Growth factor regulation of corneal keratocyte mechanical phenotypes in 3-D collagen matrices.

Stromal keratocytes play a central role in mediating the corneal response to lacerating injury or refractive surgery.1 During wound healing, quiescent corneal keratocytes surrounding the area of injury generally become activated, and transform into a fibroblastic repair phenotype.2,3 These activated fibroblasts proliferate, migrate into the provisional matrix, and generate the forces required for wound closure. In certain wound types, fibroblasts further differentiate into myofibroblasts, which generate even stronger forces and synthesize a fibrotic extracellular matrix (ECM) associated with scar formation.4,5 These wound healing responses can cause a permanent reduction in corneal clarity, and can also decrease the refractive effect of vision correction surgeries such as photorefractive keratectomy (PRK).6,7 Peptide growth factors present in the cornea and tear film, such as insulin-like growth factor (IGF), platelet derived growth factor (PDGF), FGF, IL-1α and TGFβ,8–11 are postulated to play an important role in modulating the keratocyte phenotype during corneal wound healing. In cell culture, these growth factors differentially regulate keratocyte proliferation, cytoskeletal organization, and ECM synthesis. Keratocytes cultured under serum-free conditions maintain the quiescent, dendritic phenotype normally observed in vivo.12,13 IGF stimulates keratocyte proliferation without altering cell morphology, cytoskeletal organization, or downregulating keratocan sulfate proteoglycan (KSPG) synthesis.14,15 In contrast, exposure to serum results in fibroblast differentiation, as indicated by the assumption of a bipolar morphology, formation of intracellular stress fibers, and downregulation of keratin sulfate proteoglycan expression.13,16–18 PDGF and basic FGF (FGF2) have also been shown to activate corneal keratocytes in vitro, but have distinct effects on cell morphology, cytoskeletal organization, and proteoglycan synthesis.14,15,19,20 TGFβ induces myofibroblast differentiation, as indicated by expression of stress fibers containing α-smooth muscle actin (α-SMA),21 and production of abnormal, fibrotic ECM.15,20,22 PDGF participates in TGFβ-induced myofibroblast differentiation through an autocrine feedback loop,23 whereas FGF2 has been shown to reduce expression of α-SMA.24 Most previous studies investigating the effect of these growth factors on corneal keratocyte differentiation have been performed using rigid, two-dimensional (2-D) substrates. However, keratocytes reside within a complex 3-D extracellular matrix in vivo, and significant differences in cell morphology, adhesion organization, and mechanical behavior have been identified in 2-D versus 3-D culture models.25–27 Furthermore, unlike rigid 2-D substrates, 3-D models also allow assessment of cellular force generation and cell-induced matrix reorganization; biomechanical activities that are critically involved in the migratory, contractile, and remodeling phases of wound healing. Recent studies also suggest a potential linkage between increased keratocyte contractility and altered ECM synthesis, further highlighting the potential importance of cell mechanics in the wound repair process.20,28 We have recently established 3-D culture models which support keratocyte differentiation in serum-free media, and allow changes in cell morphology, mechanical activity, and matrix reorganization to be measured using quantitative imaging techniques.29,30 In this study, we use these models to assess the effects of PDGF BB, FGF2, IGF, TGFβ1, and TGFβ2 on the keratocyte mechanical phenotype in 3-D collagen matrices, and compare these responses with those observed on a rigid 2-D substrate.