**3. Results**

#### *3.1. Synthesis of Nanotubular Coatings and Analysis of Their Surface Morphology on Different Steps of Experimental Procedure*

Titania nanotube (TNT) layers on the surface of Ti6Al4V foil samples were produced according to the earlier described electrochemical oxidation methodology (anodization process) [25] in potentials range *U* = 5–60 V at room temperature (RT). After washing them in deionized water and drying them in the stream of argon their surface morphology has been checked. SEM images presented Ti6Al4V/TNT/Ar surface nanoarchitecture are visible in Figures 1 and 2. In Table S1 diameters of formed nanotubes and their wall thickness are presented.

In the next stage, the produced Ti6Al4V/TNT5-TNT60/Ar systems were divided into two groups. The first one was autoclaved directly, leading to the production of Ti6Al4V/TNH5-TNH60, and the second one, before autoclaving was subjected to the additional drying process, using immersion in acetone and drying at 396 K for 1 h. This second procedure led to the production of Ti6Al4V/TNT5-TNT60. Figures 1 and 2 present the differences in surface morphology of Ti6Al4V/TNH5-TNH60 and Ti6Al4V/TNT5-TNT60 and their comparison with the initial nanotubes samples, after drying in Ar stream, before the autoclaving process - Ti6Al4V/TNT5-TNT60/Ar. The comparison of SEM images of TNT5/Ar, TNT10/Ar coatings with TNH5, TNH10, TNT5, and TNT10 ones proves that regardless of the method used to dry the samples before autoclaving, the surface morphology after autoclaving remains unchanged, even identical (Figure 1). In the case of TNT15/Ar, TNH15, and TNT15 coatings, the slight surface changes were noticed for the TNH15 sample, which consisted in the partial, incidentally appeared destruction of nanotubes. Analysis of SEM images of TNT20-TNT60 coatings and TNH20-TNH60 ones revealed significant differences in their morphology. The tubular surface architecture of Ti6Al4V/TNT20-60 is identical with the initial samples Ti6Al4V/TNT20-60/Ar. But the surface architecture of Ti6Al4V/TNH20-60 samples is nothing like the starting nanotubes—nanotubular architecture of Ti6Al4V/TNT20-60/Ar was completely destroyed (Figure 2).

**Figure 1.** Scanning Electron Microscopy (SEM) images of Ti6Al4V/TNT5-15/Ar, Ti6Al4V/TNH5-TNH15, and Ti6Al4V/TNT5-15 samples surface (selected destruction sites of TiO2 nanotubes were marked with circles).

A significant influence of preparing procedure of nanotubular coatings for autoclaving and the autoclaving process itself, on surface morphology changes, for nanotubes with higher potential (20–60 V), highlighted a focus for future works in terms of structure, wettability, mechanical properties, and biocompability of two groups of samples obtained at potential 20–60 V: (1) nanotubes coatings, dried after their production in ordinary way, only with Ar stream at RT—which nanotubular morphology during the autoclaving is completely destroyed (we will describe them as Ti6Al4V/TNH20-60 systems, or just TNH20-60 coatings, H—indicates that they are hydrothermally modified) and (2) nanotubes coatings dried additionally using immersion in acetone and slow drying in 396K, which nanotubular morphology during the autoclaving is not changed (we will describe them as Ti6Al4V/TNT20-60 systems or just TNT20-60 coatings).

**Figure 2.** SEM images of Ti6Al4V/TNT20-60/Ar, Ti6Al4V/TNH20-TNH60, and Ti6Al4V/TNT20-60 samples surface.

#### *3.2. Structural Studies on TNH20-60 and TNT20-60 Coatings and Their* Wettability Analysis

The Raman and diffuse reflectance infrared Fourier transform spectroscopy (IR DRIFT) methods have been used to study the eventual structural differences between Ti6Al4V/TNH20-TNT60 and Ti6Al4V/TNT20-60 systems, as we suspected that structural changes could follow the already described morphological changes (Figure 3). Analysis of Raman spectra between 300 and 700 cm<sup>−</sup><sup>1</sup> of TNT20-TNT60 coatings confirms the amorphousness of these samples (Figure 3a). Raman spectra of TNH20-TNH60 samples indicate also on the formation of amorphous layers. However, very weak bands, which were found at 450 and 611 cm<sup>−</sup><sup>1</sup> and also at 399, 516, and 639 cm<sup>−</sup><sup>1</sup> indicate on the possible phase transitions and the formation of TiO2 rutile/anatase (TNH20, TNH30, TNH60) nanocrystals (Figure 3b). The strong bands detected between 600 and 950 cm<sup>−</sup><sup>1</sup> in all DRIFT spectra of TNH20-TNH60 samples confirm the formation of TiO2 layers (Figure S1).

**Figure 3.** Raman spectra of Ti6Al4V/TNT20-60 (**a**) and Ti6Al4V/TNH20-TNT60 (**b**) samples (A—TiO2 anatase form, R—TiO2rutile form).

In order to answer the question, what is responsible for the differences in the surface morphology of Ti6Al4V/TNT20-60 and Ti6Al4V/TNH20-TNT60, and at the same time taking into account our suspicion that the reason for the differences may be the water, which is not completely dried during the traditional drying process using a stream of argon, we made detailed Raman and IR DRIFT spectra analyses of Ti6Al4V/TNT20-60/Ar and Ti6Al4V/TNT20-60/Ac systems. Analysis of DRIFT spectra of Ti6Al4V/TNT20-60/Ar samples revealed the presence of weak and very weak bands at 3320–3390 cm<sup>−</sup><sup>1</sup> and 1620–1660 cm<sup>−</sup>1, which were attributed to ν(OH) (stretching) and δ(HOH) (bending) modes of water molecules, respectively (Figures 4 and 5). Moreover, the very strong band, which was found between 450 and 1000 cm<sup>−</sup><sup>1</sup> was assigned to ν(Ti-O) modes of TiO2, which confirms the formation of titania nanotube layers. In IR spectra of Ti6Al4V/TNT20-60/Ac samples (which after anodization were immersed in acetone and dried at 396 K), the intensity of bands attributed to vibrations of water molecules significantly decreased (Figure 5). According to these data, we can assume that the use of additional drying procedure allows for the removing of water molecules from the nanotubular surface of Ti6Al4V/TNT20-60/Ar, in particular from inside the nanotubes.

**Figure 4.** Infrared (IR) spectra (DRIFT) of (**a**) Ti6Al4V/TNT20/Ar and (**c**) Ti6Al4V/TNT50/Ar samples (the samples after drying in the Ar stream) and Ti6Al4V/TNT20/Ac (**b**) and Ti6Al4V/TNT50/Ac (**d**) the samples immersed in acetone and dried at 396 K by 1 h.

**Figure 5.** IR DRIFT spectra of (**a**) Ti6Al4V/TNT20-60/Ar and (**b**) Ti6Al4V/TNT20-60/Ac systems.

The results of contact angles measurements for water and diiodomethane, and also changes of surface free energy value (SFE) of Ti6Al4V/TNH20-60 and Ti6Al4V/TNT20-60 are presented in Figure S2 and in Table S2. According to these data, it can be stated that the wettability of Ti6Al4V/TNH20-60 layers is significantly different then adequate values for Ti6Al4V/TNT20-60. However, these differences in the case of TNT60 and TNH60 are not so huge. Analysis of data presented in Figure S2(a) indicate the clear hydrophobic character of Ti6Al4V/TNH20-60 layers, whose tubular architecture was destroyed and much more hydrophilic character of Ti6Al4V/TNT20-60. In the case of Ti6Al4V/TNT20-60 their hydrophilicity decreases from TNT20 to TNT60. The free surface energy (SFE) of the produced coatings was appointed by the Owens-Wendt method [33]. This method required the contact angles measured for two liquids, i.e., water as a polar liquid (Figure S2(a)) and dispersive one such as diiodomethane (Figure S2(b)). The SFE calculations for Ti6Al4V/TNT20-60 samples showed that their values change in the narrow range, i.e. from SFE = 47.6 (mJ/cm2) up to SFE = 53.7 (mJ/cm2). In the case of Ti6Al4V/TNH20-60 samples, the SFE value increases from 28.4 to 63.8 (mJ/cm2) for TNH20-TNH40 and again decreases to 61.0 and 49.2 (mJ/cm2) for TNH50-TNH60 respectively (Figure S2(c)).

#### *3.3. Topography and Mechanical Properties of Ti6Al4V/TNH20-60 and Ti6Al4V/TNT20-60 Samples*

The studies of surface topography and mechanical properties (such us hardness, Young's modulus) were carried out on the reference Ti6Al4V specimens, Ti6Al4V/TNH20-60 and Ti6Al4V/TNT20-60 systems. The studies of adhesion were performed for the same composites without reference Ti6Al4V specimens. The purpose of the research was to determine the relations between roughness parameters Sa, of studied systems.
