Hydrogel substrate stress-relaxation regulates the spreading and proliferation of mouse myoblasts

Mechanical properties of the extracellular microenvironment are known to alter cellular behavior, such as spreading, proliferation or differentiation. Previous studies have primarily focused on studying the effect of matrix stiffness on cells using hydrogel substrates that exhibit purely elastic behavior. However, these studies have neglected a key property exhibited by the extracellular matrix (ECM) and various tissues; viscoelasticity and subsequent stress-relaxation. As muscle exhibits viscoelasticity, stress-relaxation could regulate myoblast behavior such as spreading and proliferation, but this has not been previously studied. In order to test the impact of stress relaxation on myoblasts, we created a set of two-dimensional RGD-modified alginate hydrogel substrates with varying initial elastic moduli and rates of relaxation. The spreading of myoblasts cultured on soft stress-relaxing substrates was found to be greater than cells on purely elastic substrates of the same initial elastic modulus. Additionally, the proliferation of myoblasts was greater on hydrogels that exhibited stress-relaxation, as compared to cells on elastic hydrogels of the same modulus. These findings highlight stress-relaxation as an important mechanical property in the design of a biomaterial system for the culture of myoblasts. STATEMENT OF SIGNIFICANCE: This article investigates the effect of matrix stress-relaxation on spreading and proliferation of myoblasts by using tunable elastic and stress-relaxing alginate hydrogels substrates with different initial elastic moduli. Many past studies investigating the effect of mechanical properties on cell fate have neglected the viscoelastic behavior of extracellular matrices and various tissues and used hydrogels exhibiting purely elastic behavior. Muscle tissue is viscoelastic and exhibits stress-relaxation. Therefore, stress-relaxation could regulate myoblast behavior if it were to be incorporated into the design of hydrogel substrates. Altogether, we showed that stress-relaxation impacts myoblasts spreading and proliferation. These findings enable a better understanding of myoblast behavior on viscoelastic substrates and could lead to the design of more suitable substrates for myoblast expansion in vitro.