The purpose of this study was to assess quantitatively the differences in morphology, cytoskeletal organization and mechanical behavior between quiescent corneal keratocytes and activated fibroblasts in a 3-D culture model. Primary cultures of rabbit corneal keratocytes and fibroblasts were plated inside type I collagen matrices in serum-free media or 10% FBS, and allowed to spread for 1-5 days. Following F-actin labeling using phalloidin, and immunolabeling of tubulin, alpha-smooth muscle actin or connexin 43, fluorescent and reflected light (for collagen fibrils) 3-D optical section images were acquired using laser confocal microscopy. In other experiments, dynamic imaging was performed using differential interference contrast microscopy, and finite element modeling was used to map ECM deformations. Corneal keratocytes developed a stellate morphology with numerous cell processes that ran a tortuous path between and along collagen fibrils without any apparent impact on their alignment. Fibroblasts on the other hand, had a more bipolar morphology with pseudopodial processes (P </= 0.001). Time-lapse imaging of keratocytes revealed occasional extension and retraction of dendritic processes with only transient displacements of collagen fibrils, whereas fibroblasts exerted stronger myosin II-dependent contractile forces (P < 0.01), causing increased compaction and alignment of collagen at the ends of the pseudopodia (P < 0.001). At high cell density, both keratocytes and fibroblasts appeared to form a 3-D network connected via gap junctions. Overall, this experimental model provides a unique platform for quantitative investigation of the morphological, cytoskeletal and contractile behavior of corneal keratocytes (i.e. their mechanical phenotype) in a 3-D microenvironment.
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