Growth Factor Regulation of Corneal Keratocyte Differentiation and Migration in Compressed Collagen Matrices

Stromal keratocytes play a central role in mediating corneal response to injury or refractive surgery.1 In the corneal stroma, quiescent keratocytes normally have a dendritic morphology and a cortical distribution of F-actin.2 During wound healing, quiescent corneal keratocytes surrounding the injured region differentiate into fibroblast or myofibroblast phenotypes that mediate cell migration, wound contraction, and matrix remodeling3,4; these processes have a decisive impact on a patient's corneal clarity and refractive outcome.3,5,6 Numerous cell culture studies have identified several key growth factors that play important roles in mediating keratocyte differentiation into specific wound-healing phenotypes.7–20 However, these studies have been performed primarily using 2D substrates, and keratocytes reside within a complex 3D extracellular matrix in vivo. Significant differences in cell morphology, adhesion organization, and mechanical behavior have been identified between 2D and 3D culture models, with 3D models generally more closely mimicking in vivo cell behavior.21–30 Three-dimensional models also allow investigation of cell-induced matrix degradation and remodeling, key events in corneal wound healing. Thus, an in vitro model that mimics the 3D geometry and mechanical properties of the corneal stroma would be an important new platform for investigating key aspects of the wound-healing process. Because collagen is the main ECM component of most tissues, many in vitro models of various tissues involve the use of hydrated 3D fibrillar collagen matrices. The low collagen density and high compliance of these matrices are ideal for assessing how subcellular force generation and cell-induced matrix reorganization are regulated31–41; however, unlike native corneal stromal tissue, they undergo significant deformation in response to cellular forces, which limits their use for corneal tissue engineering. To overcome this limitation, some investigators have used glutaraldehyde or other chemical cross-linking techniques to increase the rigidity of hydrated collagen matrices. However, these approaches have several limitations: (1) they are often toxic and can only be applied to acellular matrices; (2) repopulating the acellular matrices after cross-linking can only be accomplished using corneal fibroblasts (not quiescent keratocytes); (3) the collagen concentration of these matrices is much lower than the collagen density of the cornea; and (4) they do not allow normal cell-based scaffold remodeling.42–45 Another approach that has been used to generate high-stiffness tissue equivalents in vitro is to plate fibroblasts within standard fibrillar collagen matrices and then stimulate the cells to compact the matrix by contractile force generation.46 This process produces a stiffer matrix with a high density of cells and collagen fibrils (a tissue equivalent). This approach has been used to generate artificial stromas for corneal tissue engineering.47,48 However, an important limitation of these models is that the cells have to be activated to pre-contract the collagen matrix, which may alter their subsequent growth factor responses and migratory mechanics. Similarly, collagen sponges and vitrigel collagen membranes do not support the invasion and differentiation of quiescent corneal keratocytes.49,50 More recently, a self-assembly approach has been used to generate more rigid 3D collagen matrices. In this model, corneal fibroblasts are cultured for several weeks in media containing a high concentration of ascorbic acid, which results in the generation of layered cell/matrix constructs that appear to have structural similarity to the in vivo cornea. This elegant approach has the potential to provide important insights into the developmental organization of the corneal stroma.51,52 However, it takes several weeks of culture to produce an organized ECM using this method, and these matrices cannot yet be generated using quiescent corneal keratocytes. To study the phenotypic transformation and migratory response of quiescent (i.e., noncontractile) corneal keratocytes to specific wound-healing cytokines and other signals, a model is still needed that does not require the use of activated corneal fibroblasts for its generation. Recently, Brown et al.53–55 developed a novel approach to dermal and bone tissue engineering based on plastic compression of fibrillar collagen matrices. In this model, standard cell-seeded, hydrated collagen matrices are polymerized. The cell-matrix construct is then immediately compacted by placing a stainless steel block on top and allowing fluid to flow out of the matrix into a paper blotting layer underneath. This process results in the formation of a flat, cell/collagen sheet. Because the excess fluid present in collagen gels is a result of casting and not of an inherent swelling property, the matrix maintains its compressed shape after removing the load (hence, the term plastic compression). These matrices have several unique properties that may be well suited for corneal stromal tissue engineering. In this study, we investigated the behavior of corneal keratocytes within these matrices. We first established that corneal keratocytes survived the matrix compression procedure and appeared to differentiate normally and to respond to growth factors within these matrices. We then used this model to investigate the effects of key wound-healing growth factors on corneal keratocyte migration using a “nested” 3D matrix approach.