2.11.1. MTT Assay for Cytotoxicity Evaluation

All in vitro biological evaluations were performed within the guidelines of ISO 10993-5 (in vitro cytotoxicity test) and ISO 10993-12 (sample preparation). Test samples (n = 4) were immersed in complete media (material/media = 0.025 g/mL) and agitated at 37 °C for 72 h to induce the release of any leachable substances (e.g., ions, unreacted monomer and oligomers). Cells were seeded in a 96-well plate (1 × 10⁴ cells/well) and incubated for 24 h. The media was removed and replaced by the test eluants (neat, dilutions of 50, 25 and 12.5%), DMEM with FCS as the negative control, and DMEM with FCS and 10% ethanol as the positive control. The cells were exposed to the eluants and controls for 24 and 72 h.

A sterile 5% (w/v) MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution in phosphate buffer saline (PBS) was prepared and mixed with ascorbic-acid-free media at 1:10 solution to media. Following the designated exposure period, the eluant media were removed, and replaced with media containing MTT; cells were incubated for 2 h. The solution was then replaced by cell-culture-grade dimethyl sulfoxide (DMSO), and the plate agitated for 5 min to dissolve the MTT crystals, turning the solution purple. Colorimetric assay (570/620 nm) was used to quantify the relative cell viability.