2.5.1. Cell Culture

Murine fibroblast cell line L929 (American Type Culture Collection, Manassas, VA, USA) was cultured at 37 ◦C in 5% CO2 and 95% humidity in a complete RPMI 1640 medium containing 2 mM L-glutamine, heat-inactivated 10% fetal bovine serum (FBS) and antibiotics (100 μg/mL streptomycin and 100 IU/mL penicillin) (all compounds was from Sigma-Aldrich, Darmstadt, Germany). L929 cells were grown in 25 cm<sup>2</sup> cell culture flasks and the culture medium was changed every 2–3 days. The cells were passaged using a cell scraper when reaching 70–80% confluency.

Human osteoblast-like MG 63 cells (European Collection of Cell Cultures, Salisbury, UK) were plated in a 25 cm<sup>2</sup> cell culture flask and cultured with Eagle's Minimum Essential Medium containing 2 mM L-glutamine, 1 mM sodium pyruvate, MEM non-essential amino acid, heat-inactivated 10% FBS and antibiotics (100 μg/mL streptomycin and 100 IU/mL penicillin) (all reagents were purchased from Sigma-Aldrich). The cells were grown at 37 ◦C in an incubator providing a humidified (95%) atmosphere containing 5% of CO2. The culture medium was changed every 2–3 days. The cells were passaged using a 0.25% trypsin-EDTA solution (Sigma-Aldrich) when reaching 70–80% of confluency.

#### 2.5.2. Cell Adhesion and Proliferation Detected by the MTT Assay

L929 fibroblasts, as well as MG-63 osteoblasts, in a volume of 1 mL of appropriate complete culture medium were seeded onto the autoclaved tested nanolayers placed in a 24-well culture plate (Corning, NY, USA) at a density of 1 × 10<sup>4</sup> cells/well for 24, 72 or 120 h, respectively. The cell adhesion (measured after 24 h) and proliferation (evaluated after 72 h and 120 h) was studied by the MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide; Sigma Aldrich) assay using the same method as it was reported in Reference [33]. Briefly, after the respective incubation time, the plates were transferred to a new 24-well culture plate. The MTT solution (5 mg/mL; Sigma-Aldrich) in an appropriate culture medium without phenol red (RPMI 1640 medium for L929 fibroblasts or Eagle's Minimum Essential Medium for MG-63 osteoblasts; both from Sigma-Aldrich) was added to each well. After 3 h of incubation, the solution was aspirated and 500 μL of dimethyl sulfoxide (DMSO; 100% *v*/*v*; Sigma Aldrich) was added to each well. Finally, the plates were shaken for 10 min and the absorbance was measured at a wavelength of 570 nm with a subtraction of 630 nm (background) using a microplate reader (Synergy HT; BioTek, Winooski, VT, USA). All measurements were done in duplicate in five independent experiments.

#### 2.5.3. Cell Morphology Evaluated by Scanning Electron Microscopy

L929 fibroblasts and MG-63 osteoblasts (1 × 10<sup>4</sup> cells/well) were incubated on the tested specimens for 24, 72 and 120 h, respectively. Scanning electron microscopy (SEM; Quanta 3D FEG; Carl Zeiss, Göttingen, Germany) analyses were performed to study the morphology changes of the cells grown on the surface of tested plates using the same method as in Reference [33]. Briefly, after the selected incubation time, the samples were fixed in a 2.5% *v*/*v* glutaraldehyde (Sigma Aldrich) and dehydrated in a graded series of ethanol (50%, 75%, 90%, and 100%). Finally, the specimens were dried in vacuum-assisted desiccators overnight and stored at room temperature until the SEM analysis was performed.

#### 2.5.4. Statistical Analysis in the MTT Assay

All values are reported as means ± standard error of the means (SEM) and were analyzed using the nonparametric Kruskal–Wallis one-way ANOVA test with the level of significance set at *p* < 0.05. Statistical analyses were performed with GraphPad Prism 7.0 (La Jolla, CA, USA).

#### *2.6. The Evaluation of the Antibacterial Properties of the Ti6Al4V/AgNPs and Ti6Al4V/TNT5/AgNPs Samples*

The antimicrobial activity of titanium alloys, Ti6Al4V and Ti6Al4V/TNT5, coated with silver nanograins was studied against Gram-positive (*Staphylococcus aureus* ATCC 6538 and *S. aureus* ATCC 25923=PCM 2054) and Gram-negative (*Escherichia coli* ATCC 8739 and *E. coli* ATCC 25922=PCM 2057) bacteria and yeasts of *Candida albicans* ATCC 10231. Sterile sample plates (7 × 7 mm pieces, 0.2 mm thick) were placed in 1 mL of phosphate buffered saline (PBS) without ions (EURx) for 24 h, and 14 and 28 days to allow for the silver ions to be released. PBS was sterilized with cellulose filters (ø 0.2 μm) prior to use. Ti6Al4V/AgNPs and Ti6Al4V/TNT5/AgNPs plates were then removed and PBS was inoculated with the tested microorganism (final concentration of microorganism in each sample was approximately 5 × 10<sup>5</sup> c.f.u mL−1). Microbial inoculum density was estimated by colony counts. Briefly, the microbial inoculum (approximately 5 × 10<sup>5</sup> c.f.u. mL−1) in sterile PBS was diluted (1:1000) and 100 μL was then spread over the surface of Trypticase Soy Agar (TSA, Becton Dickinson, Franklin lake, NJ, USA) or Sabouraud Dextrose Agar (SDA, Becton Dickinson, Franklin lake, NJ, USA). After incubation, the presence of approximately 50 colonies indicated an inoculum density of 5 × 10<sup>5</sup> c.f.u. mL−1.

Inoculated samples were incubated at 37 ◦C for 24 h. Subsequently, serial ten-fold dilutions of each sample were prepared. Aliquots (100 μL) of each dilution was spread over the surface of Trypticase Soy Agar (TSA, Becton Dickinson, Franklin lake, NJ, USA) or Sabouraud Dextrose Agar (SDA, Becton Dickinson, Franklin lake, NJ, USA) plates, which had been dried for 15 min prior to inoculation. TSA and SDA media were used for bacterial and fungal growth, respectively. The positive control was Ti6Al4V or Ti6Al4V/TNT5 plates non-coated with AgNPs. Tests were performed in triplicate. Colony forming units were counted on the inoculated plates and compared with the appropriate control plates to estimate the reduction of bacterial or fungal growth.

The antibacterial rate was calculated using the following formula:

$$\mathcal{R} = ((B - A)/B) \times 100\%,$$

where *R* is the antimicrobial rate (%), *B* is the average number of microorganisms in PBS after the use of uncovered titanium alloys, and *A* is the average number of microorganisms in PBS after the use of titanium alloys enriched with silver nanoparticles.
