Research

Cell Mechanics

Overview

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. 

Experimental Models

Our in vitro approaches include: 1) 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 dynamic changes in ECM mechanical properties (as might be observed during development or wound healing), 3) 3-D constructs for assessing the mechanical interplay between fibroblast migration, sub-cellular force generation, and ECM patterning, and 4) engineered substrates that allow investigation of the impact of ECM protein composition, topography and elasticity on cell differentiation and mechanical behavior.

Together, these experimental models are providing unique insights into the underlying biochemical and biomechanical signaling mechanisms controlling corneal fibroblast migration, contraction, and matrix reorganization. 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. 

Example Images

 

Figure 4: Color overlay of f-actin (green) and collagen fibils (red) allows interaction between cells and the extracellular matrix to be directly visualized. Increased compaction and alignment of collagen fibrils parallel to the pseudopodial tips is generally observed at the ends of corneal fibroblasts. Collagen was visualized using reflected light confocal imaging and f-actin was visualized using fluorescent labeling (Alexaflour phallodin 488).

Figure 1: Color overlay of f-actin (green) and collagen fibils (red) allows interaction between cells and the extracellular matrix to be directly visualized. Increased compaction and alignment of collagen fibrils parallel to the pseudopodial tips is generally observed at the ends of corneal fibroblasts. Collagen was visualized using reflected light confocal imaging and f-actin was visualized using fluorescent labeling (Alexaflour phallodin 488).

 

Figure 3: FEM strain maps generated using ANSYS, showing regions of matrix tension and compression. Stress on the matrix in serum containing media (A) is reduced when the cell is switched to Y-27632, which inhibits Rho Kinase (B). Stress is reestablished after washing out the Y-27632 (C). Note the reduction in stress fibers within the cell (as indicated by GFP-α-actinin labeling) following Y-27632 treatment. Strain is shown relative to the “relaxed” matrix configuration determined by treating cells with Cytochalasin D and TritonX-100.
 
Figure 2: FEM strain maps generated using ANSYS, showing regions of matrix tension and compression generated by a living corneal fibroblast within a 3-D collagen matrix. Stress on the matrix in serum containing media (A) is reduced when the cell is switched to Y-27632, which inhibits Rho Kinase (B). Stress is reestablished after washing out the Y-27632 (C). Note the reduction in stress fibers within the cell (as indicated by GFP-α-actinin labeling) following Y-27632 treatment. Strain is shown relative to the “relaxed” matrix configuration determined by treating cells with Cytochalasin D and TritonX-100.

 

Figure 6: Cell-matrix mechanical interactions in response to PDGF, which activates Rac. Cell-induced displacement and realignment of collagen fibrils was observed during PDGF-induced spreading. Tracking of the ECM displacements showed minimal collagen displacement prior to the addition of PDGF (B, red tracks, crosses mark position at time 0:00). However, following addition of PDGF, the matrix in front of the cell was pulled inward by the extending pseudopodial processes (C), resulting in compression of the ECM (yellow arrows).
 
Figure 3: Cell-matrix mechanical interactions during cell motility in response to PDGF, which activates the signaling protein Rac. Dynamic cell-induced displacement and realignment of collagen fibrils was observed during PDGF-induced spreading. Tracking of the ECM displacements showed minimal collagen displacement prior to the addition of PDGF (B, red tracks, crosses mark position at time 0:00). However, following addition of PDGF, the matrix in front of the cell was pulled inward by the extending pseudopodial processes (C), resulting in compression of the ECM (yellow arrows).