IMIS - Marine Research Groups

VSa13 bone-derived cells were cultured according to Pombinho et al. (2004), induced for mineralisation through the supplementation of calcium, phosphate and ascorbic acid, then exposed to each three different minerals: copper sulphate, manganese sulpahte, sodium selenite. Two separate experiments were conducted: Experiment 1 (duration: ten days): the survival and proliferation rate of the cells exposed to different levels of minerals were evaluated to establish the concentrations to use for the mineralisation assay. The cytotoxic effect of each mineral was determined using the XTT assay (Applichem) in confluent cultures supplemented for ten days with different amounts of minerals. Proliferative effect was also determined using the XTT assay but in dividing cultures supplemented for ten days with different amounts of minerals. Experiment 2 (duration four weeks): The mineralogenic capacity of the different minerals was then evaluated using non-cytotoxic concentrations determined within the scope of Experiment 1. A mineralisation cocktail (4 mM calcium chloride, 10 mM beta-glycerophosphate and 50 ug/mL of ascorbic acid) was added to confluent cell cultures, and the amount of mineral deposition was evaluated after three weeks of exposure through alizarin red staining and quantification. Each treatment level was tested in five replicas. The quantification was conducted using a spectrophotometer set at a wavelength of 550-560nm.