The Role of Rho GTPases During Fibroblast Spreading, Migration, and Myofibroblast Differentiation in 3D Synthetic Fibrous Matrices

Abstract

Connective tissue repair and mechanosensing are tightly entwined in vivo and occur within a complex three-dimensional (3D), fibrous extracellular matrix (ECM). Typically driven by activated fibroblasts, wound repair involves well-defined steps of cell spreading, migration, proliferation, and fibrous ECM deposition. While the role of Rho GTPases in regulating these processes has been explored extensively in two-dimensional cell culture models, much less is known about their role in more physiologic, 3D environments. We employed a 3D, fibrous and protease-sensitive hydrogel model of interstitial ECM to study the interplay between Rho GTPases and fibrous matrix cues in fibroblasts during wound healing. Modulating fiber density within protease-sensitive hydrogels, we confirmed previous findings that heightened fiber density promotes fibroblast spreading and proliferation. The presence of matrix fibers furthermore corresponded to increased cell migration speeds and macroscopic hydrogel contraction arising from fibroblast generated forces. During fibroblast spreading, Rac1 and RhoA GTPase activity proved crucial for fiber-mediated cell spreading and contact guidance along matrix fibers, while Cdc42 was dispensable. In contrast, interplay between RhoA, Rac1, and Cdc42 contributed to fiber-mediated myofibroblast differentiation and matrix contraction over longer time scales. These observations may provide insights into tissue repair processes in vivo and motivate the incorporation of cell-adhesive fibers within synthetic hydrogels for material-guided wound repair strategies.