Corneal wound healing following lacerating injury, penetrating keratoplasty or refractive surgery involves an ordered sequence of cell-matrix mechanical interactions. In the corneal stroma, quiescent keratocytes normally have a dendritic morphology and a cortical distribution of f-actin [44
]. Following injury or surgery, keratocytes surrounding the wound transform to an activated, fibroblast phenotype characterized by a more bipolar morphology and prominent intracellular stress fibers [45
]. Corneal fibroblasts migrate into the wound, synthesize new extracellular matrix, and reorganize the ECM through the application of mechanical forces. Wound contraction and matrix remodeling by corneal fibroblasts ultimately determine a patient’s corneal clarity and refractive outcome [47
]. Understanding the underlying cellular and molecular mechanisms that regulate these biophysical processes in corneal fibroblasts could ultimately lead to more effective approaches to modulating the wound healing response in vivo.
Previous in vitro studies on rigid substrates have shown that serum induces transformation of quiescent corneal keratocytes to a wound healing, fibroblast phenotype [23
]. In this study, following culture S+ media (in which Rho is activated) corneal fibroblasts in 3-D collagen matrices had a bipolar or stellate morphology, and stress fibers were often observed along the cell body and pseudopodial processes, consistent with the in vivo corneal fibroblast phenotype. In contrast, cells treated with the Rho kinase inhibitor Y-27632 were more elongated, had a dendritic morphology, and f-actin staining that was generally limited to the cell cortex, similar to quiescent keratocyte phenotype in vivo. Consistent with previous studies using dermal fibroblasts, global matrix contraction was also significantly reduced when Rho kinase was inhibited [19
]. Overall, the data suggest that activation of Rho kinase may play a key role in mediating the transformation of quiescent corneal keratocytes to a contractile, fibroblast phenotype during corneal wound healing.
Because the attachment plane of the matrices is large compared to the matrix height (1 cm vs. 100 μm) and the top of the matrix is unconstrained, there is more resistance to collagen displacement in the x, y plane than there is along the z-axis. This is why contraction of attached collagen matrices occurs through a decrease in height, not diameter. In order to provide insights into the underlying mechanism of global matrix contraction, quantitative analysis of 3-D cell morphology and both the pattern and amount of local cell-induced collagen matrix reorganization was performed. After 24 hours of culture, cells in S+ were always aligned nearly parallel to the bottom of the culture dish on which the collagen matrix was plated, and collagen fibrils were compacted and aligned parallel to the long axis of stress fibers and pseudopodia. In contrast, cells had longer extensions along the z-axis when Rho kinase was inhibited, and collagen surrounding the cells was less compacted and more randomly organized. Thus Rho kinase dependent contraction by corneal fibroblasts leads to co-alignment of cells and collagen fibrils along the plane of greatest mechanical resistance. Interestingly, these data are consistent with previous studies which demonstrate gradual alignment of stress fibers along the axis of greatest mechanical resistance during in vivo corneal wound healing [37
Previous investigators have visualized the collagen organization surrounding isolated cells using DIC imaging or electron microscopy. Qualitative analysis of these 2-D images suggests that existing matrix fibrils generally become aligned parallel to the long axis of the cell, consistent with the quantitative results from this study [10
]. However, confocal reflection imaging allows the 3-D collagen architecture to be reconstructed, and thus provides better insights into cell-induced matrix reorganization than 2-D DIC or TEM imaging. Friedl and coworkers have used this technique to study local matrix reorganization by different cell types within 3-D collagen lattices [41
]. In fibroblasts and MV3 melanoma cells, β1 integrins are clustered predominantly at the leading edge of cells in association with aligned and compacted collagen fibers [55
]. In contrast, migrating T-lymphocytes are free of clustered β1 integrins, and local matrix reorganization is not observed. These results suggest that sustained force generation by large, slow moving cells in S+ media leads to increased structural matrix reorganization as compared to the more transient forces produced by migratory cells. The results of this study suggest that in corneal fibroblasts, these sustained forces are Rho kinase dependent.
Although global matrix contraction, and compaction and alignment of pseudopodia were significantly reduced when Rho kinase was inhibited, they were still greater than control matrices without cells. Thus cellular force generation was only partially blocked by inhibiting Rho kinase. In previous studies using time-lapse DIC imaging, we have investigated the dynamic pattern of force generation by corneal fibroblasts within 3-D collagen matrices. These studies demonstrated that in cells cultures in S+, extension of pseudopodial processes generated tractional forces on the ECM as indicated by pulling in of the collagen fibrils in front of the cell. At the same time, regions of ECM compression were produced at the base of pseudopodia. After 24 hours of culture in S+, addition of Y-27632 induced rapid cell body elongation and relaxation of matrix stress along the cell body, as well as pseudopodial and/or filopodial extension [40
]. However, smaller forces continued to be generated at the leading edge of extending pseudopodia, as indicated by transient displacement (pulling in) of collagen fibrils. Furthermore, Grinnell and coworkers have demonstrated that LPA stimulated dermal fibroblasts can displace the collagen surrounding them when Rho kinase is inhibited (as indicated by movement of embedded microspheres) [32
]. Sheetz and coworkers investigated the relationship between force and focal complex development during spreading of NIH 3T3 fibroblasts on rigid substrates by using an optical laser trap to monitor the force on beads coated with a fragment of fibronectin type III [56
]. The development of force was inhibited by dominant-negative Rac and by myosin light chain kinase inhibition, but not by dominant-negative Rho or Rho kinase inhibition. Taken together, these studies suggest that small, transient forces can be generated at the leading edge of extending pseudopodia via Rho kinase-independent myosin light chain phosphorylation. In contrast, larger forces generated along the cell body and at the base of pseudopodia are Rho/Rho kinase-dependent. Thus, the residual matrix reorganization observed following sustained Rho kinase inhibition in the current study likely results from localized tractional forces generated during formation and extension of dendritic cell processes.
The complexity of collagen fibril organization makes quantitative analysis of orientation information in 3-D difficult. Davidson et al. have used a pattern matching method to analyze fibril orientation of composite materials [57
]. To define the orientation of a fiber unambiguously in space, two angles are required; in-plane angle
and out-of-plane angle θ. A number of serial sections of images can be obtained, and the pattern matching method is then used to correlate the fiber images from each section, allowing full 3-D orientation data to be extracted. However, the measurement error at low values of θ can be large, so that smaller angles are cut to attain greater accuracy. This technique is also limited to unidirectional continuous fibril composites and relies on number of other assumptions. The Hough transform [58
] is commonly used for the detection of regular curves such as lines, circles, and ellipses but this cannot detect the ends of fibers and can become unreliable for irregular curves like collagen fibers. Thus to assess local matrix remodeling in the present study, we evaluated the alignment of collagen fibrils using 2-D Fourier transform analysis, which avoids the artifacts mentioned above. We have used this approach previously to assess stress fiber orientation during in vivo corneal wound healing [37
]. Schwartz and coworkers have used a similar FT approach to assess flow-induced collagen matrix remodeling [59
]. In their study, the peak of the FT line averages was used to define an orientation angle, and the degree of alignment at this angle was determined by using a ratio of the sum of frequencies within 20° of that angle against the sum of all angles. We instead used a previously described orientation index, which allows the degree of collagen fibril alignment along a particular angle to be expressed as a single normalized value (%). While the angle of maximum fibril alignment can also be calculated using this approach, we found the use of a single orientation index more useful in the current study. Overall, such quantitative approaches should be valuable for future investigations of the underlying mechanisms of local cell-induced matrix remodeling using confocal reflection imaging.