Regulation of Corneal Fibroblast Morphology and Collagen Reorganization by Extracellular Matrix Mechanical Properties

Cell-induced matrix reorganization and patterning are fundamental processes in developmental morphogenesis, corneal tissue engineering, and wound healing. During embryonic development, physical forces exerted by corneal fibroblasts organize extracellular matrix into a unique pattern that provides structural support while it maintains transparency.1–5 Similarly, one of the challenges in corneal tissue engineering is the development of an organized stroma with mechanical and optical properties that mimic the native tissue.6–10 Finally, after injury or refractive surgery, wound contraction and matrix remodeling by corneal fibroblasts play a central role in determining corneal clarity, mechanical integrity, and refractive properties.11–13 Despite the importance of cell-induced matrix patterning in corneal stromal biology, few studies have directly investigated the factors that regulate this process. One potential player in the regulation of cell-induced extracellular matrix (ECM) patterning is the mechanical state of the ECM itself. It is well established that mechanical stimuli play key roles in regulating growth and function in a variety of cell types.14–19 The formation of focal contacts and stress fibers by fibroblasts are tension-dependent processes,20–22 and these structures tend to align along the tensile axis under anisotropic conditions.23–26 Alignment and global force generation parallel to the axis of greatest mechanical resistance by dermal fibroblasts, myoblasts, and bone marrow stem cells have also been documented in three-dimensional (3-D) culture models.27–31 However, the effect of ECM anisotropy on corneal fibroblast behavior has not been assessed. Furthermore, these previous studies were performed using high cell densities, which does not allow detailed investigation of the underlying pattern of matrix reorganization at the cellular and subcellular levels. Thus, the effects of matrix anisotropy on the local pattern of cell-induced ECM remodeling (i.e., compaction and alignment of collagen fibrils) have not been directly assessed. Confocal microscopy allows quantitative assessment of local changes in ECM organization within 3-D collagen matrices.32–37 In the present study, we applied this technique to directly investigate the role of ECM mechanical anisotropy on corneal morphology and matrix patterning using an in vitro 3-D culture model. Human corneal fibroblasts were cultured at low density within rectangular collagen constructs with varying degrees of mechanical constraint along the long axis. Overall, significant differences in cell alignment, morphology, and matrix reorganization were observed between constrained (anisotropic) and unconstrained (isotropic) matrices. Cells were aligned nearly parallel to the long axis of the construct in constrained matrices, whereas cells in unconstrained matrices showed no preferential orientation. At the ends of cells, local collagen density and degree of cell/collagen coalignment were higher in constrained matrices. Taken together, these data demonstrate for the first time that under anisotropic conditions, isolated corneal fibroblasts tend to align and compact collagen parallel to the axis of greatest effective stiffness. This effect was observed several millimeters away from the location of the physical constraints, indicating that collagen ECM is an effective conduit for long-range transmission of mechanical forces. Interestingly, in regions of higher cell density within constrained matrices, additional bands of compacted and aligned collagen spanning between adjacent cells were often observed, suggesting mechanical coupling which leads to local amplification of matrix patterning.