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Vijay Raghuanthan, Peter Strom, Sara Thomasy, Paul Russell, Christopher Murphy, ; Elastic modulus of keratocytes and myofibroblasts differ regardless of substratum stiffness. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1636.
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Injury to the corneal stroma initiates a cascade of events resulting in the differentiation of quiescent keratocytes (K) to activated fibroblasts (F) and myofibroblasts (M). This transformation is critical for appropriate corneal wound healing and its dysregulation can result in scar and haze formation. Transforming growth factor-β1 (TGFβ1) is critical in differentiating keratocytes and fibroblasts to myofibroblasts. We investigated the influence of substratum stiffness on TGFβ1 induced K-F-M transition and measured the elastic modulus of the different cell types.
Primary rabbit corneal keratocytes were isolated and cultured in serum free media. Cells were then cultured on polyacrylamide substrates of differing stiffness (5 or 25 kPa ) or on tissue culture plastic (TCP; >1GPa) in serum free media in the absence or presence (10 ng/ml) of TGFβ1. Expression of α-smooth muscle actin (αSMA) was determined by qPCR and immunocytochemistry (ICC) to confirm myofibroblast transformation. Keratocytes and myofibroblasts were visually identified by differences in cell morphology. Keratocytes had a characteristically stellate morphology while the myofibroblasts were flatter and more spread out. Elastic moduli of keratocytes and myofibroblasts on the various substrates were measured by atomic force microscopy (AFM).
Overall, interaction with softer substrates inhibited genesis of the myofibroblast phenotype. In the presence of TGFβ1,<10%, 40-50% and 90% of cells displayed a myofibroblast phenotype on 5 kPa gels, 25 kPa gels and TCP, respectively. The elastic modulus of myofibroblasts (3.5 ± 1 kPa) was significantly greater in comparison to keratocytes (1.3 ± 0.3 kPa) regardless of the underlying substratum stiffness.
Substratum stiffness profoundly influences the number of cells transitioning from keratocytes to myofibroblasts in the presence of TGFβ1. Softer gels, approximating values for stiffness found in vivo, stabilize the corneal stromal cell phenotype. Myofibroblasts are markedly stiffer than keratocytes irrespective of the underlying substratum stiffness. This is possibly due to increased actin cytoskeletal architecture in a myofibroblast. In aggregate, results suggest that substratum stiffness modulates the molecular mechanism by which cell differentiation occurs but not the intrinsic cytoskeletal properties that define the myofibroblast phenotype.
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