Biocompatibility test—in vitro cytotoxicity test

Cell isolation and culture of L929 and human gingival fibroblasts are described in the ESI†. The cytotoxicity of the mineralizing film was assessed by a Cell Counting Kit-8 (CCK-8) assay (Beyotime, Shanghai, China). Different concentrations of the mineralizing films (0.1, 0.2, 0.3, 0.5, 1, 2, 4, 8 mg mL⁻¹) were incubated at 37 °C in Duibecco’s modified Eagle’s medium (DMEM, Gibco, USA) for 24 h to obtain leaching solutions, in which 10% fetal bovine serum (FBS, Gibco, USA) was added to the L929 cells and 15% FBS was added to the human gingival fibroblasts. Afterward, L929 cells and human gingival fibroblasts were planted into 96-well plates at 10,000 cells per well. They were incubated for 24 h to allow cell attachment. Then, the attached cells were incubated with 5% of CO₂ and 95% relative humidity in each well with the different leach solutions for 1, 3 and 5 days at 37 °C. Afterward, 10 µL CCK-8 was added to each well, and the attached cells were incubated for another 2–3 h. The measurements were performed six times at a wavelength of 450 nm using a microplate reader (Bio Tek Eon, USA).