**2.6. Cytotoxicity analysis**

Cytotoxicity was assessed by quantitative methodology. The test is based on the determination of viable cells after exposure of the cell population to the extract obtained from incubation of the samples in cell culture medium RPMI (Gibco®) supplemented with serum bovine fetal 10% and antibiotic/antimycotic (solution Gibco®) 1% at 37°C for 9 days under constant gentle stirring. The long period use was chosen to mimic if the samples were implanted. The treated surface was carefully immersed in cell culture medium to evaluate if there was debris released from the samples that can lead to a cytotoxicity effect. The extract of each sample were used to cultivate on a cell monolayer, CHO (Chinese Hamster Ovarian) cell line for 24 hours.

The analysis of the number of viable cells was performed by the colorimetric method for the metabolization of *supravital* dye, MTS, and the electron coupling agent, PMS (Promega®) were used as the supplier instructions, and subsequently reading in a spectrophotometer at 490 nm. The amount of dye metabolized by the cell population is directly proportional to the number of viable cells on the plate.
