Cytotoxicity Assessment of Biomaterial Eluates

Material cytotoxicity was assessed for the different eluates (1:1, 1:2 and 1:4) of AHP, AHPbcs and ESbcs cultured with hPDLSCs (test groups) and compared with hPDLSCs cultured in unconditioned growth medium (negative control group). This analysis was performed via a 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay, as previously reported by similar studies (Rathinam et al., 2021 (link)). In brief, hPDLSCs were seeded onto 96‐well plates with 180 μl of DMEM and stored for 24 h at 37°C, 5% CO₂ and 95% humidity. The material eluates were placed in the culture medium with 1 × 10⁴ hPDLSCs (n = 3). An MTT reagent (Sigma Aldrich) was added for 4 h, following its manufacturer's instructions. When a purple precipitate was detectable, Dimethylsulfoxide (DMSO) (Sigma‐Aldrich) was added to each well (100 μl/well), and plates were covered and kept in dark conditions for 4 h to solubilize the formazan crystals produced by viable cells, after reducing the MTT reagent. After 24, 48 and 72 h of culture, light absorbance per well was recorded by means of a microplate reader (ELx800; Bio‐Tek Instruments) at 570 nm wavelength. Culture media with fresh eluates from the respective groups were replaced every 3 days.