Comparative studies on cytotoxic effects of dental amalgams and alternative alloys according to ISO standards in vitro

Deleterious effects of dental alloys, especially those of dental amalgams, have become an important issue in current discussions on biomaterials. Cytotoxicity and further related risks of amalgams are discussed in a controversial way in the literature without leading to a final conclusion. There is still a need for basic clinical and pre-clinical research, especially with respect to the wide distribution of dental amalgams. Standardized methods of cytotoxicity testing have been established by the ISO. It was the aim of the present study to detect and compare possible cytotoxic effects of dental amalgams and alternative non-amalgam alloys in vitro. According to the ISO standards, direct contact tests and extract dilution tests were performed using the cell lines HeLa and L-929 as well as primary isolated human fibroblasts, a relevant cell type of the human gingiva. For direct contact tests the samples were fixed on thermanox discs. Zn and Ni-chloride in defined molar concentrations were used as positive controls in the extract dilution tests, while copper was the positive control in the direct contact tests. The tested amalgam was a Non-Gamma-2 amalgam. For extract dilution tests sixteen extraction dilutions were performed. The different cell types were incubated with the extracts in 96-well microtitre plates. MTT-testing was performed to evaluate the effects on cellular metabolism. The BrdU labelling index was determined with the help of EIA methods to analyse the effects of the extracts on the cellular proliferation at DNA synthesis level. The morphological status of the cells seeded on the materials (direct contact test) were studied with the help of light microscopy. No cytotoxic effects of formerly extracted dental amalgam was found, although fresh amalgam elicited a significant cytotoxic effect, in general the non-amalgams have to be regarded as non cytotoxic. The negative control and the non-amalgams elicited no measurable cytotoxicity in the indirect contact assays, independent of the number of extraction dilutions. This applied to all cell types studied. The tested amalgam also gave a significant cytotoxic effect in the MTT-assays, while in addition a significant reduction of BrdU incorporation after incubation with the extracts of the first dilution series, compared to the silicone control was found. The effects were reduced after an incubation with the extracts of the higher dilution series. It is suggested by the presented results that amalgams might have cytotoxic effects, especially when being freshly applied. The cytotoxic effects were no longer detectable after extraction procedures. Nevertheless, a negative effect around such amalgams must be considered. The insights provided by the present studies might be helpful for a rational choice of dental materials.