*2.5. Microbiological Assessment*

#### 2.5.1. Microbial Strains and Growth Conditions

Bacterial reference strains: *Staphylococcus aureus* ATCC 43300 (MRSA, methicillin-resistant *S. aureus*), *Staphylococcus aureus* ATCC 29213 (MSSA, methicillin-susceptible *S. aureus*), *Escherichia coli* ATCC 25922, *Streptococcus gordonii* ATCC 10558, and *Streptococcus mutans* ATCC 25175; and fungal reference strains: *Candida albicans* ATCC 10231 and *Candida glabrata* ATCC 90030 were used in the study. Bacteria were cultured on tryptic soy agar (TSA; BTL, Warsaw, Poland) or tryptic soy broth (TSB; BTL, Poland) containing 0.25% glucose (TSB/Glu). Fungi were culture on Sabouraud Agar (SDA; BTL, Warsaw, Poland) or Roswell Park Memorial Institute (RPMI) without phenol red (Sigma, Indianapolis, USA) containing 0.25% glucose (RPMI/Glu).

#### 2.5.2. Anti-Adhesive and Anti-Biofilm Properties of Titanium Surfaces Tested

Microbial strains were grown on appropriate liquid media for 24 h at 310 K. Then, microbial suspensions in TSB/Glu (bacteria) or RPMI/Glu (fungi) at the optical density of OD535 = 0.6 (nephelometer type Densilameter II, Brno, Czech Republic) were prepared. Biomaterial samples were added to 1 mL of microbial suspensions into the wells of 24-well tissue culture polystyrene plates (Nunc S/A, Roskilde, Denmark) and incubated for 24 h at 310 K in stable conditions to form a microbial biofilm. Microbial suspensions alone (without biomaterial) and liquid media only were used as a microbial growth control and negative control, respectively. Alamar Blue (AB; BioSource, CA, San Diego, USA) staining for bacteria and fluorescein diacetate (FDA; Sigma Aldrich Inc., MO, St. Louis, USA) staining for fungi were used to assess microbial colonization and biofilm formation on the tested biomaterials. First, the biomaterials were dipped in PBS (Biowest, MO, Riverside, USA) to gently remove microbial cells weakly bound to their surface. Then, the pieces of titanium biomaterials tested were sonicated (5 min, room temperature) in TSB or RPMI (for bacteria or fungi, respectively) to reclaim the cells forming the biofilm. The obtained microbial suspensions or medium (negative control) were added (100 μL) in quadruplicate to the tissue culture 96-well microplates (Nunc, Roskilde, Denmark) in case of bacteria and to the black 96-well microplates (Greiner Bio-One, Frickenhausen, Germany) in case of fungi. Microbial cell staining was performed as recommended by the manufacturer of AB and FDA. Finally, the fluorescence of AB at λex 550 nm/em, 585 nm, and FDA fluorescence at λex 485 nm/em, 520 nm was measured at Spectra Max i3 (Molecular Devices, CA, San Jose, USA) in the Laboratory of Microscopic Imaging and Specialized Biological Techniques at the Faculty of Biology and Environmental Protection University of Łód´z. Based on fluorescence units (FUs), a percentage of metabolically active microbial cells in the biofilms formed on modified titanium samples tested in comparison to microbial biofilm on reference Ti6Al4V, considered as 100% was calculated.

#### 2.5.3. Antimicrobial Activity of the Titanium Sample-Derived Supernatants

All titanium alloy implant samples tested were incubated separately in 1 mL of PBS without Ca2+ and Mg<sup>2</sup>+ (Biowest, MO, Riverside, USA) at 310 K for 24 h, 2 weeks, and 4 weeks. Then, biomaterial samples were removed, and to these obtained supernatants, 100 μL of microbial suspensions in TSB/Glu (bacteria) or RPMI/Glu (fungi) at the optical density of OD535 = 0.6 were added for 24 h of incubation at 310 K. Microbial suspensions (100 μL) in PBS (1 mL) were used as microbial growth controls. After incubation, microbial cultures were diluted from 10-1 to 10-6 in PBS preceded by intensive vortexing. Then, 100 μL of the suspensions (10-4-10-6) were cultured on TSA (bacteria) or SDA (fungi) and colony-forming units (CFU) were counted after 24 h of incubation at 310 K. The density of microbial suspensions after culture in the presence of titanium sample-derived supernatants was calculated using the average value of CFU counts. The experiment was performed twice, and each microbial culture was prepared in duplicate.

#### *2.6. AFM Topography and Mechanical Properties Studies*

The topography studies of implants TNT5 and TNT5/AgNPs were performed using atomic force microscopy (AFM, NaniteAFM, Nanosurf AG, Liestal, Switzerland). The measurements were performed in the non-contact mode at 55 mN force on an area 50 × 50 μm. The Sa parameters (area roughness) were calculated using the integrated software. The nanomechanical properties and nanoscratch-tests of implants TNT5 and TNT5/AgNPs were performed using Nanoindenter NanoTest Vantage (Micro Materials Ltd., Wrexham, UK). To determine the nanomechanical properties, 50 independent measurements in two di fferent areas of the implants (2 × 25 mm) of indentation were performed on each tested implant. The 3-side diamond Berkovich indenter with an angle of 124.4◦ was used. The maximum force was 10 mN; 15, 5, and 10 s of loading; and dwell with maximum force and unloading, respectively. The distance between the indentations in one section (tested area) was 20 μm in both axes. The nanomechanical properties were determined using the Oliver and Pharr method [36]. To calculate Young's modulus from the reduced Young's modulus, the Poisson's ratio value of 0.25 was used. Nanoscratch tests were performed on 500 μm with a maximum applied force of 500 mN and rate loading force of 3.3 mN/s. The 3-side diamond Berkovich indenter with an angle of 124.4◦ was used and 5 independent measurements were performed for each tested implant. The adhesion of the coatings was assessed based on the observation of an abrupt change in the frictional force during the test.

#### *2.7. Statistical Analysis in the Biological Assays*

All values are reported as means ± standard error of the means (SEM) and they 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 for immunological assays with GraphPad Prism 7.0 (La Jolla, CA, USA) and for microbiological and genotoxicity tests with the program Statistica 12.0 (Stat Soft Inc., Tulsa Shock, OK, USA).
