To fill the TiO2 nanotubes on Ti thin films, freeze drying was performed with the lyophilization method [33], and filling was adapted from the modified method in Popat et al. [34]. In brief, a solution of 1 mg/ml gentamicin was prepared in a PBS solution,
and then the specimens (1.0 × 1.0 cm2) were cleaned with distilled water before filling the PEO-fabricated TiO2 nanotubes with the drug. Before filling with the drug, the PBS solution was used to gently clean the specimen. To load the drug, 10 μl gentamicin solution was simultaneously spread around the surface and dried in air at room
temperature for 2 h. The drying and pipetting steps were repeated until a sufficient amount of gentamicin was filled in the TiO2nanotube. The specimens were quickly rinsed with 500 μl of PBS over the specimens in order to remove excess gentamicin that covered the surface in the final drying step.