*3.5. Microbiological Assessment*

The ability of implants modified with TNT and TNT/AgNPs layers to inhibit microbial colonization and biofilm formation was tested in comparison to an unmodified Ti6Al4V surface (control biomaterial) with the use of Gram-positive (*S. aureus*, *S. gordonii*, *S. mutans*) and Gram-negative (*E. coli*) bacteria, as well as fungi (*C. albicans*, *C. glabrata*). The metabolic activity of the microorganisms attached to the surfaces after 24 h of exposure to the microbial suspensions was measured using Alamar Blue. The results are presented in Figures 7 and 8 (for bacteria and fungi, respectively) as a percentage of the metabolic active microbes recovered from the biofilms formed on the tested surfaces in comparison to the biofilms formed on unmodified control biomaterial (Ti6Al4V) being considered as 100%. All tested modified titanium alloy implant surfaces were able to inhibit microbial colonization and biofilm formation; however, in the case of bacteria, the observed effect strongly depended on the strain used. Generally, well-defined anti-biofilm activity on the tested TNT and TNT/AgNPs layers was demonstrated against *S. aureus* ATCC 29213 and *E. coli* ATCC 25922 (Figure 8). The average percentage

of biofilm inhibition, compared to the control biofilm developed on unmodified Ti6Al4V, achieved the range from 41.1 ± 3.0% (*p* = 0.034) to 49.7 ± 1.5% (*p* = 0.034) for *S. aureus* ATCC 29213 and from 33.2 ± 10.7% (*p* = 0.034) to 76.3 ± 1.5% (*p* = 0.034) for *E. coli* ATCC 25922. The weakest inhibitory effect was observed for *S. gordonii* ATCC 10558 (biofilm reduction of up to 9.0% on TNT5/AgNPs and TNT15/AgNPs, *p* = 0.028 and *p* = 0.0082, respectively). The surfaces expressed no significant or moderate activity against *S. aureus* ATCC 43300 and *S. mutans* ATCC 25175, with the exception of TNT5/AgNPs, which inhibited biofilm formation by these second bacteria of 80.9 ± 1.2% (*p* = 0.021). In the case of fungi, the inhibitory effect of the surfaces tested was similar for both strains (*C. albicans* and *C. glabrata*), achieving the level of 13.3 ± 1.6% to 33.7 ± 8.5% (Figure 8, all results were statistically significant). Interestingly, there was no grea<sup>t</sup> distinction in the reduction of the microbial biofilm caused by TNT surfaces and corresponding them to the TNT/AgNPs layers.

**Figure 7.** Bacterial biofilm on TNT- and TNT/AgNPs-modified Ti6Al4V surfaces assessed using Alamar Blue staining. The results are presented as the mean percentage ± standard deviation (SD) of the bacterial biofilm formed on the tested layers compared to a control biofilm formed on the reference biomaterial (Ti6Al4V) considered as 100%. Statistical analysis was estimated with nonparametric Kruskal–Wallis one-way ANOVA test (\* significant differences, *p* < 0.05).

**Figure 8.** Fungal biofilm on TNT- and TNT/AgNPs-modified Ti6Al4V surfaces assessed using FDA (fluorescein diacetate) staining. The results are presented as the mean percentage ± standard deviation (SD) of the fungal biofilm formed on the tested layers compared to a control biofilm formed on the reference biomaterial (Ti6Al4V) considered as 100%. Statistical analysis was estimated with nonparametric Kruskal–Wallis one-way ANOVA test (\* significant differences, *p* < 0.05).

Since biologically active (biostatic/biocidal) substances can be released from modified titanium surfaces when implants are in the host tissue, we also tested the antimicrobial effect of the supernatants obtained after short- (24 h) and long-term (2 and 4 weeks) biomaterial incubation in PBS to simulate such conditions. Four microbial strains were used for these studies (*S. aureus* ATCC 43300, *S. aureus* ATCC 29213, *E. coli* ATCC 25922, and *C. albicans* ATCC 10231) and the results are presented in Figure 9a–c as the mean density of microbial suspensions cultured for 24 h in the presence of biomaterial-derived supernatants (s). As expected, the type of titanium surfaces and the time of their incubation in PBS were the most important factors determining the release of biologically active substances from biomaterial samples and thus the antimicrobial activity of the supernatants tested. After a short (24 h) incubation, the supernatants showed almost no activity against bacterial strains (Figure 9a).

**Figure 9.** Antimicrobial effect of the supernatants obtained after 24 h (**a**), 2 weeks (**b**), and 4 weeks of (**c**) TNT- and TNT/AgNPs-modified Ti6Al4V surfaces' incubation in PBS, tested using the culture method and colony forming unit (CFU) counting. The results are presented as the mean microbial suspension density [CFU/mL] ± standard deviation (SD) after 24 h of culture in the presence of the tested supernatants.

The number of bacteria in the presence of the compounds released from AgNP-modified layers was reduced in the range 2.6%–27.9% for the TNT5/AgNPs supernatant and 0%–24.6% for the TNT15/AgNPs supernatant, in comparison to the number of the bacteria exposed on the control Ti6Al4V-derived supernatant. Whereas, *C. albicans* cells proved to be the most sensitive to the antimicrobial activity of the compounds released from the modified biomaterial samples. The reduction of the yeas<sup>t</sup> cell number caused by the TNT15/AgNPs 24-h supernatant reached 99.9% (Figure 9a), which means it has strong biocidal activity against fungi. By extending the incubation time of the biomaterial samples, the bactericidal properties of the supernatants obtained from AgNP-containing layers increased significantly. However, the compounds released from TNT5/AgNPs demonstrated the strongest antibacterial activity after two weeks (Figure 9b) while those from TNT15/AgNPs after four weeks (Figure 9c). The two-week supernatant of TNT5/AgNPs significantly reduced the number of all tested microbial strains (both bacteria and fungi), with the reduction levels reaching 61.5%, 91.4%, 78.3%, and 99.9% for *S. aureus* ATCC 43300, *S. aureus* ATCC 29213, *E. coli*, and *C. albicans*, respectively (Figure 9b). The two-week TNT15/AgNPs-derived supernatant activity was similar only against *C. albicans* (99.8% reduction of fungal viability; Figure 9b). However, during 4 weeks of biomaterial incubation in PBS, the antimicrobial potential of the TNT15/AgNPs-derived supernatant increased significantly, causing complete elimination of most of the tested microorganisms. The reduction of the *S. aureus* ATCC 43300, *E. coli*, and *C. albicans* populations exceeded 99.9% after exposition of this supernatant (Figure 9c). The effect on *S. aureus* ATCC 29213 was a little bit weaker (71.3% of eradication) but still very strong (Figure 9c) while the supernatants derived from the TNT5 and TNT15 samples (both 2 and 4 weeks) did not exhibit killing activity against the microorganisms tested (Figure 9b,c).

#### *3.6. AFM Topography and Nanomechanical Properties Studies*

The topography images and the Sa parameter values of TNT5 and TNT5/AgNPs samples (systems whose surface shows the best biological properties) using atomic force microscopy (AFM) are presented in Figure 10. Analysis of these data showed that the implant surface has a much more extensive surface topography before the AgNP deposition process. The roughness parameters, Sa, decrease about 57% from 0.89 for TNT5 before silver deposition to 0.39 for implant TNT5/AgNPs with nanosilver on the surface. A significant decrease in the roughness value was probably caused by the deposition of silver nanoparticles in the surface cavities, which led to their smoothing.

**Figure 10.** The topography of TNT5 and TNT5/Ag implants with Sa (Average Roughness) parameter values, which was determined using atomic force microscope (AFM).

The nanomechanical and nanoindentation properties of the tested implants (TNT5 and TNT5/AgNPs) for the two tested areas of each surface are presented in Table 4. In the case of the TNT5, no such significant differences in the mechanical properties (nanohardness and Young's modulus) were observed between the tested area surfaces (I and II) as in the case of the tested areas (I and II) of the implant TNT5/AgNPs. The presence of silver nanoparticles resulted in an increase of the nanohardness and Young's modulus and as a consequence, as increase of parameter H/E (Hardness to

Young's Modulus ratio), which determines the resistance to wear of the tested specimens. The relation between parameter H/E and wear resistance were reported [35]. The significant standard deviation values confirm the credibility and diligence of the presented results and their value in studies on the nanoindentation on titanium dioxide nanotube layer were reported previously by Jemat et al. [41] and Rayon et al. [42]. Moreover, using small values of force (10 mN) on surfaces with a high surface roughness causes significant differences between individual measurements. The 3D distribution of nanomechanical properties, such as the nanohardness and Young's modulus, for TNT5/AgNPs (tested area II) are presented in Figure 11. The presented results confirm the value of the standard deviation presented in Table 4. The presented results show the heterogeneity of the distribution of the mechanical properties and the relationship between the hardness and Young's modulus because the distributions are similar to each other.


**Table 4.** Nanomechanical and nanoindentation properties of the tested implant samples.

**Figure 11.** The nanomechanical properties (nanohardness and Young's modulus) of TNT5/Ag implant in the studied area II.

The nanoscratch test results for TNT5 and TNT5/AgNPs are presented in Table 5. Nowadays, the nanoscratch test method is a dedicated method to assess the adhesion of thin coatings or layers, as in the case of the presented tests. In Table 5, two different types of force obtained during nanoscratch test measuring for the tested coatings are presented. The critical force is the maximum applied force between the coating and the indenter during full delamination of the coating from the metallic substrate (Ti6Al4V) and the critical friction force is the maximum friction force registered during full delamination of the coating. The presence of silver nanoparticles in the composite coating (TNT5/AgNPs) caused an increased critical force (from 79.70 to 173.40 mN) and critical friction force (from 130.77 to 212.34 mN). The results obtained from nanoindentation tests determined the wear resistance of the tested surfaces (H/E ratio) correlated with the nanoscratch test results. The H/E parameter for the TNT5/AgNPs surface was significantly higher than for the TNT5 surface. The same trend was reported in the nanoscratch tests results.


**Table 5.** Adhesion properties of the titanium dioxide nanocoatings to the titanium alloy surfaces.
