**4. Discussion**

The studies on the relationship between the number of silver nanoparticles (AgNPs) deposited on the surface of Ti6Al4V and Ti6Al4V/TNT5 substrates, their size and their distribution, and the wettability and bioactivity of the produced systems were the purpose of our investigations. The following two factors determined the choice of the substrate used in our research: (a) the common use of the Ti6Al4V alloy as a material in implantology, and (b) the use of titania nanotube coatings (TNT) to modify the titanium/titanium alloys surfaces and to provide them with biocompatible properties. The electrochemical anodization method, with the use of constant potential (U = 5 V), was applied in the TNT5 coatings production. The results of our earlier works revealed that the TNT5 layer consists of densely packed titania nanotubes of diameters 35–45 nm and length c.a. 150 nm. Simultaneously, this type of coating exhibited optimal biointegration properties [34]. The above-mentioned coating enrichment with AgNPs using the CVD technique lad to the deposition of the dispersed metallic grains mainly on their surface, as in the case of the pure alloys substrates [36]. To achieve better control over the dispersion and growth of deposited AgNPs, low flow values of precursor vapors over the substrate surface were used during the CVD process. Depending on the precursor mass applied in the CVD experiments (i.e., 5, 10, 20, 50 mg) and the defined carrier gas flow, the amounts of the precursor which flow above the substrate surface, were 0.2, 0.3, 0.7, and 1.7 mg·min−1, respectively. The analysis of the SEM images confirmed the clear influence of the experimental conditions on the size and density of the deposited AgNPs (Table 2 and Figures 1 and 2). Moreover, it should be noted that the type of used substrate also affects the increase of the deposited AgNPs' size and density. The diameter of the AgNPs deposited on the surface of Ti6Al4V/TNT5 was bigger than the ones, which were deposited on the surface of the Ti6Al4V substrates (Table 2).

In our work, the biointegration of the studied samples was evaluated using two cell lines: mouse L929 fibroblasts and human osteoblasts-like MG-63 cells. According to earlier reports, the long-term success of an implant placement depends not only on the integrity of osseointegration, but also on the contact with the surrounding soft tissue [40]. In recent years, the MG-63 cell line has become a standard model for bone research in addition to primary human osteoblasts and this cell line is also well-established for studying the effects of surface nanotopography on osteoblast-like cells [41]. In addition, established permanent cell lines of soft tissue, such as L929 fibroblasts, are widely used to test the cytotoxicity of dental materials when employing in vitro methods of experimentation [42]. Moreover, fibroblasts are the most common cells in connective tissue, one of the main components of peri-implant soft tissue, which is key to the formation of the peri-implant mucosal seal and helping to prevent epithelial ingrowth [40]. Therefore, the study of the biointegration of the nanomaterials using two selected cell lines allowed for a comprehensive examination of implant biocompatibility.

It is well-established that cellular behavior, such as cell adhesion and proliferation or morphologic change (including the formation of filopodia) is determined by the surface properties of nanomaterials, thus, the cellular response measured by these parameters are required to assess the biointegration of implants [43]. In our study, the biointegration level of the tested specimens was examined using an MTT assay (for evaluation of cell adhesion and proliferation) and scanning electron microscopy images analysis (for assessment of cell adhesion, proliferation and morphology). The results of the MTT assays related to the cell adhesion (measure after 24 h) and proliferation (measured after 72 h and 120 h) revealed that there were no differences in the MG-63 osteoblasts adhesion and proliferation between the reference Ti6Al4V layers and Ti6Al4V/AgNPs (Figure 8B). On the other hand, the Ti6Al4V/AgNPs induced a significant decrease in the L929 fibroblast proliferation, especially after 120 h of incubation for the all tested concentration of nanosilver (Figure 8A). Surprisingly, the different results were obtained for Ti6Al4V/TNT5/AgNPs samples. L929 fibroblasts cultured on the surface of Ti6Al4V/TNT5 samples enriched with all tested concentration of AgNPs showed a higher rate of cell proliferation after 120 h of incubation than the cells that grow on the Ti6Al4V reference layers (Figure 9A). In contrast, the same nanolayers induced a decrease in the level of proliferation of the MG-63 osteoblasts after 120 h of incubation (Figure 9B). However, it should be clearly emphasized that for the all tested samples, we have noticed an increase in the L929 fibroblast and MG-63 osteoblast proliferation over time, which is confirmed not only by the MTT assay results, but also through the analysis of the comparative SEM micrographs (compare the images in Figures 10 and 11). Importantly, the cells, especially the MG-63 osteoblasts, have almost overgrown the entire surface of the nanolayers enriched with AgNPs (Figure 11f,i,j). The high level of biocompatibility of the tested nanomaterials was also confirmed by the cellular behavior associated with the formation of filopodia by the fibroblasts, as well as the osteoblasts, between the cells (arrows in Figure 10j,k and Figure 11k, respectively). These actin-based cell protrusions also attached the cells to the coating's surface (arrows in Figures 10l and 11l, respectively) by penetration inside the porous nanolayer, functioning as anchorage points enhancing cell proliferation. Filopodia are regarded as one of the most important cellular sensors, collecting information on whether the surface is suitable for cell attachment and proliferation, cell-cell interacting and allowing for cell migration toward the destination [44]. Therefore, filopodia formation is evidence of the biocompatible properties of tested nanomaterials.

Although it is believed that silver has cytotoxicity to some cells at certain concentrations [45], it is well-known that eukaryotic cells exhibit a far bigger target for attacking silver ions than prokaryotic cells and that they show more structural and functional redundancy. Therefore, a higher silver ion concentration is required to achieve comparable toxic effects, relative to bacterial cells [46]. Similar to our results, Reference [47] demonstrated that Ag-decorated TiO2 nanotubes exhibited monotonically increasing trend in the fibroblasts' cell line proliferation and, at the same time, these specimens may cause a decrease in osteoblast proliferation [48]. Importantly, in our study, the viability of MG-63 osteoblasts cultured on the TNT5 samples enriched with nanosilver was 70% or more after 120 h of incubation in comparison to the Ti6Al4V references alloy (Table below Figure 9B). According to the ISO 10993-1:2018 standards [49] (Biological evaluation of medical devices: Part 1: evaluation and testing within a risk managemen<sup>t</sup> process), if the cell viability was reduced to <70% of the blank, it would have a cytotoxicity potential. Therefore, our results indicate reasonable biocompatibility of TNT5 enriched with all tested concentration of nanosilver. As we have described above, Ti6Al4V alloy foils enriched with all tested concentrations of silver nanograins induced a slight decrease in L929 fibroblast proliferation without affecting the proliferation of osteoblasts. However, these results also demonstrate reasonable biocompatibility of the tested nanomaterials because the viability of the cells after 120 h of incubation was 85% or more compared to the reference Ti6Al4V specimens (Table below the Figure 8A). Our results corresponding with the findings from the other authors who have shown that silver deposited titanium reduced fibroblasts proliferation by 20% in comparison with titanium control samples [50] or titanium samples coated with the silver alloys, which did not have a cytotoxic effect on osteoblast cells [51].

To summarize, our results from the MTT assay and analysis of comparative SEM micrographs, including an increase in the cell proliferation over time, filopodia formation and viability of cell higher than 70% for the all tested samples, clearly demonstrate the biocompatible properties of the tested nanomaterials that can be used in dentistry or maxillofacial surgery. This conclusion is based on the statement that a favorable cellular interaction with the biomaterial's surface is critical for the long-term success of the implants [52].

Implant-associated infections are one of the critical issues for dental and maxillofacial implantology and can result in serious complications, such as the need for complex revision procedures, as well as poor prognoses, patients suffering, and even death [53,54]. The infections associated with implants are caused mainly by bacterial colonization and biofilm formation on the surface of the implanted specimen, which affects the adjacent tissues [55]. It is known that the most effective way to prevent biofilm buildup on implants is to prohibit the initial bacterial adhesion because the biofilms are quite difficult to remove after formation. One of the approaches is to directly impregnate an implant device with antibiotics to prevent the initial adhesion of bacteria onto the implant surface [53,56]. Although these antibiotic-impregnated surfaces displayed significant therapeutic effects, the potential toxicity and increased microbial drug resistance through the slow-release doses have become increased risks in surgery [53,55]. Therefore, postoperative infection rates could be greatly reduced by improving the antimicrobial properties of the implant surface by its modifications with metal ions such as Ag and Zn [54,55,57]. Silver-containing coatings have attracted increasing attention due to the nontoxicity of the active Ag+ to human cells and its antimicrobial activity [53,58]. Thus, the surface-modification of titanium alloys with silver coating, which was also performed in the present study, is considered a strategy to prevent the development of peri-implant infections [54]. Based on the results obtained from ICP-MS, which showed a significant release of silver ions from all Ti6Al4V/AgNPs and Ti6Al4V/TNT5/AgNPs immersed into a PBS solution after 14 and 28 days, we presumed that such ions could be responsible for antimicrobial properties. Although superior microbiocidal activity was also observed for all the studied samples after a 24-h ion release time, the ICP-MS did not confirm the presence of silver ions in the case of nanotubular modified titanium alloy surfaces enriched with the smallest amount of silver (Figure 5). This might be due to the low content of released ions, which was not detectable. Thus, the limited sensitivity of ICP-MS is not without meaning. A high biocidal effect of Ti6Al4V/AgNPs and Ti6Al4V/TNT5/AgNPs was observed even at the lowest concentrations of Ag deposited on the surface of the tested alloys. We assert that the required Ag dose in the implants is typically low, which makes it possible to introduce Ag into biocompatible coatings [55]. Therefore, the incorporation of a sufficient amount of Ag to enhance the antibacterial ability of porous coatings could lead to the production of a surface that retains biocompatible and relatively long-term antibacterial activity. On the other hand, the optimization of the fabrication of Ti-Ag specimens by a decrease of the Ag amount on their surface might also improve the adhesion and proliferation of fibroblasts and osteoblasts, thus affecting the better integration of implants with human tissues.

We assert that the inhibitory effect of silver nanoparticles is mainly associated with silver ions present in nanoparticles, but it is not the sole mechanism of antimicrobial activity induced by nanosilver [59]. The major difference between the effectiveness of silver nanoparticles and silver ions against bacteria is that AgNPs act in nanomolar concentrations, while ions act in micromolar ranges [60]. Silver nanoparticles, due to their small size, can easily penetrate and disrupt the membranes of bacteria. Both silver species (nanoparticles and ions) may react with protein thiol groups (key respiratory enzymes) and/or phospholipids of the bacterial membrane [61–63]. This leads to an increase in the membrane permeability and may cause more pronounced effects such as the loss (by leakage) of cellular contents, including ions (mainly K+), proteins and reducing sugars and a decrease of the ATP level [60,64,65]. Silver species may also interact with nucleic acids, which may probably result in the impairment of DNA replication [59,60,66,67]. All of these effects may culminate in the loss of cell viability [60,68]. It is also suggested that silver ions generate free radicals inside cells, which are

involved in the antimicrobial activity of silver nanoparticles and released silver ions [69]. Some authors claimed that the thickness of the peptidoglycan layer of gram-positive bacteria might prevent the action of the silver ions as they found a higher inhibitory activity from the silver ion solution against *E. coli* than against *S. aureus* [70]. However, the microbiocidal activity of silver nanoparticles has been found against both Gram-positive (e.g., *Staphylococcus aureus*) or Gram-negative (e.g., *Escherichia coli*) and even yeasts [69,71,72], which is consistent with our results. The results of our study, where surface modified titanium alloys affected the inhibition of the growth of both Gram-positive and Gram-negative bacteria or fungi, are in good accordance with a previous report [58] where TiO2 nanotubes enriched with Ag demonstrated superior bactericidal properties against the planktonic bacteria. Similar findings were reported by Reference [54]. The authors showed the strong bactericidal activity that titanium specimens incorporated with silver against *Staphylococcus aureus*. Moreover, the number of bacteria decreased as the dosage of the incorporated Ag increased, suggesting that the antibacterial ability increased with the content of deposited Ag [54].
