The media were filtered through 0.2 μm PTFE membrane syringe filters following incubation to remove the particles before use in cell culture. Group 2 (particle): Sterilised grade 4 and 5 Ti particles were suspended in DMEM at concentrations of 0.75, 1.5 and 3 mg ml⁻¹ and used for cell culture without filtering. Basal DMEM and DMEM containing unprocessed grade 4 and 5 implants were used as controls. One millilitre of aliquots was taken at day 0, 3 and 10 for the analysis of pH and ionic concentration.

A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) metabolic activity assay was performed to assess cellular metabolic activity of HGFs in response to group 1 and 2 test samples. Briefly, HGFs were seeded at 3000 cells cm⁻² in 24-well plates and allowed to grow for 24 h in basal DMEM. Basal medium was then replaced with the test samples supplemented with 10% FBS and cultured for a further 3, 7 or 10 days. At each time point, culture medium was replaced with 1 mg ml⁻¹ MTT in plain DMEM. MTT solution was removed after 3 h, and dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals formed by living cells. The luminescence of the resulting solutions was measured at 570 nm in a plate reader.

Inductively coupled plasma optical emission spectrometer (ICP-OES)

The elemental concentration of titanium, aluminium, iron and vanadium in the following media was determined using ICP-OES (iCaP6300 Duo, Thermo Fisher Scientific, UK) (1) SBF; (2) dissolution products of the particles from immersion in DMEM for 3 days (dissolution), and (3) DMEM sampled during the cell culture studies where cells were cultured with the particles (particle). Mixed standards of titanium, aluminium, iron and vanadium ions were prepared at 0, 2, 5, 20 and 40 ppm for calibration. All samples were run in triplicates, and an acid blank was incubated under the same conditions and used as a control.

Statistical analysis

Results were presented as mean ± standard deviation (S.D.). Mann–Whitney U test (2 groups) or Mann–Whitney U test with Bonferroni correction (> 2 groups) was performed using OriginPro 2019. Results were deemed significant if the probability of occurrence by random chance alone was less than 5% (i.e. p < 0.05).