2.6. Cytocompatibility

The cytotoxicity test was performed using the murine fibroblast cell line L929 in accordance with ISO 10993-5. The L929 cells were grown in alpha-minimum essential medium (α-MEM, Gibco, Gaithersburg, MD, USA) containing nonessential amino acids and supplemented with 10% horse bovine serum, 100 mg/mL of streptomycin, and 100 U/mL penicillin. The L929 cells were maintained in a humidified atmosphere at 5% CO₂ and 37 °C. The culture medium was renewed two times a week. The water-soluble tetrazolium salt (WST) assay was performed to evaluate cell proliferation at 1 day after the initial seeding of 5 × 10⁴ L929 cells on the cement surface in a 24-well plate. The L929 cells were subcultured using α-MEM supplemented with 10% horse bovine serum and incubated at 37 °C under 5% CO₂. The L929 cells of the tenth passage were detached using 0.25% trypsin in phosphate-buffered saline and resuspended in α-MEM until experimental use. Cell proliferation was investigated using the WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] assay. WST-8 is reduced by dehydrogenases in cells to yield a yellow product (formazan), which is soluble in the tissue culture medium, and the amount of formazan generated by dehydrogenase activity in cells is directly proportional to the number of living cells. The OD450 values of the WST-8-treated medium (10% v/v) was determined after a 2 h incubation by using an enzyme-linked immunosorbent assay reader.