**1. Introduction**

The design and manufacture of implants, which are safe and highly accepted as being biocompatible with the human body, is a priority of modern medicine [1,2]. Works aimed at solving this issue are supported by the intense investigations on novel biomaterials and the development of modern technologies. The application of additive technologies (e.g., selective laser sintering, selective laser melting, commonly called 3D printing), which, allow for bone implant fabrication with anatomical accuracy, and lead to the shortening of the surgery duration and postoperative recovery, is a good example [3–5]. Titanium and titanium alloy powders are materials widely used in the aforementioned above-mentioned additive technologies due to the fact that implants fabricated using these powders show desirable mechanical properties, allowing them to transfer large loads. Therefore, these materials offer grea<sup>t</sup> potential for applications in orthopedics, dentistry, and spine surgery [6–8]. The advantage of the additive technology is its ability to fabricate porous systems, which can increase the ingrowth of bone and the anchorage of the implants [8,9]. However, low osteoconduction and integration of titanium-based implants with the bone for long-term survival, their weak anti-inflammatory properties, and the possibility of toxic components releasing into the human body requires surface modification and the formation of a layer, which significantly eliminates these above-mentioned adverse factors. These surface modifications can be carried out into two ways: (a) The roughness and wettability changes of the titanium implants' surface, which can stimulate a durable connection between the implant and the bone [9–11]; and (b) the formation of bioactive coatings, which accelerate bone formation (e.g., hydroxyapatite layers [12,13]) or increase their biocidal activity (e.g., bio-functional magnesium coating, as well as silver nanoparticles [14–16]). The formation of an oxide layer (passivation layer) on the surface of titanium/titanium alloy implants, which is practically insoluble and largely responsible for their high corrosion resistance and biocompatibility, is an important way to approach implants' surface modification [17]. The implants' surface oxidation process control lead to the fabrication of titania coatings of defined architecture, porosity, and microstructure, on titanium-based implants' surface, which may contribute to an improvement of their mechanical properties and to their bioactivity increase [18–21].

From a practical point of view, the anodic oxidation of titanium-based implants' surface in the HF solution, leading to the formation of first-generation TiO2 (TNT) nanotube coating, seems to be particularly interesting [22–25]. Depending on the value of the applied potential [ *U*], this method allows the following to be obtained: (a) Ordered porous layers ( *U* = 3–10 V), consisting of nanotubes with common walls; (b) ordered tube layers ( *U* = 10–30 V), composed of separated titania nanotubes; and (c) oxide coatings with a sponge-like structure (above *U* = 30 V) [24,26]. Produced TNT coatings, as obtained, are amorphous and form a uniform oxide layer of a thickness c.a. 150 nm on the entire surface of the substrate. The type of produced coating has a direct impact on the surface wettability, its porosity, and roughness, as well as on the mechanical properties. Moreover, it was found that the substrates covered with the TNT layer are characterized by more vigorous cell growth (fibroblasts) and better integration of bone with the implant surfaces [20,25,26]. The enrichment of TNT coatings with silver nanoparticles (AgNPs) using chemical vapor deposition (CVD) and atomic layer deposition (ALD) techniques, allowing control of their size and dispersion, was another direction of our works [27–30]. Forming a TNT/AgNPs system, we exploited the antimicrobial properties of silver nanoparticles without exceeding the potentially acceptable and safe dose of silver ions [16,28–30]. The composite systems produced in this way could prevent the formation of bacterial biofilms that form on the implant surface, thus being di fficult to eradicate.

Our previous research [20,21,24–31] focused on the development of technology to produce the bioactive coatings on the surface of Ti6Al4V alloy substrates, i.e., widely used material in the construction of orthopedic implants. However, in order to implement the developed nanocoatings into implant fabrication, it is necessary to estimate their bioactivity in detail. Therefore, we focused on the wide-ranging immunological studies on selected coatings, i.e., TNT5 (porous one produced at *U* = 5 V), TNT15 (tubular one produced at *U* = 15 V), TNT5/AgNPs, and TNT15/AgNPs (TNT5 and TNT15

coatings enriched with silver nanoparticles), as well as on studies intended to exclude their potential genotoxicity. Studies on the antimicrobial potential of produced coatings that counteract the colonization and biofilm formation by selected bacterial and fungal strains on TNT- and TNT/AgNPs-modified Ti6Al4V surfaces were especially important for us. The results of all of these investigations are presented and discussed in this paper.

#### **2. Materials and Methods**

#### *2.1. The Modification of the Ti6Al4V Implant Surface and the Characterization of Titania Coatings*

The studied Ti6Al4V implants were modified by the fabrication of titania coatings on their surface using the anodization oxidation method, in accordance with a previously described procedure [25]. The implants were produced by 3D technology using selective laser sintering (SLS; EOS M 100; EOS GmbH Electro Optical Systems, Krailling, Germany) of Ti6Al4V powder, the chemical composition of which was consistent with ASTM F136-02a (ELI Grade 23) [32]. The crystallographic structure of the produced implants was confirmed by the XRD di ffraction pattern (Figure S1) [33]. The anodization of the implants' surface was carried out at room temperature using 0.3 wt% aqueous HF solution as an electrolyte, the anodization time t = 30 min, and potentials of *U* = 5 V (TNT5) and 15 V (TNT15). After the anodization, the samples of the Ti6Al4V/TNT5 and Ti6Al4V/TNT15 systems were dried in a stream of argon at room temperature (RT), and additionally immersed in acetone and dried at 396 K for 1 h. Half of the TNT5 and TNT15 samples were enriched with silver nanoparticles using the CVD technique (metallic silver precursor—[Ag5(O2CC2F5)5(H2O)3]) under earlier described conditions [27,30]. The morphology of the produced coatings was studied using quanta field-emission gun scanning electron microscope (SEM; Quanta 3D FEG; Carl Zeiss, Göttingen, Germany). A 30.0 kV accelerating voltage was chosen for SEM analysis and the micrographs were recorded under high vacuum using a secondary electron detector (SE). The structure of the produced oxide layers was analyzed using X-ray di ffraction (XRD; PANalytical X'Pert Pro MPD X-ray di ffractometer, PANalytical B.V., Almelo, The Netherlands, using Cu-K α radiation; the incidence angle was equal to 1 deg) and raman spectroscopy (RamanMicro 200 PerkinElmer, PerkinElmer Inc., Waltham, MA, USA). X-ray photoelectron spectroscopy (XPS) spectra of the investigated samples were obtained with monochromatized Al K α-radiation (1486.6 eV) at room temperature using an X-ray photoelectron spectrometer (PHI 5000 Versaprobe, Physical Electronics, Inc., Chanhassen, MN, USA). The sample surface was sputtered using an Ar+ ion beam for 3 times. Energy of 2.5 keV was used for each sputter and the duration of each sputter was 2 min. All surface-modified implants (named for the publication needs as TNT5, TNT15, TNT5/AgNPs, and TNT15/AgNPs) as well as non-modified Ti6Al4V and silver-enriched Ti6Al4V/AgNPs were cut into 8 × 8 × 2 and 10 × 10 × 2 mm pieces and used in all biological experiments.

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

The wettability and surface free energy of the produced titania-based nanocoatings were determined using earlier described methods [25,34,35]. The contact angle was measured using a goniometer with drop shape analysis software (DSA 10 Krüss GmbH, Hamburg, Germany). Each measurement was repeated three times.
