Modification of the agar overlay assay: assessment of the influence of acrylics used in orthodontics on proliferation and differentiation of primary and transformed fibroblasts.

In orthodontics, removable acrylic appliances are preferably produced from methylmetacrylates. However, in the adjacent oral mucosal tissue, cell damage may be caused by the evaporation of residual monomer. The aim of this study was to modify the classic agar overlay assay in order to apply histochemical methods by comparing conventionally used transformed mouse fibroblasts (L-929) with primary human gingival fibroblasts to further elucidate the term toxicity. While proliferation was assessed via the incorporation of the base analogon bromdesoxyuridine, differentiation was investigated by detection of the fibroblast-specific intermediate filament vimentin. After the monomers had taken effect, reduced proliferation extending over the inhibition area was observed by indirect immunofluorescence, independent of cell type. In contrast to this observation, a few cells within the inhibition area which could not be clearly detected by neutral red staining still exhibited mitotic activity. Detection of the differentiation-specific intermediate filament vimentin was comparable with the degree of neutral red fading visible in the classic agar overlay assay. The study showed that the inhibition areas with primary gingival fibroblasts were smaller (approximately 1/3) after monomer action compared with conventionally applied transformed fibroblasts. The results indicate that the modified assay is comparable with the classic method. Evaluation of neutral red staining with respect to toxic material influence can moreover be supplemented by histochemical studies of typical cell properties.