Tese_TÃ¢nia Vieira.pdf - Ubi Thesis

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Chapter II – Materials and Methods 2.2.8. Proliferation of cells in the presence of the produced nanoparticles Human osteoblast cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM- F12) supplemented with heat-inactivated FBS (10% v/v), penicillin G (100 units/mL), streptomycin (100 μg/mL) and amphotericin B (0.25 μg/mL). Cells were seeded in 75 cm 3 T-flasks until confluence was obtained. Detachment of confluent cells was achieved by a 3 min incubation in 0.18% trypsin (1:250) and 5 mM EDTA. Then, an equal volume of culture medium was added to the free cells in order to stop the reaction. The cells were centrifuged, the pellet was resuspended in culture medium, and the cells were then seeded in new 75 cm 3 T-flasks (Ribeiro et al. 2009). To verify the influence of the presence of the produced nanoparticles in cell adhesion and proliferation, cells were seeded with nanoparticles in a 96-well plate at a density of 15×10 3 cells/well, for 24 and 48 h. Before this procedure, plates and the materials were UV sterilized for 30 min. Cell growth was monitored using an Olympus CX14 inverted light microscope (Tokyo, Japan) equipped with an Olympus SP-500 UZ digital camera. 2.2.9. Evaluation of the cytotoxic profile of the produced nanoparticles The MTS assay was performed in order to evaluate nanoparticles toxicity. Human osteoblast cells, at a density of 15×10 3 per well, were seeded in a 96-well plate and cultured with DMEM-F12. At the same time, in another 96-well culture plate, DMEM-F12 was added to the produced nanoparticles that were previously irradiated with UV light for 30 min, in order to be sterilized. The nanoparticles were left in contact with medium for 24 and 48 h. After the period of incubation, the cell culture medium was removed and replaced with 100 µL of medium that was in contact with the nanoparticles. Then, cells were incubated at 37°C, in a 5% CO 2 humidified atmosphere for more 24 h. The cell viability and proliferation was assessed through the reduction of MTS into a water-soluble brown formazan product. Briefly, the medium of each well was removed and replaced with a mixture of 100 µL of fresh culture medium and 20 µL of MTS/PMS reagent solution. Then, cells were incubated for 4 h at 37 °C, in a 5% CO 2 atmosphere (Ribeiro et al. 2009). The absorbance was measured at 490 nm using a Biorad Microplate Reader Brenchmark (Tokyo, Japan). Wells containing cells in the culture medium without biomaterials were used as negative control (K - ). EtOH 96% was added to wells containing only cells and was used as positive control (K + ). 24
Chapter II – Materials and Methods 2.2.10. Statistical Analysis of MTS results Statistical analysis of the results of cell viability assays was performed using one-way analysis of variance (ANOVA) with Bonferroni t-test, with at least three independent results. A value of p
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