Positive reference material for cytotoxicity testing of dental restorative materials

Objectives: For cytotoxicity testing of dental restorative materials a positive (= toxic) reference material is necessary, which is handled like the test materials, elicits a standard toxic reaction and can easily be produced from commercially available chemicals. It was the aim of the present study to develop such a material and to compare its cytotoxicity to a so far used material (Vitrebond). Methods: Three groups of materials were assessed: (1) resin-based formulations (bis-GMA/TEGDMA) with 0.1 % to 2 % diphenyliodoniumchloride (DICl); (2) a glass ionomer cement powder (GIC) (Chemfil) with different water/HEMA mixtures, with/without DICl; (3) a glass powder (Schott) with different water/HEMA mixtures, with/without DICl. Curing was initiated using camphorquinone/ethyl-4-dimethylaminobenzoate at 780 mW/cm² irradiation for 40 seconds. Standard specimens in teflon rings were eluted in nutrient medium (MEMα) with 10% FCS for 24 hours at 37ºC (91.6 mm²/ml medium). Monolayers of SV40-transfected human pulp cells were exposed to the extracts for 24 hours and cell reaction was assayed by the crystal violet test (CVT). A selected material from group (3) was tested in the dentin barrier test (DBT) using three-dimensional cultures of SV40-transfected bovine pulp cells. After 24 hours exposure the cell reaction was determined by the MTT test. The measured ODs were transformed into relative cell viability with the unexposed cultures being 100%. Medians were subjected to Mann-Whitney tests (α=0.05). Results: DICl did not influence the toxicity of the resin-based formulations (CVT). Mixing water/HEMA to the GIC (instead of water alone) significantly increased the toxicity, with no difference to Vitrebond (CVT). Mixing the glass powder with water/HEMA revealed the same cytotoxicity as Vitrebond (CVT, DBT), DICl had no influence. Conclusion: The glass powder based material, which can easily be produced from commercially available chemicals, is a suitable positive reference material for cytotoxicity testing of dental restorative materials.