Hydrogel Degradation Assay Protocol

The degradation assay of the hydrogels from six samples (coming from three different batches) at the different conditions (cECMH, and cECMH with PEG at 3, 6, and 12 mg/mL) was performed as previously described, with slight modifications [20 (link)]. Briefly, the solubilized matrix (20 µL) was gelled at 37 °C for 24 h in a 1.5 mL Eppendorf. A total of 20 µL of collagenase type II (200 units/mL, LS004176, Worthington) dissolved in 0.1 M Trizma base buffer (T1503, Sigma-Aldrich) pH 7.4 and 0.25 M CaCl₂ were added, and the samples were further incubated at 37 °C for 5, 24, and 48 h. A total of 20 µL of collagenase in 20 µL of PBS was used as blanks. Following incubation, the samples were centrifuged at 15000 rpm for 5 min, and 10 µL of the supernatant was mixed with an equal volume of 2% ninhydrin reagent solution (N7285, Sigma-Aldrich). The samples were boiled at 10 min in a water bath, and then 380 µL of distilled water was added. In total, 100 µL (in triplicate) was transferred into a 96-well plate and the OD at 570 nm was measured using an EMax^® Plus Microplate Reader (BioteK, Winooski, VT, USA). With the ninhydrin assay, a higher OD at 570 nm is indicative of more soluble amines.