2.12. Cytotoxicity Test of PUA and the Resins by MTT Assay

To evaluate the in vitro cytotoxic effects of any leachable by-products from the polymerized PUA, the extract test was carried out using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay according to ISO 10993-5. Briefly, mouse fibroblasts (L929) were cultured in minimum essential medium (MEM) (Gibco) containing 10% fetal bovine serum (SBF) and 1% penicillin streptomycin. Following ISO standards 10993-12 the extract was prepared by soaking samples in culture medium at a concentration of 3 cm² mL⁻¹. The extraction was carried out at 37 °C for 24 h. Cells were seeded with the concertation of 10⁴ cells 100 μL⁻¹ in a 96-well plate. To ensure the cytotoxic response of the specimens provides an appropriate response of the cells, the negative control and the positive control were also analyzed. Cells cultured in 0.4% phenol in culture medium were the positive control which provides a reproducible cytotoxic response and cells in culture medium only were the negative control which provides a background response of the cells. After cells seeded for 24 h, all the culture mediums were removed and replaced with 100 μL extract. Finally, the culture medium was removed, and 50 μL MTT solution was added in each well. After incubation at 37 °C for 3 h, the MTT solution was aspirated and 100 μL isopropanol was added to each well to dissolve the purple formazan. After shaking for 30 min, absorbance was measured using Multiskan Spectrum ELISA reader (Thermo Fisher Scientific, Waltham, MA, USA) at 570 nm. The cell viability was calculated according to the following equation:

where OD_570t is the value of the measured optical density of the test well, OD_570n is the value of the measured optical density of the negative control, and OD_570b is the mean value of the measured optical density of the blanks.