Individual versus collective fibroblast spreading and migration: Regulation by matrix composition in 3D culture

Abstract

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 3-D 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 in 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 3-D culture. Similar processes may also influence cell behavior during wound healing, development, tumor invasion and repopulation of engineered tissues.

Fibroblast-fibronectin patterning and network formation in 3D fibrin matrices

“…We previously demonstrated that cells interacting with collagen matrices develop dendritic processes and move independently, whereas cells interacting with fibrin matrices develop stress fibers and form an interconnected meshwork [24, 25]. In constructs in which cell-seeded compressed collagen matrices are nested within acellular fibrin matrixes, interconnected streams of cells are formed during migration into fibrin (Supplemental Movie 1).…”

“…Specifically, whereas corneal fibroblasts generally move independently within 3-D collagen matrices, fibrin induces a switch to an interconnected, collective mode of cell spreading and migration which is independent of differences in ECM stiffness [24, 25]. Fibronectin is an adhesive protein secreted by cells that facilitates binding to other ECM proteins [32].…”

“…In order to study the pattern and amount of 3-D cell migration, cell-populated compressed collagen constructs were nested within acellular uncompressed fibrin matrices as previously described [24, 25, 55, 66]. In this model, 2ml of a neutralized collagen solution (4 mg/ml) is poured into a rectangular metal mold (3 cm length, 2 cm width, 1 cm height) and placed in a humidified incubator (37°C, 5% CO 2 ) for 30 minutes for polymerization.…”

“…In recent studies, we demonstrated that ECM composition has a profound impact on the pattern of corneal fibroblast spreading and migration in 3-D culture. Specifically, cells interacting with collagen matrices develop dendritic processes and move independently, whereas cells interacting with fibrin matrices develop stress fibers and form an interconnected meshwork [24, 25]. Interestingly, corneal fibroblasts form an interconnected mesh as they migrate into the wound space following incisional surgery, and these interconnections are hypothesized to mediate force transduction during wound contraction [26, 27].…”

“…Fibronectin is an adhesive protein secreted by cells that facilitates binding to other ECM proteins (including fibrin) [32–35], and it is expressed by corneal fibroblasts during clustering on top of fibrin ECM [25]. Fibronectin synthesis has been shown to contribute to vascular endothelial cell network formation and tubulogenesis in vitro [36, 37].…”