*3.1. Surface Characterization*

All prepared TiO2-based coatings were blue in color, homogeneous, without any cracks on the surface, and they exhibited good adhesion to the substrate. Surface characterization carried out with scanning electron microscopy revealed fine crystalline structure of all TiO2-based coatings (results not shown). In case of coatings doped with silver ions, SEM analysis revealed small white points on the surface, of which the amount was increasing with increasing concentration of silver. The SEM method does not allow conclusions to be drawn about the convexity or concavity of the surface elements, thus the topography of these white points was analyzed by atomic force microscopy. In addition, the AFM analysis made it possible to determine the roughness (by Rq parameter) of the synthesized coatings. Figure 1 presents the general view of the coated samples, and AFM images (scan sizes of 5 μm × 5 μm and 1 μm × 1 μm) for all types of coatings.

The AFM results (AFM 2D images and values of Rq) presented in Figure 1 are in good agreemen<sup>t</sup> with SEM results. For every sample, the coating is uniform and it reflects the topography of the substrate regardless of the coating composition. Based on AFM images, it can be observed that coatings are applied even inside the surface scratches. In addition, in case of coatings doped with calcium and silver ions, especially with the increasing amount of silver ions, holes appear on the surface of the coatings. These holes correspond to the white points observed on the SEM images. They are the result of the thermal decomposition of calcium nitrate and silver nitrate used in doping procedure. According to "CRC Handbook of Chemistry and Physics" edited by Lide [40], both nitrates undergo decomposition during heat treatment, but at different temperatures. Calcium nitrate tetrahydrate decomposes at a temperature of 132 ◦C, but this decomposition is not total, it only involves the removal of water molecules. The total thermal decomposition of alkaline earth metal nitrates leading to the formation of nitrogen dioxide undergoes at temperatures higher than 500 ◦C. Whereas, in the case of silver nitrate, such total decomposition undergoes at a temperature of 444 ◦C according to Equation (1):

$$2\text{AgNO}\_3 \rightarrow 2\text{Ag} + 2\text{NO}\_2\uparrow \text{ } + \text{O}\_2\uparrow. \tag{1}$$

The presence of these holes (pores) results in different coating roughness. Coatings with an increasing amount of silver ions are characterized by a greater surface development (higher Rq).

**Figure 1.** The general view of the coated samples, and atomic force microscopy (AFM) images (scan sizes of 5 μm × 5 μm and 1 μm × 1 μm) for all types of coatings.

The same coloration of the coatings implies similarity in the coating thickness. According to Velten et al. [2], the thickness of TiO2 coatings that are blue in color should be in the range of 50–80 nm. The verification of this statement was performed via XRR analyses. Values of thickness of the investigated coatings were determined based on the Fourier transform analysis of the registered X-ray reflectivity curves. The obtained XRR results are presented in Table 2.

**Coating Thickness**/**nm** TiO2 77 ± 4 Ca\_TiO2 74 ± 4 75Ca25Ag\_TiO2 80 ± 4 50Ca50Ag\_TiO2 75 ± 4 25Ca75Ag\_TiO2 77 ± 4 Ag\_TiO278±4

**Table 2.** The thickness of TiO2-based coatings doped with Ca and Ag ions in different molar ratios.

The determined values are in good agreemen<sup>t</sup> with the literature-based predictions. Furthermore, the analysis of the results allowed to conclude that if the constancy of the sol composition (the ratio of individual reagents in doping procedure) is maintained, then the doping procedure does not significantly affect the thickness of the sol-gel coating.

The phase composition of the investigated coatings was determined by X-ray diffraction method. Figure 2 shows the XRD patterns obtained for TiO2-based coatings. The results reveal that every single coating exhibits the anatase structure of TiO2 (Ref. 00-064-0863). This is confirmed by peaks centered at 2theta, 25.37◦, broad peak being a superposition of three peaks (centered at 2theta equal to 36.93◦, 37.96◦, 38.64◦), 48.06◦, 54.02◦ and 55.03◦ (marked with asterisks on the chart). The comparison of the intensity of the peaks for particular coatings shows positive influence of silver onto the crystallization process of titanium dioxide. The most intensive and well defined peaks were registered for TiO2 coatings with the highest concentration of silver. In the case of calcium, no noticeable difference between TiO2 and Ca\_TiO2 XRD spectra was observed, which means the incorporated Ca does not alter the crystallization process of anatase. Therefore, it can be stated that silver promotes the crystallization of titanium dioxide in the form of anatase. Such a finding corresponds to the report of García-Serrano et al. [41].

**Figure 2.** XRD patterns for TiO2-based coatings doped with Ca and Ag ions in different molar ratios.

## *3.2. Corrosion Tests*

Anticorrosion properties of TiO2-based sol-gel coatings were determined via electrochemical methods based on polarization near the corrosion potential and polarization in wide anodic range. Such measurements allowed for the evaluation of the resistance of the samples against general and pitting corrosion in PBS solution.

The linear polarization measurements performed in a narrow scanning range (± 20 mV vs. Ecor), allowed for the calculation of the values of corrosion rate, CR, based on determined polarization resistance, Rp, values (according to the assumptions of standard ASTM G102-89 [42]). The mean values of Ecor, Rp, and CR with standard deviations for all investigated TiO2-based coatings are given in Figure 3. In order to confirm the protective properties of TiO2-based coatings, the results for uncoated M30NW alloy substrate are also included in Figure 3.

**Figure 3.** Values of (**a**) corrosion potential, Ecor, (**b**) polarization resistance, Rp, and corrosion rate, CR, determined for TiO2-based coatings doped with Ca and Ag ions in different molar ratios.

It can be observed that the Ecor value remains constant (of ca. 0.20V) for undoped TiO2 coating and coatings with predominant calcium content (i.e., Ca\_TiO2, 75Ca25Ag\_TiO2, 50Ca50Ag\_TiO2). Whereas, when the silver content is predominant and its concentration increases in the coating, the corrosion potential progressively decreases up to 0.09V. This is attributed to the increasing amount of Ag metallic nanoparticles in the coating.

As can be seen in Figure 3b, the Rp of the Ca-doped TiO2 coating is higher than that of the undoped TiO2 coating, suggesting that calcium incorporation into TiO2 coating has a significant effect in improving its corrosion resistance. However, as the silver addition in the films increases, the Rp of the coatings decreases gradually from 50 to 9.6 M Ω·cm2. This probably means that a larger amount of silver ions is released from Ag-doped TiO2 coatings with higher silver content, resulting in a higher corrosion rate (see CR diagram in Figure 3b). Analogous observations were reported by X. Zhang et al. [43]. While, some other researchers [24,35] reported opposite corrosion behavior of Ag-incorporated TiO2 coatings—with an increased amount of silver content the corrosion resistance was improved. This tendency was; however, attributed to the fewer surface defects [35] or the presence of an Ag-TiO2 nanocomposite [24]. Nevertheless, in terms of polarization resistance and corrosion rate, all our doubly-doped coatings act as corrosion protective—the samples with those coatings exhibit better corrosion resistance than the uncoated alloy substrate. According to the Rp and CR results, the Ca\_TiO2coating provides the best anticorrosion protection for the steel substrate.

Pitting corrosion resistance of M30NW alloy samples coated with TiO2-based coatings was examined through the potentiodynamic anodic polarization. The potentiodynamic curves of undoped TiO2- and Ca,Ag-doped coatings were recorded in PBS solution within the wide anodic potential range (up to ca. 1.7V) in order to study the passivation and breakdown behavior of all types of coatings, and are shown in Figure 4. Table 3 gives values of corrosion quantities determined from potentiodynamic curves: current density in passive range (at arbitrary chosen potential of 0.2V) and breakdown potential Eb.

**Figure 4.** Potentiodynamic polarization curves of TiO2-based coatings in phosphate bu ffered saline (PBS) solution (scan rate 1 mV·s<sup>−</sup>1).


**Table 3.** Values of corrosion quantities determined from potentiodynamic characteristics.

Based on the potentiodynamic characteristics shown in Figure 4 and data presented in Table 3, it can be stated that an increasing amount of silver in the TiO2 coatings results in higher electrochemical activity of the coatings. For the sample with Ca/Ag molar ratio of 1:3 (25Ca75Ag\_TiO2), the current density in passive range is two times higher when compared to the Ag-free coatings (TiO2, Ca\_TiO2) and coatings with predominant or equal calcium content (75Ca25Ag\_TiO2, 50Ca50Ag\_TiO2). However, for the coating doped only with silver ions (Ag\_TiO2), the value of current density in the passive range is the highest, and is about 23 nA/cm2, which is about seven times higher than for the undoped coating. This fact can be related to the previously found higher porosity of the coatings containing silver ions. The deep pores present in the coating can facilitate the penetration of the corrosion solution through the coating toward the substrate and thus increase the reactivity of the sample.

The breakdown potential Eb value was determined as the potential at which there is a sharp increase in current on the potentiodynamic curve. As shown in Figure 4 and Table 3, prepared materials are characterized by relatively high values of Eb potential of ca. 1.6 V regardless of doped ions. In order to confirm the veracity of such high Eb values, an additional experiment was also performed for each sample, and polarization was stopped at 1.5 V, just after the earlier increase in current recorded on the characteristic curve. Nevertheless, post-polarization microscopic analysis showed no pits on the surface, which proves that, in the case of investigated samples, pitting corrosion occurs at potentials higher than 1.5 V. Such high values of Eb may result from the surface finishing degree (polishing to mirror surface), passivation in mixture of HF and HNO3 acids, as well as the nature of the titanium dioxide. Based on the corrosion tests results, it can be stated that the M30NW alloy samples with TiO2-based coatings doped with calcium and silver ions belong to the group of high pitting corrosion-resistant materials.

On the surface of all tested TiO2-based coatings, anodic polarization caused the formation of corrosion damages as pits, di ffering in morphology, depth, and width. In many cases, these pits were spherical-ish in shape and covered with corrosion sludge. Moreover, for coatings containing the addition of silver ions (75Ca25Ag\_TiO2, 50Ca50Ag\_TiO2, 25Ca75Ag\_TiO2, Ag\_TiO2), the destruction of the coating in close proximity to the pits can be observed. This is most likely the result of the greater reactivity of these samples. The SEM images shown in Figure 5 indicate that the pitting mechanism starts with the breakdown of the coating, followed by under-film corrosion (dissolution) of the substrate material. In subsequent stages, the damaged fragments of the coating wrap become detached and reveal the corroded substrate (pit). The interiors of the pits reveal the dissolved intergranular edges of the alloy grains.

## *3.3. Immersion Test*

Studies on in vitro bone-bonding ability (referred to as bioactivity) of materials were started by Kokubo and co-workers dozens of years ago [44]. They proposed that the essential requirement for an artificial material to bond to living bone is the formation of bone-like apatite on its surface when implanted in the living body. In laboratory conditions, this ability can be assessed by immersion test in a simulated body fluid (SBF) with ion concentrations nearly equal to those of human blood plasma [39]. The evidence of the bioactive properties of the biomaterial is the formation of an apatite layer as a result of exposure to the SBF solution. Thus, an immersion test in SBF allows for the prediction of the material's in vivo bone bioactivity.

In order to study the e ffect of calcium and silver ions doping on bioactivity of TiO2-based coatings, the M30NW alloy samples with five types of coatings, undoped TiO2, Ca\_TiO2, 75Ca25Ag\_TiO2, 50Ca50Ag\_TiO2, 25Ca75Ag\_TiO2 and Ag\_TiO2, were immersed in SBF solution for 28 days, and then the samples' surfaces were examined using SEM-EDS. Figure 6 shows SEM micrographs (magnitude of 50,000×) of apatite formed on undoped TiO2 (a), Ca\_TiO2 (b), 75Ca25Ag\_TiO2 (c), and Ag\_TiO2 (d) after soaking for 28 days in SBF solution. All these SEM images are in the same scale, thus direct comparison of the results is possible. In addition, the results of qualitative elemental analysis in the form of Ca/P molar ratios estimated for these samples are given as insets in Figure 6.

**Figure 5.** Post-polarization SEM images (1000× mag., bar 50 μm) and optical microscopic images (50× mag., bar 1000 μm).

SEM analysis revealed new particles of different morphologies existing on the samples' surfaces after 28 days of exposure to SBF. In case of undoped TiO2 coating the randomly distributed agglomerates with Ca/P molar ratio of ca. 1.2 can be observed (Figure 6a). A deposit of a completely different morphology can be seen in Figure 6b for the coating doped with calcium ions (Ca\_TiO2). In this case, needle-like particles, fully covering the surface, with Ca/P molar ratio of ca. 1.5 were formed. As the content of calcium ions in the coating decreased the Ca/P ratio also decreased, and it was ca. 1.2 for 75Ca25Ag\_TiO2, ca. 1.2 for 50Ca50Ag\_TiO2, ca. 1.0 for 25Ca75Ag\_TiO2, and finally ca. 1.1 for Ag\_TiO2

sample. Apart from the low values of Ca/P molar ratio for coatings doped with both calcium and silver ions, the resulting deposits did not completely cover the surfaces; on the part of the surface they formed large agglomerates, while the remaining part of the surface was uncovered. The size and morphology of the particles deposited onto Ag-doped coating (Ag\_TiO2) were larger in average diameter and more interconnected comparing with undoped and Ca-Ag co-doped surfaces. No apatite aggregates were observed on Ag\_TiO2 surface.

**Figure 6.** SEM micrographs of apatite formed on: (**a**) undoped TiO2, (**b**) Ca\_TiO2, (**c**) 75Ca25Ag\_TiO2, and (**d**) Ag\_TiO2 after soaking for 28 days in simulated body fluid (SBF) solution (detector TLD, mag 50,000<sup>×</sup>, bar 1 μm).

According to Zhang et al. [45], the apatite particles nucleate spontaneously onto bioactive surfaces by consuming calcium and phosphate ions from the SBF solution (Equations (2) and (3)):

$$6H^{2+} + 8OH^- + 6HPO\_4^{2-} \rightarrow Ca\_{10}(PO\_4)\_6(OH)\_2 + 6H\_2O \tag{2}$$

$$2\text{ }10\text{Ca}^{2+} + 2\text{OH}^- + 6\text{PO}\_4^{3-} \rightarrow \text{Ca}\_{10}(\text{PO}\_4)\_6(\text{OH})\_2 \tag{3}$$

In case of coatings doped with calcium, the apatite nucleation is enhanced due to the presence of positively-charged calcium ions in the coating, which react with phosphate anions to form an amorphous calcium phosphate. Since this phase is metastable, it is eventually transformed into stable crystalline bone-like apatite [45]. Results obtained in this study indicate that the more Ca2+ that is incorporated in TiO2 coating, the easier and quicker is the apatite nucleation on the surface. The Ca/P ratio of the apatite formed on most of the coatings fabricated in this study is in the range 1.1–1.2, which indicates that the apatites are calcium-deficient HA [33]. Only for that coating, the Ca/P ratio was approaching the value of 1.67 characteristic of the stoichiometric hydroxyapatite. It is a very important factor for orthopedic implants, since literature data indicate that the newly formed bone tissue closely adheres to the implanted element when the Ca/P molar ratio is in the range of 1.67–2.0.
