It is well established that mechanical stimuli play a key role in regulating growth and function in a variety of cell types (Sadoshima and Izumo, 1997
; Tummina et al., 1998
; Liu et al., 1999
; Brown, 2000
; Shyy and Chien, 2002
; Guo et al., 2006
). During embryonic development, physical forces exerted by mesenchymal cells organize extracellular matrix (ECM) into a wide variety of spatial patterns, and feedback between cell and matrix mechanics has long been thought to be a key factor regulating this process (Bard and Hay, 1975
; Bard and Higginson, 1977
; Stopak and Harris, 1982
; Stopak et al., 1985
; Krieg et al., 2008
). For example, the stiffness of planar subtsrates in 2-D cell culture models plays a central role in directing human mesenchymal stem cells along neuronal, muscle or bone lineages (Engler et al., 2006
), and these effects are mediated by cellular force generation via nonmuscle myosin II. As shown by Bard and Hay, migrating mesechymal cells are in close proximity during corneal stromal development, thus localized alterations in effective matrix stiffness due to the activity of nearby cells are likely to occur (Bard and Hay, 1975
). In the current study, we use small microneedles (Femtotips) to allow precise local control over 3-D ECM deformation, and investigate the cellular responses to changes in localized ECM tension along different directional axes.
Changing local ECM stress using femtotips induced rapid and reproducible response patterns in both rabbit and human corneal fibroblasts. Reducing effective ECM stiffness by pushing the needle parallel to the long axis of a cell resulted in rapid cellular shortening with corresponding ECM compression along the cell body. This initial shortening is likely due to the release of pre-existing cellular contractile forces, since the response was blocked by treatment with cytochalasin D, or Rho kinase inhibition. These data demonstrate that there is dynamic feedback between cytoskeletal forces and local ECM stress that regulates corneal fibroblast mechanical behavior within 3-D matrices. Brown and coworkers used a culture force monitor (Eastwood et al., 1994
) to measure how dermal fibroblasts within 3-D collagen matrices respond to global
changes in tensional loading (Brown et al., 1998
). Their data suggests that cells within 3-D matrices alter their contractility in response to changes in mechanical loading in a way that maintains “tensional homeostasis” (constant tension) in their surrounding matrix. Our data on local
feedback between cells and ECM is also consistent with the tensional homeostasis model, since following the reduction of tension induced by pushing the needle toward the cell, fibroblasts attempt to re-establish baseline tension by actively pulling in the ECM.
In contrast to ECM compression parallel to the long axis of cells, compressing the ECM perpendicular to the long axis had little effect on cell morphology or mechanical activity. This is also consistent with the tensional homeostasis model, since the cytoskeleton, focal adhesions and contractile forces are all aligned parallel to the long axis of bipolar cells, and therefore reducing the effective stiffness of the ECM alongside of the cell should have little impact on cellular tension.
We observed similar response patterns from corneal fibroblasts derived from elderly human tissue as well as those from young rabbit eyes. Many aspects of the mechanical behavior of these ocular cells have been shown previously to be similar to that of dermal fibroblasts (Roy et al., 1999a
; Jester and Chang, 2003
; Kim et al., 2006
; Karamichos et al., 2007
; Kim and Petroll, 2007
). Furthermore, the morphological changes these cells undergo during initial spreading and migration in 3-D culture are nearly identical to that of chick embryo fibroblasts. Thus we hypothesize that tensional homeostasis may be a conserved mechanism that regulates mesenchymal cell behavior both during developmental morphogenesis and adult wound healing. Additional experiments using embryonic-derived cells are clearly needed to test this hypothesis.
The Rho-family of small GTPases such as Rho, Rac, and Cdc42 play a central role in regulating the cytoskeletal changes associated with cell spreading, migration and contraction (Hall, 2005
; Jaffe and Hall, 2005
; Bustelo et al., 2007
). These GTP binding proteins function as molecular switches; alternating between the active GTP-bound state and the inactive GDP-bound state. Previous studies suggest that Rho and Rac may be involved in the cellular response to a variety of mechanical signals. For example, in vascular smooth muscle cells, non-cyclic uniaxial mechanical stretching was shown to downregulate Rac and suppress cell spreading, whereas decreasing mechanical tension (by inhibiting Rho kinase or myosin light chain kinase) increased cell spreading through upregulation of Rac (Katsumi et al., 2002
). A reduction in mechanical tension and increased spreading by corneal fibroblasts has also been demonstrated following Rho kinase inhibition in 3-D matrices (Vishwanath et al., 2003
). In the current study, cell spreading and tractional force generation by corneal fibroblasts was observed after reducing ECM tension by pushing with microneedles. A similar spreading response was induced by local ECM microinjection of PDGF, which activates Rac (Sander et al., 1999
; Grinnell, 2000
). Taken together, the data suggest that the interplay between Rho and Rac activation may play a central role in the fibroblast response to local mechanical stimulation. Additional studies more specifically targeting Rho and Rac signaling pathways are needed to clarify the molecular mechanisms underlying these important processes.
The cellular response to transient mechanical stimulation using microneedles has been investigated previously using planar elastic polyacrylamide substrates (Lo et al., 2000
; Wang et al., 2001
). The advantage of this 2-D model is that the mechanical properties of the substrate can be fully characterized, and precise mapping of cellular forces in response to mechanical or biochemical stimulation can been achieved. In their model, pushing the substrate toward the leading edge of a cell caused the cell to retract its leading edge and migrate away from the needle, a phenomenon termed “durotaxis”. We did not observe this type of migratory behavior in response to mechanical stimulation in the current study. Instead, cell spreading toward the microneedle was observed, even when the ECM was compressed at the rear of a migrating cell. This disparity is likely due, in part, to differences in the geometry and mechanical properties of planar elastic substrates and 3-D fibrillar matrices, as well as the fact that corneal fibroblasts are generally less migratory in our 3-D model. While durotaxis likely plays a role in modulating cell migration within collagen matrices, tensional homeostasis appears to dominate cell behavior under the conditions used in the current study.
Localized application of cytokines or peptides can be a useful tool for assessing mechanical behavior at the subcellular level. For instance, Wang and coworkers compared the effects of local application of the GRGDTP peptide to selectively disrupt substrate adhesions at the front and rear of migrating 3T3 fibroblasts on planar substrates (Munevar et al., 2001
), and cell matrix interactions were found to be distinctly different at the leading and trailing edge of the cells. In the current study, we investigated whether microinjection needles could be used to study the effects of localized application of a cytokine on dynamic cell behavior in 3-D collagen matrices. Femtotips could be inserted axially into the matrix without breaking the tip or significantly deforming the collagen organization surrounding the cells, and injection of control solution did not alter cell mechanical activity. In contrast, injection of PDGF BB induced rapid cell spreading and elongation, similar to that observed following matrix compression along the cell axis. Overall, local microinjection into 3-D collagen matrices may be a promising new approach for investigating mechano-regulation of corneal fibroblasts at the subcellular level.
Elizabeth Hay was a pioneer in live-cell imaging, and was the first to use DIC imaging to visualize corneal embryonic fibroblast migration in situ and within 3-D collagen matrices. While important insights into the mechanisms of cell migration were gained in these studies, recent advances in microscope optics and digital imaging technology allow these processes to be studied with much higher temoral and spatial resolution using current microscope systems. In this study, some cells underwent significant translocation either spontaneously in serum containing media, or following local microinjection of PDGF into serum-free media. In both cases, extension of pseudopodia and tractional force generation at the leading edge of migrating cells was observed. At the rear of the cells, apparent rupture of cell-matrix adhesions led to elastic recoil of cell processes and release of ECM tension (as indicated by collagen movement away from the cell). Extension of existing processes or protrusion of a new process was often observed at the front of the cell simultaneously with a large retraction at the rear, consistent with the concept that reducing cellular tension stimulates cell spreading. Surprisingly, small extensions and retractions of pseudopodia and associated collagen fibril displacements were detected at the trailing edge of the cell during cell migration. Furthermore, when the needle was pushed toward the trailing edge of a migrating cell, both the initial contraction and secondary spreading response were still observed. These novel findings suggest that similar cytoskeletal machinery and/or signaling networks may be present to some extent at both the front and rear of migrating cells, facilitating remarkable plasticity and rapid responses to mechanical stimuli at either end.