**3. Results**

#### *3.1. Ti6Al4V Implants Modified by Titania Nanotube Coatings*

The implants used in our investigations were produced by the selective laser sintering method, using Ti6Al4V ELI powder (Figure 1a). Analysis of SEM images of the implant, as obtained, revealed the presence of the non-melted or partially melted powder grains (Figure 1b). Therefore, before electrochemical modification, the surfaces of the implants were mechanically ground and sandblasted (Figure 1c). The anodization of Ti6Al4V alloy substrates using 0.3 wt% aqueous HF solution as an electrolyte enabled the production of uniform amorphous titanium dioxide layers (Figure 1d) on their surface. The electrolytic processes were performed using potentials of 5 and 15 V, which allowed the formation of nanoporous (TNT5) and nanotubular (TNT15) coatings (Figure 1e,f). Based on the SEM image analysis, the *pore diameters of TNT5 coatings* were c.a. 21 ± 4 nm and the tube diameters of TNT15 were c.a. 51 ± 9 nm. The thickness of the walls in both cases was c.a. 8 ± 1.5 nm. The part of the above-mentioned coatings was enriched with AgNPs using the CVD technique [27–30]. According to the results of our previous works, the AgNPs filled the interiors of the TNT5 nanoporous layer (Figure 1g) while in the case of TNT15, the spherical nanoparticles of diameters c.a. 10 ± 2.0 nm were located mainly on the surface of the separated nanotube walls (Figure 1h).

Analysis of the XPS depth profiles of the Ti6Al4V/TNT5 and Ti6Al4V/TNT15 systems allowed changes in the titanium oxidation states between the TNT surface layer and substrate for nano-porous and nano-tubular coatings to be traced (Tables 1 and 2, Figure S2). According to these data, the surface of the TNT5 nano-porous layer consists entirely of oxides in which the Ti oxidation state is +4, which was confirmed by the presence of peaks 2p3/2 at the binding energy (BE) at c.a. 458.9 eV and 2p1/2 at c.a. 464.6 eV (Figure S2). Simultaneously, peaks of O1s at 530.2 and 531.9 eV were assigned to the O2- of Ti–O and OH− groups, respectively. The high-resolution XPS spectra registered after the first, second, and third sputtering revealed the splitting of the Ti 2p3/2 and 2p1/2 peaks, which shows the presence of Ti components for the different valence states. To confirm the valence state of Ti in the titanium oxides (Ti<sup>2</sup>+, Ti3+, or Ti4<sup>+</sup>), the differences in the BE (Δ(O–Ti)) of lines assigned to the oxygen (O1s) and Ti2p3/2 component were determined. Atuchin et al. [37] and Chinh et al. [38] showed that values of the Δ(O–Ti) criterion in the Ti2+, Ti3+, and Ti4<sup>+</sup> valence state amount to 75.0–76.7, 72.9–73.1, and 71.4–71.6 eV, respectively. According to these data, Δ(O–Ti), which for TNT5 is equal 71.3 eV, corresponds to Ti4<sup>+</sup> and suggests that TiO2 is the main component of this surface layer. The sputtering of the TNT5 sample revealed the presence of nonstoichiometric titanium oxides: After the first sputter, the layer consisted of Ti4<sup>+</sup> (58%), Ti3<sup>+</sup> (24%), and Ti2<sup>+</sup> (18%); after the second, Ti2<sup>+</sup> (12% + 55%) and Ti<sup>0</sup> (33%); and after the third, Ti2<sup>+</sup> (35%) and Ti<sup>0</sup> (65%) (Tables 1 and 2).

**Figure 1.** (**a**) Photography of the orthopedic implant produced using selective laser sintering of Ti6Al4V powder, SEM images of (**b**) the implant surface obtained, (**c**) implant surface after grinding and polishing, (**d**) surface modification of the implant by anodic oxidation using a 5 V potential, (**e**) the morphology of the TNT5 coating, (**f**) the morphology of the TNT15 coating, (**g**) the morphology of the TNT5/AgNPs coating, and (h) the morphology of the TNT5/AgNPs coating.

The calculated values of Δ(O–Ti) after the second sputtering were 75.3 and 76.6 eV, which, according to Atuchin et al. [37], confirm the presence of the titanium on the second oxidation state. Therefore, in Tables 1 and 2, both values are presented as Ti2+. The XPS studies of the non-sputtered layer, which consists of separated tubes (TNT 15), revealed the presence of dual 2p3/2 and 2p1/2 peaks at a binding energy (BE) of c.a. 459.0 and 457.8, and 464.7 and 463.4 eV, respectively (Figure S2). The calculated Δ(O–Ti) values of 71.2 and 72.2 eV, respectively, indicate the formation of oxides, in which titanium occurs at the +4 (86%) and +3 (14%) oxidation state. After the third sputtering of TNT15, it is possible to see the layer consisting of Ti4<sup>+</sup> (30%), Ti3<sup>+</sup> (23%), and Ti2<sup>+</sup> (37%) oxides, and Ti<sup>0</sup> (10%) (Tables 1 and 2, Figure S2).


**Table 1.** Changes in the position of O1s and Ti2p core levels in TNT5 and TNT15 coatings (BE, binding energy) and values of the spectral energy differences between oxygen bonded to Ti2+, Ti3+, and Ti4<sup>+</sup> ions (Δ(O–Ti) = O1s–Ti2p3/2) during Ar<sup>+</sup> sputtering.

**Table 2.** XPS depth profile of TNT5 and TNT15.


#### *3.2. Wettability and Surface Free Energy of Biomaterials*

The wettability of TNT and TNT/AgNPs sample surfaces was studied by measuring the contact angles of water (polar liquid) and diiodomethane (dispersion liquid) and the surface free energy values (SFEs) were calculated (Table 3). According to these data, the surfaces of Ti6Al4V implants after sintering and machining are hydrophobic while the anodization of titanium alloy leads to an increase of its hydrophilic character. It should be noted that the type of the TNT layer (i.e., nanoporous (TNT5) and nanotubular (TNT15) is an important factor influencing the wettability of the studied coatings. The enrichment of the studied layers by AgNPs was associated with increases of the hydrophobic character of the TNT/AgNPs surfaces.


**Table 3.** Results of the wetting angle measurements and results of the surface free energy (SFE) measurements of the materials.
