*2.4. Sample Preparation*

Titanium alloy grade 5 (Ti6Al4V) plates with dimensions of 15 cm × 15 cm and a thickness of 0.1 cm were purchased from William Gregor Ltd., London, UK, and cut into 1 cm × 1 cm plates. Then, the plates were mounted in conducting Bakelite and polished to mirror finish. Three steps were used during polishing of the plates. First, Bakelite-mounted Ti6Al4V plates were ground with MD-Piano of 220 grit, with water used as a lubricant. Then, a DiaDuo-2 diamond of 9 μm grain size suspended in water and an MD-Largo polishing plate were used. Finally, a colloidal suspension (OP-S) activated with ammonia solution was used and polished on MD-Chem polishing disc. All the polishing materials and equipment were purchased from Struers Ltd., Rotherham, UK. After polishing the plates to a mirror finish, the highly polished surfaces were secured with electrical tape to prevent the introduction of scratches and Bakelite was broken down to release the mounted Ti6Al4V plates. The tape was then removed from the plates and any impurities introduced on the Ti6Al4V surfaces during the previous steps were removed by cleaning the plates in an ultrasonic bath with water (15 min) and acetone (15 min). The plates were dried overnight in a desiccator and were used within 48 h after cleaning.

Having been cleaned and polished to a mirror finish, Ti6Al4V plates were placed inside a 24-well cell culture plate with the polished side facing upwards. Dopamine, purchased from Sigma-Aldrich, was dissolved to a final concentration of 5 mg/mL in 50 mM Tris buffer (Fisher Scientific UK Ltd., Loughborough, UK) at pH = 8.5. Then, 1.5 mL of dopamine solution was transferred into the cell culture plates containing the Ti6Al4V plates. Subsequently, the prepared plates were placed in the dark for 24 h without a cover to allow simultaneous dopamine polymerisation in air and metallic surface coating. Ti6Al4V plates coated with polydopamine (pDA) were washed to remove any loose pDA particles and placed in a new set of 24-well cell culture plates. The AMP solution was then prepared by dissolving peptides in a concentration equal to the MIC value for each peptide in 50 mM Tris buffer at pH 7.4. Then, 1.5 mL of this solution was transferred into the cell culture plates with the pDA-coated Ti6Al4V plates. The cell culture plates were then kept in the dark without a cover for 24 h to allow conjugation of the peptides with the pDA. Finally, the plates were washed several times with dH2O to remove unconjugated peptides. The prepared plates were stored in the dark in a desiccator to dry and used within 7 days of preparation.
