Tissue-Mimetic Hydrogel Synthesis

Various concentrations of ldECM hydrogels were prepared following a previously published protocol but with slight modifications [20 (link)]. Lyophilized dECM was crushed into a powder using a freezer mill (6775 Freezer/Mill; SPEX SamplePrep, Metuchen, NJ, USA). ldECM powders (1, 2, and 3 w/v%) were solubilized using 0.5 M acetic acid with pepsin (1% w/w for ldECM) (Sigma-Aldrich, St. Louis, MO, USA) for 48 h. After complete solubilization of ldECM, 10X Phosphate-Buffered Saline (PBS) was added at a 1:10 ratio to the ldECM solution, and the pH was increased to 7.0, using 5M sodium hydroxide (Sigma-Aldrich). Tissue-mimetic hybrid hydrogels were prepared by mixing 1% ldECM hydrogel with 3 mg/mL of a high concentration of rat tail collagen type I solution (9.41 mg/mL; Corning, NY, USA). 1% ldECM and collagen solutions were varied in volume ratios of 25/75, 50/50, and 75/20 to optimize the tissue-mimetic hybrid hydrogel composition. Moreover, fibrinogen (3 mg/mL; Sigma-Aldrich) solution in PBS (Gibco) and the type I collagen solution were thoroughly mixed at a 1:1 ratio, and the collagen/fibrinogen solution was mixed with thrombin (50 U/mL, Sigma-Aldrich) at a volume ratio of 50:1 to use the collagen/fibrin hydrogel as a control group.