**3. Results**

#### *3.1. The Fabrication of Ti6Al4V/AgNPs and Ti6Al4V/TNT5/AgNPs Systems*

Silver nanoparticles were deposited on the surface of Ti6Al4V and Ti6Al4V/TNT5 substrates using the CVD method (*hot wall* reactor, precursor: Ag5(O2CC2F5)5(H2O)3) in the conditions listed in Table 1. The use of the following CVD precursor masses, *m* = 5, 10, 20, 50 mg, made it possible to produce coatings of the AgNPs content: c.a. 0.9, 1.1, 1.3, 2.3 wt% on the surface of Ti6Al4V substrates and 0.6, 1.0, 1.6, 2.3 wt% on the surface of Ti6Al4V/TNT5, respectively (wt% of AgNPs was determined based on the mass sample difference before and after CVD process). Considering the wt% of deposited silver, the studied specimens were marked as Ti6Al4V/0.9–2.3AgNPs and Ti6Al4V/TNT5/0.6–2.3AgNPs.

#### *3.2. Surface Morphology and Topography*

SEM images of the Ti6Al4V/0.9–2.3AgNPs and Ti6Al4V/TNT5/0.6–2.3AgNPs samples are presented in Figures 1 and 2. Analysis of these data shows that the precursor mass applied in the CVD experiments and the substrate type are two main factors directly impacting the size and distribution of the deposited nanoparticles (Table 2).


**Table 2.** The weight % and the diameters of the AgNPs deposited on the surface of the Ti6Al4V and Ti6Al4V/TNT5 substrates using the CVD technique.

The analysis of the SEM images of Ti6Al4V substrates (Figure 1a–d), whose surfaces have been enriched with AgNPs, shows that this surface is evenly covered by dispersed silver grains, whose densities increase with the increase of the nanoparticles weight percent on the substrate surface. The diameter of the nanosilver grains ranges from 18 ± 8 nm up to 53 ± 18 nm. The smallest diameter of nanosilver is observed when 5 mg of the precursor was used. The use of 20 mg of the CVD precursor in the same deposition conditions led to the formation of AgNPs with significant differences in the diameter (from 45 up to 90 nm) and shape (from spherical to rods) of silver grains (Figure 1c). These deposition conditions are probably suitable for the nucleation of spherical grains and their later growth in one direction (formation of rods). The increase of the CVD precursor concentration in vapors (50 mg) caused the deposition of AgNPs of similar diameter and shape, but their arrangemen<sup>t</sup> is characterized by a significantly higher density (Figure 1d).

**Figure 1.** The scanning electron microscopy (SEM) images of Ti6Al4V/0.9AgNPs; *d* = 18 ± 8 nm (**a**), Ti6Al4V/1.1AgNPs; *d* = 45 ± 15 nm (**b**), Ti6Al4V/1.3AgNPs; *d* = 68 ± 32 nm (**c**), Ti6Al4V/2.3AgNPs; *d* = 53 ± 18 nm (**d**).

SEM images of the Ti6Al4V/TNT5/AgNPs systems are presented in Figure 2a–d. Their analysis shows that on the surface of the TNT5 coating (tubes diameter allow c.a. 28 ± 11 nm), the diameter of the deposited AgNPs changes from 38 ± 14 nm up to 115 ± 49 nm. Both the size of the nanoparticles' diameters and their density on the surface of nanotubes grow along with the increase of the number of silver precursor used in the deposition process. Compared to the growth of the silver nanoparticles on the unmodified surface of the titanium alloy, the silver nanoparticles' growth on the nanotubes is more rational, predictable and does not show any anomalies.

**Figure 2.** Scanning electron microscopy (SEM) images of Ti6Al4V/TNT5/0.6AgNPs; *d* = 38 ± 14 nm (**a**), Ti6Al4V/TNT5/1.0AgNPs; *d* = 43 ± 10 nm (**b**), Ti6Al4V/TNT5/1.6AgNPs; *d* = 57 ± 24 nm (**c**), Ti6Al4V/TNT5/2.3AgNPs; *d* = 115 ± 49 nm (**d**).

The surface roughness parameter (Ra) of the studied samples was measured in the 2 × 2 μm area using software, which is an integral part of the atomic force microscopy (AFM, Veeco (Digital Instruments), Figure 3). The values of the Ra parameters determined for Ti6Al4V and Ti6Al4V/TNT5 samples were used as reference samples. The analysis of these data revealed a significant increase of the roughness parameter value with the increase of the density and size of the AgNPs deposited on the surface of both types of substrates. Moreover, the comparison of the Ti6Al4V/TNT5/AgNPs samples and the Ti6Al4V/AgNPs ones indicate the clear influence of the nanotubular architecture on the increase of the surface roughness, e.g., the value of a Ra parameter of Ti6Al4V/TNT5/1.0AgNPs is about 42% higher than that of Ti6Al4V/1.1AgNPs.

In order to confirm the deposition of silver nanograins on the surface of studied substrates, energy dispersive X-ray spectroscopy (EDS) was applied. The low intense lines, which are found in the EDS spectra of Ti6Al4V/0.9AgNPs and Ti6Al4V/TNT5/0.6AgNPs, show the presence of dispersed silver nanoparticles on the surface of the used substrates (Figure 4). The increase of AgNPs' density on the substrates' surface and their size caused a significant increase of the integral intensity of the Ag lines in the EDS spectra.

**Figure 3.** Atomic forces microscopy (AFM) images and Ra parameters determined for the Ti6Al4V, Ti6Al4V/AgNPs, Ti6Al4V/TNT5, and Ti6Al4V/TNT5/AgNPs samples.

**Figure 4.** *Cont.*

**Figure 4.** The energy dispersive X-ray spectroscopy (EDS) spectra and maps images of Ti6Al4V/0.9AgNPs, Ti6Al4V/2.3AgNPs, Ti6Al4V/TNT5/0.6AgNPs, and Ti6Al4V/TNT5/2.3AgNPs (AgNPs are marked as the green dots on the presented map images).

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

In order to estimate the value of the surface free energy based on mathematical calculations, which were performed using the Owens–Wendt method, the contact angle of two different liquids (one of them, polar, and the other one, dispersional) had to be used in the analyses [39]. Therefore, polar water and dispersional diiodomethane were chosen as measuring liquids. The obtained contact angles measurements results, as well as the calculated SFE values, are presented in Figures 5 and 6 and in Table S1. According to these data, the hydrophobic character of the Ti6Al4V surface slightly decreases after depositing small amounts of AgNPs (0.9 wt%), however, with the increase of their density, the coatings' hydrophobicity increases (Figure 5, Table S1). The fabrication of the titania nanotubes layer on the surface of Ti6Al4V leads to the formation of a hydrophilic surface, which becomes more hydrophobic when it is enriched with silver nanoparticles (Figure 5, Table S1).

**Figure 5.** The contact angles values for Ti6Al4V, Ti6Al4V/AgNPs, Ti6Al4V/TNT5, and Ti6Al4V/TNT5/AgNPs.

**Figure 6.** The surface free energy values for Ti6Al4V, Ti6Al4V/AgNPs, Ti6Al4V/TNT5, and Ti6Al4V/TNT5/AgNPs.

The values of the surface free energy (SFE) decreases for all Ti6Al4V/AgNPs samples in comparison to the adequate value for pure Ti6Al4V sample (Figure 6, Table S1). For the sample Ti6Al4V/1.3AgNPs, this is more than two times lower. A different situation is noticed for the Ti6Al4V/TNT and Ti6Al4V/TNT/AgNPs samples. Here, with the exception of the first two silver-enriched systems (Ti6Al4V/TNT/0.6AgNPs and Ti6Al4V/TNT/1.0AgNPs) for which the SFE values are lower than for Ti6Al4V/TNT, two consecutive ones, i.e., Ti6Al4V/TNT/1.6AgNPs and Ti6Al4V/TNT/2.3AgNPs, are characterized by a similar value of free surface energy (Figure 6, Table S1).

#### *3.4. Silver Ion Release in the Body Fluid Environment*

The bioactivity of Ti6Al4V and Ti6Al4V/TNT5 samples enriched with silver nanoparticles associated with silver ions releasing from their surface can be estimated on the basis of the Ag+ ion concentration change studies versus time (5 weeks) for the samples immersed in the phosphate-buffered saline (PBS) solution at human body temperature (37 ◦C) [36]. Ti6Al4V and Ti6Al4V/TNT5 samples, whose surfaces were enriched with 1.0–1.1 and 2.3 wt% of AgNPs, deposited using 10 and 50 mg of Ag CVD precursor, respectively (Ti6Al4V/1.1AgNPs, Ti6Al4V/2.3AgNPs, Ti6Al4V/TNT5/1.0AgNPs, and Ti6Al4V/TNT5/2.3AgNPs), were used in our studies on the silver ions release effect (Figure 7). The analysis of these data proved the lack of significant differences in the Ag+ ion release effect for samples containing a high content of AgNPs on their surface, i.e., Ti6Al4V/2.3AgNPs and Ti6Al4V/TNT5/2.3AgNPs. In both cases, the rapid increase of Ag+ concentration in the PBS solution was noticed in the first 10 days of the experiment, and then the concentration changes remained at the level of 1.7–2 ppm. The deposition of nearly a 2-fold smaller amount of AgNPs on the surface of the studied substrates caused a significant reduction in the concentration of silver ions released. In the case of the Ti6Al4V/1.1AgNPs sample, the maximum Ag+ release was achieved after 7 days and, in the long-term, it remains at the level of 0.9–1.1 ppm. In the first 10 days, the release of silver ions from the surface of the Ti6Al4V/TNT5/1.0AgNPs sample immersed in the PBS solution was lower than 0.1 ppm. The highest concentration of silver ions was

observed after 34 days, i.e., 0.4 ppm. The results of the study showed a significant effect of the number of AgNPs dispersed on the surface of TNT layers on the concentration of silver ions released.

**Figure 7.** The release amount of Ag+ ions from Ti6Al4V/AgNPs and Ti6Al4V/TNT5/AgNPs samples (containing 2.3 and 1.0–1.1 wt% of AgNPs) immersed in a phosphate buffered saline (PBS) and measured by inductively coupled plasma ionization mass spectrometry (ICP-MS).

#### *3.5. The Evaluation of the Biocompatibility of the Produced Titanium Alloy-Based Materials*

The biocompatibility of the studied substrates, whose surfaces were enriched with different amounts of dispersed silver nanoparticles, were evaluated on the basis of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and Scanning Electron Microscopy (SEM) micrographs. The assays were made for Ti6Al4V, Ti6Al4V/AgNPs and Ti6Al4V/TNT5/AgNPs, using two cell lines: the murine fibroblast cell line L929 and human osteoblast-like MG-63 cells. The level of adhesion (measured after 24 h) and proliferation (assessed after 72 h and 120 h) of the cells growing on the surface of Ti6Al4V/AgNPs and Ti6Al4V/TNT5/AgNPs was compared to that which was observed for the cells cultured on the reference Ti6Al4V alloy foil. As it can be seen, with an increase of the incubation time, more L929 fibroblasts, as well as MG-63 osteoblasts, proliferated on the surface of all the tested biomaterials (Figures 8 and 9). Analysis of the MTT assay data revealed the lack of significant differences in the MG-63 osteoblasts adhesion and proliferation to the surface of the reference Ti6Al4V sample and Ti6Al4V/AgNPs samples, whose surfaces were enriched with various amounts of AgNPs (Figure 8). In contrast, we have noticed that the Ti6Al4V alloy foils covered with nanosilver provoked a slight decrease in the L929 fibroblasts' proliferation after 72 h of incubation ((for Ti6Al4V/1.3AgNPs and Ti6Al4V/2.3AgNPs) Ag nanolayers; *p* < 0.05) and 120 h of incubation for the all tested concentration of nanosilver (relative L929 cells' viability compared to the Ti6Al4V reference sample and expressed as a percentage was presented in the Table below Figure 8A). However, we did not observe any differences in the fibroblasts adhesion measured after 24 h of incubation (Figure 8A).

The results of the MTT assay for Ti6AlV/TNT5/AgNPs are presented in Figure 9. The results were compared to that which was observed for the cells cultured on the reference Ti6Al4V alloy foils. Analysis of these data showed no differences in the cell adhesion (measured after 24 h) for both the tested cells lines. Moreover, L929 fibroblasts cultured on the surface of TNT5 nanotubes' coatings doped by the all tested concentrations of nanosilver showed a higher rate of cell proliferation after 120 h of incubation than the cells that grew on the Ti6Al4V reference layers (*p* < 0.001).

This phenomenon was also observed after 72 h of incubation, but only for the samples enriched with 0.6 wt% of AgNPs (*p* < 0.001, Ti6Al4V/TNT5/0.6AgNPs). On the other hand, MG-63 osteoblasts cultured on the TNT5 nanotubes enriched with silver nanoparticles provoked a decrease in the level proliferation of MG-63 osteoblasts after 120 h of incubation (*p* < 0.001) in comparison to the Ti6Al4V reference alloy (*p* < 0.001) (the relative MG-63 cells viability compared to the Ti6Al4V reference sample and expressed as a percentage was presented in the Table below the Figure 9B). However, it should be clearly emphasized that these samples also showed an increase in the level of proliferation over time.

**Figure 8.** The L929 murine fibroblasts (**A**) and human osteoblasts MG-63 (**B**) adhesion (measured after 24 h) and proliferation (evaluated after 72 h and 120 h) on the surface of Ti6Al4V/AgNPs, detected by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The absorbance values are expressed as means ± SEM of five independent experiments. Hash marks indicate significant differences at the appropriate incubation time between the cells incubated on the reference Ti6Al4V alloy foils (Ti6Al4V) compared to the specimen coatings doped by the different concentrations of Ag (# *p* < 0.05, ## *p* < 0.01). Tables below Figure 8A,B presented relative L929 cells or MG-63 cell viability (%) compared to the Ti6Al4V reference sample measured after 120 h of incubation.

Biointegration of titania nanolayers enriched with silver nanoparticles were also assessed by the analysis of SEM micrographs. Comparative SEM images showing the morphology, adhesion and proliferation of the cells growing on the surface of Ti6Al4V alloy and Ti6Al4V/AgNPs, as well as on Ti6Al4V/TNT5/AgNPs, are presented in Figures 10 and 11, respectively. These data clearly demonstrate the high biocompatibility of both types of tested nanolayers, supporting the results from the MTT assay. As it can be seen, L929 fibroblasts cultured on the surface of Ti6Al4V/AgNPs, as well as on the surface of Ti6Al4V/TNT5/AgNPs, showed the increase in cell proliferation over time (compare micrographs in Figure 10d–i). A similar phenomenon was also observed for the MG-63 osteoblasts (compare micrographs in Figure 11d–i). Importantly, the cells, especially MG-63 osteoblasts, start to grow in layers on top of each other (Figure 11k), and moreover, the cells grow with a multilayer structure on most of the surfaces of the nanolayers after 120 h of incubation time (see micrographs in Figure 11f,i,j). Finally, the SEM images also show that L929 fibroblasts, as well as MG-63 osteoblasts, form numerous filopodia which attach the cells to the surface of specimens by penetrating deep into the nanolayers (arrows in Figures 10l and 11l, respectively). These thin, actin-rich plasma-membrane protrusions were also generated between the cells (arrows in Figures 10j–k and 11k, respectively).

**Figure 9.** The effect of TNT5/AgNPs coatings on the L929 fibroblasts (**A**) and MG-63 osteoblasts (**B**) adhesion (measured after 24 h) and proliferation (evaluated after 72 h and 120 h), detected by the MTT assay. The absorbance values are expressed as means ± SEM of five independent experiments. Asterisks indicate significant differences at the appropriate incubation time when the level of cell proliferation on the surface of specimens coating doped by the different concentrations of Ag was higher compared to the reference Ti6Al4V alloy foils (Ti6Al4V) (\*\*\* *p* < 0.001). Hash marks denote significant differences at the appropriate incubation time when the level of cell proliferation on the samples enriched with AgNPs was lower in comparison with the reference Ti6Al4V alloy foils (### *p* < 0.001). Tables below Figure 9A,B presented relative L929 or MG-63 cells viability (%) compared to the Ti6Al4V reference sample measured after 120 h of incubation.

**Figure 10.** The scanning electron microscopy (SEM) images presenting adhesion (after 24 h) and proliferation (after 72 h and 120 h) of the murine L929 fibroblasts growing on the surface of reference Ti6Al4V alloy foils (**<sup>a</sup>**–**<sup>c</sup>**), (Ti6Al4V/0.9AgNPs) (**d**–**f**) or Ti6Al4V/TNT5/0.6AgNPs (**g**–**i**). Arrows in the micrographs indicate numerous filopodia spreading between the fibroblasts (**j**–**k**) or filopodia, which attached the cells to the surface of nanocoatings (**l**).

**Figure 11.** The scanning electron microscopy (SEM) micrographs showing the human osteoblast-like MG-63 cells adhesion (after 24 h) and proliferation (after 72 h and 120 h) growing on the surface of references Ti6Al4V alloy foils (**<sup>a</sup>**–**<sup>c</sup>**), (Ti6Al4V/0.9AgNPs) (**d**–**f**) or Ti6Al4V/TNT5/0.6AgNPs (**g**–**i**). The micrograph (**j**) presents the multilayer growth of cells on the surface of Ti6Al4V/TNT5/0.6AgNPs sample. Arrows indicate numerous filopodia, which attached the osteoblasts to the nanocoatings surface (**l**) and filopodia spreading between the cells (**k**).

#### *3.6. Antimicrobial Activity of Silver-Coated Titanium Alloys*

The antimicrobial activity of silver nanoparticles is widely known. Titanium alloys (surface non-modified and nanotubular modified), enriched with various amount of nanosilver grains (Ti6Al4V/AgNPs and Ti6Al4V/TNT5/AgNPs) were found to be extremely biocidal against the tested bacteria and fungi when compared to silver non-coated titanium alloy. Biocidal activity was found after 24 h, 14 days and 28 days of silver ion release into PBS. The Ti6Al4V/AgNPs and Ti6Al4V/TNT5/AgNPs composites reduced more than 99% of the growth of all tested microorganisms, independently from the number of silver nanoparticles deposited on their surface (Tables 3–5). The number of bacterial colonies after treatment with Ti6Al4V/AgNPs and Ti6Al4V/TNT5/AgNPs was reduced at least 100-fold when compared to Ti6Al4V, as presented in Figure S1 for *E. coli* ATCC25922 7.0 × 10<sup>5</sup> c.f.u. mL−<sup>1</sup> (**a**) and 3.8 × 10<sup>5</sup> c.f.u. mL−<sup>1</sup> (**b**), respectively.

**Table 3.** The reduction of microbial growth (%) in PBS after the use of Ti6Al4V/AgNPs and Ti6Al4V/TNT5/AgNPs alloys for 24 h of ion release.


Key: -; no reduction (control). PBS: phosphate buffered saline solution.

**Table 4.** The reduction of microbial growth (%) in PBS after the use of Ti6Al4V/AgNPs and Ti6Al4V/TNT5/AgNPs alloys for 14 days of ion release.


Key: -; no reduction (control).


**Table 5.** The reduction of microbial growth (%) in PBS after the use of Ti6Al4V/AgNPs and Ti6Al4V/TNT5/AgNPs alloys for 28 days of ion release.

Key: -; no reduction (control).
