**1. Introduction**

Silver as an antibacterial agen<sup>t</sup> was known and applied in antiquity [1–3], but its wide use in different fields of our contemporary life is a result of more and more detailed studies on the mechanisms of Ag antimicrobial activity [4–6]. Moreover, the development of modern technologies, which allow for the production of silver nanoparticles (AgNPs) of different sizes, shapes, and properties, is of grea<sup>t</sup> importance [7–9]. The bactericidal and fungicidal activity of AgNPs has not been fully explained yet, but the three most probable mechanisms are proposed [10–13]. The first of them assumes the capture of free silver ions, which interferes with ATP production and DNA replication. The second one assumes the generation of reactive oxygen species (ROS) by nanoparticles and silver ions. The produced ROS may also have an adverse effect on DNA, the cell membrane and the membrane proteins [14]. The third

mechanism takes into consideration the damage of the bacteria cell membrane as a result of its direct contact with AgNPs. In this case, silver nanoparticles joining the proteins of the cell membrane through the connection with sulfur cause a change in its structure [13,15–18]. Extensive use of silver nanoparticles, especially in medicine, forces us to think about the potential toxicity of silver to the human body. In some cases, nanoparticles may be toxic for human organisms and the prolonged use of specimens containing silver may cause argyria [19,20]. The potential risk of AgNPs lies in their ability to bioaccumulation in the body [21]. The harmful effects of nanosilver on human cells act according to similar mechanisms, which was mentioned earlier for bacteria. AgNPs accumulate outside the mitochondria, leading to ROS production, which causes mitochondrial damage and interruption of ATP synthesis. Moreover, the interaction of nanosilver with DNA leads to cell cycle stopping [22–25]. The analysis of previous reports exhibited that the potential toxicity of silver nanoparticles to the human body depends on their size and shape. According to these data, nanoparticles of a diameter smaller than 25 nm and a silver concentration higher than 60 mg/L are considered to be cytotoxic to mammalian cells. Simultaneously, it should be noted that a harmful effect of ionic silver on eukaryotic organisms was noticed at a concentration of 1 mg/L [26,27]. On the other hand, the increase of the AgNPs diameter above 25 nm caused these nanoparticles to be toxic mainly to microorganisms and not harmful to the human body [28,29]. Particles of such sizes are used in different technologies, in which the necessity of a bacteria-free environment exists. One of the interesting directions in the using of AgNPs is combining them with other materials, e.g., polymeric or ceramic ones, in order to form nanocomposites of unique physicochemical properties and bioactivity [30–32]. This approach is developed by us in the design and fabrication of new generation surgical titanium/titanium alloy implants, whose surface is enriched with AgNPs as the anti-inflammatory factor [33,34]. To avoid the inflammation effect after implantation, we enriched the titanium alloy implants' surface with dispersed silver nanoparticles of appropriate diameters using the chemical vapor deposition (CVD) or atomic layer deposition (ALD) techniques. The monitoring of the influence of silver nanoparticles on the adhesion and proliferation of fibroblasts on the implant surface revealed the very diverse biointegration properties of AgNPs-enriched surfaces [35,36]. It should be noted that in our work, two types of Ti6Al4V implants surface were studied, i.e., pure metallic implants and implants with a surface modified by the titania nanotube coating. Analysis of the results of our earlier works revealed that the different surface properties of the tested implants influence the different AgNPs deposition courses and, thereby, their different bioactivities.

Therefore, determining the direct impact of the deposited silver nanoparticles amount, the form in which they act and their size on their biointegration properties and antimicrobial activity is an important issue. Such an analysis is crucial for the production of implants with a surface, which will be microbiocidal enough but at the same time, optimally biointegral. For this purpose, the surfaces of Ti6Al4V and Ti6Al4V/TNT substrates were coated with different amounts of silver using the CVD technique. This technique enables for the deposition of pure silver nanoparticles on the surface of substrates with complex shapes [37]. We set ourselves the goal of enriching the surface of the titanium alloy implant (non-modified and nanotubular modified) with the smallest number of silver nanoparticles, which will have high antimicrobial properties, and which will not interfere with the adhesion and proliferation of fibroblasts and osteoblasts. We wanted, in this way, to create a biomaterial with the desired bioactivity (microbiocidal and biocompatible), minimizing the number of silver precursor used in the CVD process, thereby minimizing the number of silver nanoparticles on the surface of the layer and minimizing the potential silver toxicity. The results of our works are discussed in this paper.

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