| Wound healing comprises a complex interaction of cellular, biomechanical, and biochemical phenomena. Fibroblasts play a central role in wound healing by producing the extracellular matrix (ECM) molecules that ultimately replace the damaged tissue. Fibroblasts migrate into the wound site, proliferate (fibroplasia), deposit new matrix, and restructure the tissue by exerting traction on it.; We apply a number of in vitro assays to assess the role of both the ECM and soluble growth factors in modulating fibroblast traction and migration. Specifically, we study the effects of fibronectin, hyaluronic acid, wound fluid, and platelet-derived growth factor (PDGF) BB on fibroblast random migration in adherent collagen and fibrin gels. Fibronectin, wound fluid, and PDGF BB promote fibroblast migration in our test system. We also assess the role of mechanical stress and ECM stiffness in modulating the effects of PDGF BB on fibroblast traction and migration in fibrin and collagen gels. While PDGF BB increases migration in all cases studied, the effect on fibroblast traction varies depending on the stress state and/or stiffness of the matrix.; While useful in their ability to provide simple, quantitative assessments of cellular phenomena, in vitro assays are limited in their physiological relevance. Therefore, we develop an assay where a fibroblast-seeded collagen gel is enclosed in a semi-permeable tube that is inserted into an animal wound model and, thereby, exposed to the dynamic soluble wound environment. Fibroblast traction and proliferation rates are found to be different when fibroblast-seeded gels are implanted in the wound versus maintained in culture, as are markers of cell phenotype such as morphology and actin expression. The assay can be extended to study the roles of various growth factors in wound healing by seeding the collagen gels with cells that are unresponsive to selected growth factors. We apply the dominant negative receptor technique in an attempt to genetically alter fibroblasts so that they are unresponsive to PDGF BB. While we are unable to block all PDGF BB signaling, signaling via the PDGF receptor β appears to be blocked. The cells' migratory response to PDGF BB stimulation is eliminated while the proliferative response is not. |
