2.3. Mechanical characterization of alginate hydrogels and rat muscle

The stiffness and stress-relaxation properties of alginate gels were assessed from compression tests using an Instron 3342 single-column mechanical tester[18]. The gels used for mechanical testing were 2 mm thick, 15 mm diameter and were equilibrated in DMEM for 24 h before measurements. For the stiffness measurements, the gels were compressed to 15% strain with a deformation rate of 1 mm/min. The Young’s modulus (corresponding to the elastic modulus) was then calculated as the slope of the linear stress strain curve between 5–10 % strain. For the stress-relaxation measurements, the gels were compressed to 15 % strain with a deformation rate of 1 mm/min, consequently the strain was held constant and then stress was recorded as a function of time. The stress-relaxation rate was calculated as the half stress-relaxation time. Stress relaxation measurements of biological tissues were performed using the same procedure. Sprague Dawley Rats (male, 7 weeks of age, Charles River Lab) were euthanized in compliance with National Institutes of Health and institutional guidelines. Rat hindlimb muscles were collected after euthanization and tested immediately with the Instron 3342 single column apparatus.

Stress relaxation tests on biological tissues are typically conducted between 10–30% strains. With relation to the relevance for interactions with cells, various levels of strain have been reported. In cell mechanotransduction studies, strains of a few percent to 30% have been observed [27], [28]. The 15 % strain used here is within the strain range generated by cells and has been used in previous mechanotransduction studies[19]. A compression rate of 1 mm/min was used here so that a 15% strain could be applied on the soft, thin hydrogel samples quickly but without generating excessive strain or large noise at the beginning of the test due to the inertia from the heavy arm and load cell of the Instron mechanical tester.