Matching material and cellular timescales maximizes cell spreading on viscoelastic substrates
Proceedings of the National Academy of Sciences of the United States of America
Recent evidence has shown that, in addition to rigidity, the viscous response of the extracellular matrix (ECM) significantly affects the behavior and function of cells. However, the mechanism behind such mechanosensitivity toward viscoelasticity remains unclear. In this study, we systematically examined the dynamics of motor clutches (i.e., focal adhesions) formed between the cell and a viscoelastic substrate using analytical methods and direct Monte Carlo simulation. Interestingly, we observe
... stingly, we observe that, for low ECM rigidity, maximum cell spreading is achieved at an optimal level of viscosity in which the substrate relaxation time falls between the timescale for clutch binding and its characteristic binding lifetime. That is, viscosity serves to stiffen soft substrates on a timescale faster than the clutch off-rate, which enhances cell−ECM adhesion and cell spreading. On the other hand, for substrates that are stiff, our model predicts that viscosity will not influence cell spreading, since the bound clutches are saturated by the elevated stiffness. The model was tested and validated using experimental measurements on three different material systems and explained the different observed effects of viscosity on each substrate. By capturing the mechanism by which substrate viscoelasticity affects cell spreading across a wide range of material parameters, our analytical model provides a useful tool for designing biomaterials that optimize cellular adhesion and mechanosensing. mechanotransduction | viscoelasticity | cell spreading | focal adhesion | timescales M ounting evidence has demonstrated that cells can sense and react to the physical properties of the extracellular matrix (ECM), an ability that plays a key role in processes such as cell migration (1-3), spreading (4, 5), and proliferation (6-8). It is commonly believed that focal adhesions (FAs), which anchor the cell to the ECM as well as serving as hubs for the exchange of biological and mechanical stimuli (9, 10), are responsible for such mechanosensitivity of cells. It has been recognized that cells probe the stiffness of their surroundings by gauging the resistance of FAs to actin retrograde flow generated by intracellular myosin contractions (11) (12) (13) (14) . FAs, acting like molecular clutches, alter the movement of actin intracellular structures by providing a tunable connection to the ECM (13, 15). Based on this picture, the well-known motor-clutch model (15-17) was developed, which predicted a biphasic dependence of cell adhesion traction (and consequently cell spreading) on ECM rigidity. However, recent experiments have found a monotonic increase of cell spreading with the ECM rigidity (18, 19) , which can be attributed to reinforcement mechanisms including, for example, the activation of adhesion proteins under high surrounding stiffness/load level or the recruitment of integrins into the FAs (20, 21). Beyond substrate rigidity, most natural ECM materials such as collagen, fibrin (22), and tissues (23-25) are viscoelastic in nature and exhibit a strong frequency-dependent mechanical re-sponse. Interestingly, it was reported recently that cell spreading can be enhanced by stress relaxation of a cell culture substrate (e.g., alginate, polyacrylamide), which was dependent on the elastic modulus of the substrate (4, 26). This was explained by local remodeling (leading to increased ligand density) of the matrix during deformation (4), which corresponds to a plastic rather than viscous response. On the other hand, our own experiments (to be discussed in Model Predicts Cells Spreading for Several Different Cell Types Across a Wide Range of Elastic and Viscous Substrate Properties) suggest that viscosity has a negligible effect on how cells spread. However, it is unclear how a purely viscoelastic substrate can have different effects on cell spreading, largely due to the lack of a theoretical model capable of revealing the physical mechanisms governing the cellular response to viscoelasticity. To address this critical issue, we systematically examined how cell spreading is influenced by the viscoelastic components of the ECM via an analytical mean-field analysis and direct Monte Carlo simulations. Specifically, by treating the ECM as a standard linear viscoelastic solid, we found that an intermediate level of viscosity can promote cell spreading when the ECM rigidity is relatively low, reflecting the fact that the substrate relaxation time under such circumstance falls between the timescale Significance It is well known that cell proliferation, differentiation, and migration depend strongly on the mechanical stiffness of the extracellular matrix (ECM). Natural ECMs also exhibit dissipative (i.e., plastic, viscoelastic) properties, which can modulate cellular behavior. However, to fully utilize this information in bioengineering applications, a systematic understanding of the role of substrate viscosity on cell function is needed. Using combined theoretical and experimental approaches, we demonstrated that viscous dissipation can be as important as elasticity in determining cell response. Specifically, we found that intermediate viscosity maximizes cell spreading on soft substrates, while cell spreading is independent of viscosity on stiff substrates. This information can now be used to design dissipative biomaterials for optimal control of cell behavior.