*2.8. Coating Stability*

In order to prepare the simulated body fluid (SBF), sodium chloride (NaCl), sodium bicarbonate (NaHCO3), potassium chloride (KCl), potassium phosphate dibasic trihydrate (K2HPO4·3H2O), magnesium chloride hexahydrate (MgCl2·6H2O), hydrochloric acid (HCl) 32%, calcium chloride (CaCl2), sodium sulfate (Na2SO4), and tris(hydroxymethyl)aminomethane (Tris, (CH2OH)3CNH2) were purchased from Sigma Aldrich, Merck Life Science UK Limited, Dorset, UK. The SBF was prepared as described by Kokubo and Takadama [20]. The stability of the AMPs on the surface of Ti6Al4V was performed in SBF by submerging and keeping AMP-coated plates in SBF at 37 ◦C for a total of 30 days. Analysis of the released AMPs to the solution was performed using two methodologies. Firstly, the change in green light intensity of the pDA Ti6Al4V surfaces coated with 5(6)-FAM-labelled peptides was analysed after 1 and 6 h, then at 1, 3, 7, 14, and 30 days of submersion in SBF, imaged under fluorescence microscopy. Secondly, plates with no labelled peptides were immersed in SBF solution and analysed by HPLC at the same time intervals. However, in this method, 100 μL of the SBF solution was withdrawn to determine the cumulative absorbance at the same retention time for every individual peptide.
