Hydrogel degradation test

The in vitro enzymatic degradation property of hydrogels was evaluated by exposing them to enzymes to assess degradation rate. Collagenase has been used previously as a mimic for some of the protease secreted by cells (Mazzeo et al., 2019), and trypsin is often used in cell isolation and culture. The material degradation process of these proteases must be evaluated to provide a basis for cellular inoculation and digestion on hydrogels. The pre-weighed hydrogels (w₀) were then immersed in 0.1% collagenase type I and 0.25% trypsin/0.01% EDTA for 12 h. At each time point (0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 h), the liquid was removed completely. The hydrogels were weighed again (w_t). The degree of degradation (D) was calculated as follows:

Three repeated measurements were performed for each type of hydrogel.

The hydrolytic and cellular degradation of hydrogels were performed as described by our previous study (Yang et al., 2016). In brief, hydrogels were prepared into 35 mm culture dishes with ∼1 mL for each dish. To exclude the influence of swelling behavior of hydrogels, 2 mL of PBS was added into each dish and PBS was removed completely after 12 h; the hydrogels were weighed (w₀). The hydrogels were incubated in PBS at 37 °C with 5% CO₂ for two weeks. PBS was changed every 2 days. At different time points (0, 2, 4, 6, 8, 10, 12, and 14 d), three samples of each kind of hydrogel were weighed (w_t). The degree of degradation was calculated as above. Cell-mediated degradation was measured by seeding 1.0 × 10⁶ ADSCs (see ‘Primary culture of ADSCs’) on each hydrogel scaffold. The rest of the steps were the same as above, except that the PBS was changed to cell culture medium (high-glucose DMEM, 15% FBS, and 1% P/S).