The organization of extracellular matrices by cells through the exertion of mechanical forces drives fundamental processes such as developmental morphogenesis, wound healing, and the organization of bioengineered tissues. Historically, our ability to investigate cell mechanical behavior has been limited by the technical challenges associated with measuring the sub-cellular origins of cellular force generation and local matrix patterning in a 3-D environment. Thus, our understanding of these fundamental processes is limited, especially in ocular tissues. Over the last several years, our research team has addressed these challenges through the development of new experimental models, use of emerging imaging technologies, and the application of quantitative analysis techniques. An overview of some of these approaches is provided below and additional examples are provided separately in the form of time-lapse movies and 3-D reconstructions.
We first developed an experimental model for dynamic investigation of cell-matrix mechanical interactions by plating corneal fibroblasts transfected to express GFP-tagged cytoskeletal or adhesive proteins inside fibrillar collagen matrices, and performing high-magnification time-lapse differential interference contrast (DIC) and fluorescent imaging. As shown in Figure 1, DIC imaging allowed detailed visualization of the cells and the fibrillar collagen surrounding them.
A corneal fibroblast transfected to express GFP-zyxin (which labels focal adhesions) is shown in Figure 2.
Color-coded reconstructions demonstrate that adhesions were present on both the ventral (red) and dorsal (green) surfaces of cells (2D); whereas overlaying of GFP-zyxin maximum intensity projections and individual DIC images allowed the organization of focal adhesions and collagen fibrils to be directly compared (2F). This unique approach allows dynamic changes in the sub-cellular organization of cytoskeletal and adhesive proteins to be quantitatively correlated with the pattern of cell-induced matrix deformation. By tracking matrix deformation and applying Finite Element Modeling (FEM), maps of ECM strain can be also generated. (Figure 3)
In subsequent studies, we further extended this paradigm to quantify the detailed 3-D pattern of local collagen matrix remodeling by using confocal reflection imaging. Using this approach, the 3-D structural organization of the fibrillar collagen can be directly correlated with temporal changes in the organization of key cytoskeletal, adhesive or regulatory proteins (Figure 4).
By using Fourier Transform analysis, quantitative assessment of cell-induced collagen remodeling can be performed (Figure 5).
Overall, the combination of DIC and reflected light confocal imaging allows us to assess both rapid changes in cellular contractility which can occur within minutes, and more gradual changes in cell-induced compaction and alignment of ECM which can occur over hours or even days. These novel approaches are being used to investigate the roles of the small GTPases Rho and Rac in regulating the sub-cellular pattern of force generation and extracellular matrix reorganization by corneal fibroblasts (Figure 6), as well as the cellular responses to local changes in mechanical stress (see Publications).
Recently, we have expanded our techniques to include: 1) experimental models for assessing how specific growth factors expressed following injury or surgery alter the mechanical differentiation of quiescent corneal keratocytes within 3-D collagen matrices, 2) novel approaches for assessing the response of fibroblasts to large scale changes in ECM mechanical properties (as might be observed during development or wound healing), and 3) 3-D constructs for assessing the mechanical interplay between fibroblast migration, sub-cellular force generation, and ECM patterning. Together, these experimental models are providing unique insights into the underlying biochemical and biomechanical signaling mechanisms controlling corneal fibroblast migration, contraction, and matrix reorganization in response to specific growth factors. This is fundamental information which can not be obtained using standard 2-D culture models, and may eventually lead to improved strategies for modulating cell mechanical activity during wound healing, and for designing artificial matrices and directing cell behavior during corneal tissue engineering.