Viscoelastic gels were formed following a modified internal calcium carbonate gelation protocol[28 (link)]. Alginate in PBS was mixed with (1) CaCO3 at 50 mg/mL and (2) glucono-δ-lactone (GDL; Sigma Aldrich, St. Louis, MO) at 150–200 mg/mL with a volume ratio of 8:1:1. The variation in GDL content controlled the amount of calcium ions liberated for crosslinking via lowering the pH, thereby varying the stiffness of the resulting gel. The mixture was pipetted into molds of 8 mm diameter and 1.5 mm height and allowed to gel at 37°C for 3 h.
Elastic gels underwent carbodiimide covalent crosslinking as described previously[21 (link)]. Alginate in MES buffer was mixed with (1) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC; Sigma Aldrich) at 100 mg/mL and (2) a solution of adipic acid dihydrazide (AAD) and 1-hydroxybenzotriazole (1-HOBT; Sigma Aldrich), both at 25–75 mM. The mixture had a volume ratio of 8:1:1 alginate:EDC:AAD/1-HOBT. We controlled the number of crosslinks and resultant stiffness of the gel by variation in AAD content. The mixture was pipetted into identical molds as for viscoelastic hydrogels and allowed to gel under the same conditions. In both cases, the final concentration of alginate within the gels was 20 mg/mL.
Elastic gels underwent carbodiimide covalent crosslinking as described previously[21 (link)]. Alginate in MES buffer was mixed with (1) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC; Sigma Aldrich) at 100 mg/mL and (2) a solution of adipic acid dihydrazide (AAD) and 1-hydroxybenzotriazole (1-HOBT; Sigma Aldrich), both at 25–75 mM. The mixture had a volume ratio of 8:1:1 alginate:EDC:AAD/1-HOBT. We controlled the number of crosslinks and resultant stiffness of the gel by variation in AAD content. The mixture was pipetted into identical molds as for viscoelastic hydrogels and allowed to gel under the same conditions. In both cases, the final concentration of alginate within the gels was 20 mg/mL.