Quantifying Titanium Cell Adhesion

Cell adhesion was evaluated on 648 titanium samples using a 48-well plate (BD, Milan, Italy). Cells were detached using trypsin for 3 min, carefully counted, and seeded at 3 × 10³ cells/well in 1 mL of growth medium on the different samples. The 48-well plates were kept at 37 °C, 0.5% CO₂ for 20 min, 24 h and 72 h. Before and after fixation in 4% paraformaldehyde in PBS for 15 min at room temperature, cells were washed two times with PBS and then stained with 1 μM DAPI (Molecular Probes, Eugene, CA, USA) for 15′ at 37 °C to detect cell nuclei. Samples were analyzed using a Nikon Eclipse T-E microscope with a 4X objective. Cell nuclei were then counted by using ImageJ (NIH) software with the tool “Analyze particles” [51 (link),52 (link)].