**1. Introduction**

Titanium and its alloys (Ti6Al4V, Ti5Al25Fe, and Ti6Al7Nb) are commonly used as biomaterials for orthopedic, dental, or neurosurgery applications [1–3]. This choice is determined by the beneficial properties of titanium, which is characterized by the excellent corrosion resistance, light weight, high strength, chemical stability, and modulus of elasticity much closer to that one of bone, compared to other metals. Unfortunately, its surface does not form a direct connection with the living bone [3–5]. And the most significant factor influencing the efficiency of implantology is the interaction between the body cells and the implanted artificial biomaterial [4–6]. In a wide range of clinical applications, implant rejections still occur mainly due to osseointegration defects and infection [7,8]. Many techniques are used to modify the surface topography of implants in order to improve cell adhesion and

osteogenic differentiation on them. It has been concluded that the surface roughness is conducive to the osseointegration process, influences the adhesion of osteoblasts, increases their enzymatic activity and determines the amount and type of proteins they synthesize [6,7,9–11]. The appropriate porosity of the implant is ensured, by scaffolds manufactured on its surface, which are characterized by the specific structure, morphology, and wettability [12,13]. According to previous reports, the fabrication of the titanium dioxide nanotubes coating (TNT) on the surface of titanium or titanium alloys implants, improves the adhesion and proliferation of osteoblast cells, and promotes the faster growth of bone and vascular tissue [14,15]. TNT coatings on the surfaces of medical devices, fabricated of titanium or titanium alloys, are commonly produced by their electrochemical anodization [5,7,16–20]. The main advantages of this technique are: the mild processing conditions, low costs, possibilities of large-scale production, and, which is the most important, very good control of structural and morphological properties [16,21]. By changing the type and composition of the electrolyte solution, the anodizing voltage, the temperature and duration of anodizing, it is possible to control the diameter and length of the nanotubes [17,21]. For medical applications, it is particularly important to control the diameters of the produced tubes, as their size influences on the coating biocompatibility degree. Considering earlier reports it should be noted that TNT coatings, which consist of nanotubes with different tube sizes (30–100 nm) could enhance osteoblast cell functions [22,23]. Park et al. sugges<sup>t</sup> something opposite - that nanotubes of smaller sizes than 15 nm strongly enhance cell activities and cell functions deteriorate with increasing tube sizes [24]. Results of our works confirm this suggestion. However, this applies to TNT coatings consisted of nanotubes with 25–35 diameters [25]. Zaho et al. noted that the observed divergences might be caused by the used sterilization method and its possible influence on morphology, structure and the biocompatibility of titania nanotubes [26]. Among the sterilization procedures, which are commonly applied in the clinical practice, i.e., gamma radiation, plasma sterilization, ethylene oxide (EO) sterilization, autoclaving, ultraviolent (UV) irradiation, and ethanol immersion [26–30]—autoclaving is the one, we have used in our research. In this method, the medical device's surface is treated by the hot steam under increased pressure. In the case of implants coated with bioactive TNT layer, such treatment may lead to changes in their surface morphology, structure, mechanical properties, and biocompatibility.

In our works, we have focused on studies on autoclaving procedure influence on the surface architecture rearrangement, mechanical properties and biointegration of nanotubular systems produced via anodic oxidation of titanium alloy at different potentials (5–60 V). Despite intense works on the application of TNT coatings in clinical practice as well as the wide use of autoclaving as the main sterilization procedure, above-mentioned issues have not been sufficiently investigated. Therefore, the results discussed in this paper can be important both for the design and fabrication of Ti6Al4V implants coated with bioactive coating and also for their practical use for clinicians.
