi: 10.1016/j.exer.2012.03.015.
Extracellular matrix (ECM) supplies both physical and chemical signals to cells and provides a substrate through which fibroblasts migrate during wound repair. To directly assess how ECM composition regulates this process, we used a nested 3D matrix model in which cell-populated collagen buttons were embedded in cell-free collagen or fibrin matrices. Time-lapse microscopy was used to record the dynamic pattern of cell migration into the outer matrices, and 3D confocal imaging was used to assess cell connectivity and cytoskeletal organization. Corneal fibroblasts stimulated with PDGF migrated more rapidly into collagen as compared to fibrin. In addition, the pattern of fibroblast migration into fibrin and collagen ECMs was strikingly different. Corneal fibroblasts migrating into collagen matrices developed dendritic processes and moved independently, whereas cells migrating into fibrin matrices had a more fusiform morphology and formed an interconnected meshwork. A similar pattern was observed when using dermal fibroblasts, suggesting that this response is not unique to corneal cells. We next cultured corneal fibroblasts within and on top of standard collagen and fibrin matrices to assess the impact of ECM composition on the cell spreading response. Similar differences in cell morphology and connectivity were observed – cells remained separated on collagen but coalesced into clusters on fibrin. Cadherin was localized to junctions between interconnected cells, whereas fibronectin was present both between cells and at the tips of extending cell processes. Cells on fibrin matrices also developed more prominent stress fibers than those on collagen matrices. Importantly, these spreading and migration patterns were consistently observed on both rigid and compliant substrates, thus differences in ECM mechanical stiffness were not the underlying cause. Overall, these results demonstrate for the first time that ECM protein composition alone (collagen vs. fibrin) can induce a switch from individual to collective fibroblast spreading and migration in 3D culture. Similar processes may also influence cell behavior during wound healing, development, tumor invasion and repopulation of engineered tissues.
细胞外基质（ECM）为细胞提供物理和化学信号，并为成纤维细胞在伤口修复过程中迁移提供基质。为了直接评估 ECM 组成如何调节这个过程，我们使用了嵌套的 3D 基质模型，其中细胞填充的胶原按钮嵌入在无细胞胶原或纤维蛋白基质中。延时显微镜用于记录细胞迁移到外层基质的动态模式，3D 共聚焦成像用于评估细胞连接和细胞骨架组织。与纤维蛋白相比，PDGF 刺激的角膜成纤维细胞更快地迁移到胶原中。此外，成纤维细胞迁移到纤维蛋白和胶原 ECM 的模式差异非常明显。迁移到胶原基质中的角膜成纤维细胞发育出树突状突起并独立移动，而迁移到纤维蛋白基质中的细胞具有更梭形的形态并形成相互连接的网状结构。使用真皮成纤维细胞时观察到类似的模式，表明这种反应不是角膜细胞所特有的。我们接下来在标准胶原和纤维蛋白基质内部和顶部培养角膜成纤维细胞，以评估 ECM 组成对细胞扩展反应的影响。观察到细胞形态和连接性的相似差异——细胞在胶原上仍然分离，但在纤维蛋白上聚集成簇。钙黏蛋白定位于相互连接的细胞之间的连接处，而纤连蛋白存在于细胞之间和延伸的细胞突起的尖端。纤维蛋白基质上的细胞也比胶原基质上的细胞形成更明显的应力纤维。重要的是，这些扩展和迁移模式在刚性和顺应性基底上都一致观察到，因此 ECM 机械刚度的差异不是根本原因。总的来说，这些结果首次表明，仅 ECM 蛋白组成（胶原与纤维蛋白）就可以在 3D 培养中诱导从单个到集体成纤维细胞扩展和迁移的转变。类似的过程也可能影响伤口愈合、发育、肿瘤侵袭和工程组织再填充过程中的细胞行为。
