PURPOSE: We previously reported that extracellular matrix composition (fibrin versus collagen) modulates the pattern of corneal fibroblast spreading and migration in 3-D culture. In this study, we investigate the role of thrombin and cell contractility in mediating these differences in cell behavior.
METHODS: To assess cell spreading, corneal fibroblasts were plated on top of fibrillar collagen and fibrin matrices. To assess 3-dimensional cell migration, compacted collagen matrices seeded with corneal fibroblasts were embedded inside acellular collagen or fibrin matrices. Constructs were cultured in serum-free media containing platelet-derived growth factor (PDGF), with or without thrombin, the Rho kinase inhibitor Y-27632, and/or the myosin II inhibitor blebbistatin. We used 3-dimensional and 4-dimensional imaging to assess cell mechanical behavior, connectivity and cytoskeletal organization.
RESULTS: Thrombin stimulated increased contractility of corneal fibroblasts. Thrombin also induced Rho kinase-dependent clustering of cells plated on top of compliant collagen matrices, but not on rigid substrates. In contrast, cells on fibrin matrices coalesced into clusters even when Rho kinase was inhibited. In nested matrices, cells always migrated independently through collagen, even in the presence of thrombin. In contrast, cells migrating into fibrin formed an interconnected network. Both Y-27632 and blebbistatin reduced the migration rate in fibrin, but cells continued to migrate collectively.
CONCLUSIONS: The results suggest that while thrombin-induced actomyosin contraction can induce clustering of fibroblasts plated on top of compliant collagen matrices, it does not induce collective cell migration inside 3-D collagen constructs. Furthermore, increased contractility is not required for clustering or collective migration of corneal fibroblasts interacting with fibin.
- 3-D culture
- cell mechanics
- corneal keratocytes
- extracellular matrix
ASJC Scopus subject areas
- Sensory Systems
- Cellular and Molecular Neuroscience