Cell attachment and proliferation

Cells (passage 4−5) were seeded onto the variously prepared experimental and control titanium disks at a density of 2.5·10⁴ per sample. To assess cell attachment, samples were removed from culture after 3 hours and rinsed with PBS to remove non-adherent cells before being transferred to new 24 well plates containing 500 μl of fresh media. Subsequently 100 μl of MTS assay solution (Cell-Titer 96 AQueous One Solution Cell Proliferation assay; Promega, Southampton, UK) was added to each well and cells were incubated for 2 h at 37 °C. 100 μl aliquots of the culture solution from each sample were then transferred to a 96 well plate and the absorbance was measured at 595 nm using a plate-reader (Opsys MR, DINEX Technologies, UK). To assess cell viability, the samples were incubated for 24 h after which they were rinsed and transferred to new 24 well plates containing 500 μl of fresh media. Cells were incubated for specific time points and cell viability was determined using MTS assay.