Quantitative analysis of stress relaxation in polyacrylamide hydrogels for mechanobiological studies

Purpose: The mechanical environment of the extracellular matrix strongly influences how cells behave - affecting their adhesion, migration, growth, and differentiation. While stiffness has been widely studied, recent research highlights the importance of viscoelasticity, especially the stress relaxation timescale, in how cells sense and respond to their surroundings. According to the widely accepted motor-clutch model, optimal cell spreading occurs when the stress relaxation timescale is similar to the timescale of molecular clutch binding. Methods: Polyacrylamide (PAAm) hydrogels, due to their tunable mechanical properties and bioinert nature, are commonly used as model substrates in mechanobiology. In this study, we investigated how changing the concentrations of crosslinker (N,N'-methylenebisacrylamide) and initiator (ammonium persulfate) affects the viscoelastic behavior of PAAm hydrogels. Using creep-recovery tests and fitting the data to the Standard Linear Solid model, we extracted mechanical parameters and calculated the stress relaxation timescale. Results: We found that the relaxation timescale increases with crosslinker concentration up to 0.05%, then decreases - suggesting an optimal crosslinking density. At a fixed 0.05% crosslinker, increasing initiator concentration reduced the relaxation timescale, likely due to faster gelation and less organized network formation. Conclusions: These findings demonstrate how simple adjustments in polymerization parameters can tune hydrogel relaxation behavior for mechanobiological applications.