Abstract
We previously reported that corneal fibroblasts within 3D fibrin matrices secrete, bind, and organize fibronectin into tracks that facilitate cell spreading and migration. Other cells use these fibronectin tracks as conduits, which leads to the development of an interconnected cell/fibronectin network. In this study, we investigate how cell-induced reorganization of fibrin correlates with fibronectin track formation in response to two growth factors present during wound healing: PDGF BB, which stimulates cell spreading and migration; and TGFβ1, which stimulates cellular contraction and myofibroblast transformation. Both PDGF BB and TGFβ1 stimulated global fibrin matrix contraction (p < 0.005); however, the cell and matrix patterning were different. We found that, during PDGF BB-induced cell spreading, fibronectin was organized simultaneously with the generation of tractional forces at the leading edge of pseudopodia. Over time this led to the formation of an interconnected network consisting of cells, fibronectin and compacted fibrin tracks. Following culture in TGFβ1, cells were less motile, produced significant local fibrin reorganization, and formed fewer cellular connections as compared to PDGF BB (p < 0.005). Although bands of compacted fibrin tracks developed in between neighboring cells, fibronectin labeling was not generally present along these tracks, and the correlation between fibrin and fibronectin labeling was significantly less than that observed in PDGF BB (p < 0.001). Taken together, our results show that cell-induced extracellular matrix (ECM) reorganization can occur independently from fibronectin patterning. Nonetheless, both events seem to be coordinated, as corneal fibroblasts in PDGF BB secrete and organize fibronectin as they preferentially spread along compacted fibrin tracks between cells, producing an interconnected network in which cells, fibronectin and compacted fibrin tracks are highly correlated. This mechanism of patterning could contribute to the formation of organized cellular networks that have been observed following corneal injury and refractive surgery.
Highlights
Following injury in most vascularized tissues, one of the first steps in wound healing is the formation of a provisional extracellular matrix (ECM), which is rich in fibrin
The amount of fibrin matrix contraction was similar between Platelet-derived growth factor BB isotype (PDGF BB) or TGFβ1, and was significantly greater than that produced by basal media (* p < 0.005)
We previously demonstrated that corneal fibroblasts form interconnected cell-fibronectin networks when cultured in 3-D fibrin matrices
Summary
Following injury in most vascularized tissues, one of the first steps in wound healing is the formation of a provisional extracellular matrix (ECM), which is rich in fibrin. This matrix is essential to promote cell migration and invasion into the wounded area [1]. In the case of wound healing in the cornea, which is avascular, the early stage of wound repair can involve the deposition of a provisional matrix that functions as scaffold for cells to migrate [6], and fibrin and fibronectin are among the proteins present in provisional ECMs [7,8,9]
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