ECM Stiffness Regulates the Myofibroblastic Activation and Contractility of Cultured Primary Corneal Keratocytes
Following surgery or traumatic injury, corneal wound healing can cause a scarring response that impairs ocular function. This fibrosis is caused, in part, by the activation of corneal keratocytes from a native quiescent state to an activated myofibroblastic state. Signaling pathways downstream of transforming growth factor beta 1 (TGF-β1) have been shown to be key regulators of this transformation. During wound healing, the activated corneal keratocytes begin to secrete fibrotic extracellular matrix (ECM) proteins and to exert contractile forces to close the wound. These behaviors, however, can disrupt the organization of the ECM in the corneal stroma and reduce the transparency of the tissue. Previous studies have suggested that biophysical cues can modulate keratocyte behavior, but it remains unclear how the activation and contractility of corneal keratocytes is regulated by changes in ECM stiffness. Here, to better understand how ECM stiffness interacts with TGF-β1 signaling to regulate myofibroblastic activation, we cultured primary rabbit corneal keratocytes on compliant, collagen-coated substrata of varying stiffness in either the presence or absence of TGF-β1. Polyacrylamide hydrogels were fabricated on glass coverslips and functionalized to perform two-dimensional (2D) cell culture and traction force microscopy (TFM). The gels were then plated with primary corneal keratocytes, isolated from rabbit eyeballs, as described previously, and cultured at 37°C in either serum-free media or media containing exogenous TGF-β1. Time-lapse fluorescence microscopy was used to assess changes in cellular morphology, as well as to track motion of the fluorescent microspheres embedded within the gel. The tracked bead motion was then used to estimate the traction stresses exerted by the cultured keratocytes under different experimental conditions. In other experiments, cells were fixed after 5 days of culture and stained for molecular markers of either contractility or myofibroblastic transformation. Treatment with TGF-β1 elicited distinct cellular phenotypes when keratocytes were cultured on gels of different stiffness. Keratocytes cultured on either 10 kPa (stiff) gels or glass coverslips had broad cellular processes, formed abundant stress fibers, exhibited elevated levels of alpha smooth muscle actin immunofluorescence, and exerted large traction forces. Cells cultured on 1 kPa (soft) gels, however, formed few stress fibers, exerted small traction forces, and retained an elongated morphology, indicative of a more quiescent phenotype. Confocal images of phosphorylated-myosin light chain (pMLC) immunofluorescence, moreover, revealed stiffness-dependent variations in the spatial distributions of sub-cellular contractility. Our computed traction force maps correlated strongly with the observed spatial patterns of pMLC immunofluorescence. Taken together, these data suggest that changes in ECM stiffness, often associated with tissue fibrosis, can modulate the contractility and differentiation of corneal keratocytes during wound healing.