Cell counter

MG63 cells lines (Rio de Janeiro Cell Bank, APABCAM, Rio de Janeiro, RJ, Brazil) were seeded on the scaffolds at a density of 5000 cells/cm² and cultivated for one and three days. After each time, the samples were washed in PBS (three times) to remove non-adherent cells. Then, samples were chemically fixed in paraformaldehyde (4%) at room temperature (20 min). Afterward, an ascending series of ethanol was used to dehydrate the samples (10, 30, 50, 70, 90, and 100%). Before characterization, a thin gold layer was sputtered onto the scaffolds. Then, cells were stained using trypan blue (Sigma-Aldric) and counted using a Cell Counter (Countess^®, Invitrogen). All results were expressed as mean using the total cells for each group. Cellular adhesion and proliferation were further evaluated by FE-SEM (Zeiss - EVO MA10).

The toxicity of PAE on VSMCs was determined by the trypan blue exclusion test. After 12, 24 and 48 h incubation with PAE of different concentrations (50, 100, 200 µM), the VSMCs were removed from culture and the cells that excluded 0.4% trypan blue (37°C for 3 min) were counted in an automated Cell Counter (Invitrogen; Thermo Fisher Scientific, Inc.); 3 slides were counted in each group, with 3 fields of view from each.

The Annexin V-FITC cell apoptosis detection kit (Beyotime Biotechnology, Shanghai, China) was used to detect cell apoptosis. The MC3T3-E1 cells were incubated with or without DEX and various concentrations of VK₂ (10⁻⁵, 10⁻⁶ and 10⁻⁷ M) for 6 days, collected, resuspended in 200 µl of Annexin V-FITC and 10 µl of propidium iodide, and incubated for 20 min at room temperature. Subsequently, flow cytometry was used to evaluate the number of apoptotic cells. The early apoptotic cells are labeled green and the dead and late apoptotic cells are labeled red, while the live cells are not stained.
trypan blue staining was performed to evaluate cell viability. The MC3T3-E1 cells were treated with both DEX and various concentrations of VK₂ (10⁻⁵, 10⁻⁶ and 10⁻⁷ M) in FBS-free medium for 6 days and then collected. Ten microliters of trypan blue (Invitrogen, Carlsbad, CA, USA) were mixed with 10 µl of the cell suspension, and 10 µl of the mixture were then added to the cell counting plate. The cell death rates were automatically calculated with a cell counter (Invitrogen). A ReadyProbes^® Cell Viability Imaging kit (Life Technologies, Gaithersburg, MD, USA) was also used to detect cell viability at 6 days after the MC3T3-E1 cells were incubated under the different conditions. The blue dye stained all living cells, and the green dye stained the dead cells.