Biocompatibility of five occlusal splint materials produced by four fabrication methods: an in vitro comparative study

Abstract Background To evaluate the cytotoxicity, cell death, interleukin-6 (IL-6) release, and surface roughness of five occlusal splint materials produced by four different fabrication methods on L929 mouse fibroblasts. Methods 240 disc-shaped specimens (15 × 2 mm) were fabricated from milled polymethyl methacrylate (PMMA), milled polyetheretherketone (PEEK), 3D-printed photopolymer resin, thermoformed polyethylene terephthalate glycol (PET-G), and auto-polymerizing PMMA. Extracts were prepared following ISO 10993-12 and collected on days 1, 3, and 7; at each time point the extracts of four specimens were pooled and applied to three replicate wells. Cell viability (XTT), total cell death (Annexin V-FITC/PI flow cytometry), IL-6 release (ELISA), and surface roughness (Ra, contact profilometer) were assessed. As the replicate wells were technical replicates of a single pooled extract, the biological data are reported descriptively; surface roughness was analyzed by one-way ANOVA with Tukey’s HSD test ( p < 0.05). Results On day 7, the auto-polymerizing PMMA group showed the lowest cell viability (68.44 ± 3.48%) and was the only material to fall below the 70% threshold of ISO 10993-5. Cell viability remained above the 70% threshold in all other groups at all time points. Total cell death was low throughout (1.40–3.20%; untreated control 1.50%), with the highest day-7 values in the thermoformed PET-G (3.20 ± 0.30%) and auto-polymerizing PMMA (3.07 ± 0.86%) groups. IL-6 was below the limit of detection (18.75 pg/mL) in most conditions; only the auto-polymerizing PMMA group on day 7 yielded quantifiable IL-6, in two of three wells (33.78 and 42.55 pg/mL). The milled-PEEK and thermoformed PET-G groups showed the lowest Ra values, whereas the milled-PMMA group showed the highest ( p = 0.005). Conclusions In this in vitro extract model, the auto-polymerizing PMMA group was the only material falling below the 70% viability limit of ISO 10993-5 and the only one with quantifiable IL-6 on day 7. All other groups remained above this threshold throughout, and total cell death remained low in all groups. The milled and printed materials evaluated in the present study may therefore represent biologically more favorable options than the auto-polymerizing PMMA group. These findings are descriptive, based on a single pooled extract per condition, and apply only to the specific products tested.