Cyclic Stress at mHz Frequencies Aligns Fibroblasts in Direction of Zero Strain

Abstract

Recognition of external mechanical signals is vital for mammalian cells. Cyclic stretch, e.g. around blood vessels, is one such signal that induces cell reorientation from parallel to almost perpendicular to the direction of stretch. Here, we present quantitative analyses of both, cell and cytoskeletal reorientation of umbilical cord fibroblasts. Cyclic strain of preset amplitudes was applied at mHz frequencies. Elastomeric chambers were specifically designed and characterized to distinguish between zero strain and minimal stress directions and to allow accurate theoretical modeling. Reorientation was only induced when the applied stretch exceeded a specific amplitude, suggesting a non-linear response. However, on very soft substrates no mechanoresponse occurs even for high strain. For all stretch amplitudes, the angular distributions of reoriented cells are in very good agreement with a theory modeling stretched cells as active force dipoles. Cyclic stretch increases the number of stress fibers and the coupling to adhesions. We show that changes in cell shape follow cytoskeletal reorientation with a significant temporal delay. Our data identify the importance of environmental stiffness for cell reorientation, here in direction of zero strain. These in vitro experiments on cultured cells argue for the necessity of rather stiff environmental conditions to induce cellular reorientation in mammalian tissues.

A thermodynamically motivated model for stress-fiber reorganization

“…Again, since imposed strain-rates are zero in the = 90 o direction there is no variation of ̃9 0 and ̂9 0 at steadystate. Consistent with the observations of Faust et al (2011), the model predicts isotropic stress-fiber distributions when max = 0.1 but the distribution of becomes more anisotropic with increasing max with a clear alignment of stress-fibers perpendicular to the loading direction predicted in the max = 0.4 case. To understand this strain dependence at these very slow imposed cyclic frequencies, let us consider the max = 0.4 case corresponding to = 0.025 Hz, i.e.…”

“…This hypothesis was further reinforced by the experiments of Tondon et al (2012) who demonstrated reduced sensitivity of cells subjected to fixed strain amplitudes but reduced frequencies of applied loading. However, this explanation contrasts with the observations of Faust et al (2011) who performed experiments with a fixed (low) value of applied strain-rate and varied the strain amplitude. The cells they investigated showed an increased strain avoidance when the imposed strain amplitude was increased beyond a critical value suggesting a sensitivity to strain amplitude rather than strain-rate.…”

“…This suggests that with decreasing imposed strain-rate the sensitivity of cells to cyclic loading decreases. However, Faust et al (2011) demonstrated that for umbilical cord fibroblasts subjected to cyclic straining at low frequencies (on the order of mHz), the stress-fibers reorient in a direction perpendicular to the imposed loading when the applied strain amplitude exceeds a critical value. This suggests these fibroblasts develop sensitivity to cyclic loading at large applied strain amplitudes even at applied strain-rates ≪ 0 .…”

Prediction of Cell Alignment on Cyclically Strained Grooved Substrates

“…For instance, cells orient perpendicular to cyclic strain when cultured on stiff substrates or on biaxially constrained collagen gels (1)(2)(3)(4)(5)(6)(7). This phenomenon, called strain avoidance (SA), depends on the profile of the cyclic strain because the degree of cellular (re)orientation is proportional to the amplitude and frequency of the strain (1,2).…”

“…In fact, cellular alignment generally corresponds to the main SF direction (2), and experiments have shown that these acto-myosin bundles are essential for the reorientation of cells in response to cyclic strain (3,5). On the other hand, FAs are crucial to sense and interact with topographical stimuli, because they form the link between cells and their surroundings (34).…”