Cytotoxicity assay

Hydrogel extract was prepared according to ISO 10993-5. Briefly, the hydrogel was immersed into sterile water for sufficient swelling and then weighed. After removing sterile water, Dulbecco Modified Eagle Medium (DMEM, Gibco, USA) was added at the percentage of 0.1 g/ml (hydrogel/DMEM) and placed at 37°C for 48 h. NIH/3T3 and RAW 264.7 cells were inculcated into a 96-well plate (1.0 × 10⁴ cells/well). After cell adhesion, the hydrogel extract with 10% fetal bovine serum (Ginimi, USA) was added for cell culture. After culturing for 1, 2 and 3 days, cell viability was detected by CCK-8 kit (Beyotime, Shanghai) according to the instructions. The absorption value at 450 nm was determined by using a microplate reader (Spectra Maxi3, USA) to evaluate hydrogel cytotoxicity.