S 450

Pieces of the metal payload structure with a size of 1×1 cm were cut out and the cutting edge was polished with sandpaper of different grain size (200, 400, 600, 800, 1000 µm). The payload structure pieces were cleaned in distilled water containing detergents and sonicated in an ultrasonic bath 3 times for 5 min. Each metal piece was rinsed at least 3 times in distilled water and subsequently 3 times in 96% ethanol and 3 times in aceton. 50 µg of pEGFP-C3 plasmid DNA or 50 µl of 10 mM Tris buffer were applied and the solutions were dried in a hot air stream. The samples were mounted on the sample holder with carbon adhesive Leit Tabs G3347 (Plano GmbH, Germany), inserted into the sample chamber and observed with the Hitachi S450 scanning electron microscope.

The in vitro release profile of IND from the LbL coatings was evaluated in PBS solution at 37 °C. The PLA/HA substrates with LbL coatings were immersed in 25 mL of 0.01 M PBS (pH 7.4 or 6.0). PBS (2.5 mL) was withdrawn for UV detection at different time intervals, and another 2.5 mL of fresh PBS was fed back into the original solution. All experiments were run in triplicate. The pH-sensitive release profiles of IND from the LbL coatings in response to different pH conditions were also obtained. The PLA/HA substrates with LbL coatings were immersed in PBS solution against stepwise pH changes between 6.0 and 7.4, which alternated hourly over a 10-h period. The amount of IND released during each hour was measured using 2.5 mL PBS taken from the immersion solution. All experiments were run in triplicate. The surface morphology of the LbL coatings was studied by scanning electronic microscopy (SEM) using a Hitachi S-450 (10 kV, Japan). The PLA/HA substrates with Coating-A/S and Coating-AP/S were fabricated following the previous procedure. The original coating, the coatings after incubation in PBS at pH 7.4 for 1 h, the coatings after incubation in PBS at pH 6.0 for 1 h and the coatings after 5 cycles of pH challenge between pH 6.0 and 7.4, were all observed by SEM.

The interface region of bone and TNTZ on the sliced implanted sample was observed using a scanning electron microscopy (SEM) (Hitachi S 450, Japan) with an acceleration voltage of 15 kV. To confirm any metal ion diffusion from the TNTZ alloy into surrounding tissue, the interface region of the bone and TNTZ alloy on the sliced implant was analyzed using an energy-dispersive X-ray spectrometer (EDX, JEOL JSM-5410LV, Japan). Then, elemental mappings of Ca, Ti, Nb, Ta, and Zr were conducted. For surface chemistry analysis, X-ray photoelectron spectroscopy (XPS) was performed in a PerkinElmer 5500MT spectrometer. XPS data were acquired using Al Kα X rays with pass energy 8 kV. Sputter cleaning was done with Ar⁺ sputter gun. MaltiPack software was used for the analysis.