*2.5. Wettability*

Measurements of contact angle of M30NW alloy samples modified with TiO2-based coatings were carried out using the DSA25 Drop Shape Analyzer goniometer- (Krüss GmbH, Hamburg, Germany). Each time, the measurement was performed for a minimum of three drops of water at an ambient temperature of approximately 20 ± 2 ◦C. The amount of deionized water drops applied by microsyringe was 5 μL. The values of the contact angle were determined using Advance software.

The surface free energy values were calculated based on two polar liquids-water and glycerine, and one apolar liquid-diiodomethane. Calculations were performed using van Oss Chaudhury–Good method.

#### *2.6. Biological Evaluation (Cell Viability Assays)*

Before biological evaluation, all samples were cleaned in ethanol and ultrapure water (0.055 μS/cm) for 10 min using ultrasonic cleaner. Then the steam sterilization was performed (121 ◦C, 31 min) using an autoclave. In order to conduct the biocompatibility assessment of the samples, the cell viability and proliferation assays were conducted. The human osteoblast cell line Saos-2 (ATCC, Manassas, VA, USA) was selected as a biological material for this purpose. Saos-2 cells were grown in McCoy's 5A medium (ATCC, Manassas, VA, USA) containing 15% fetal bovine serum (Biological Industries), 100 units/mL penicillin and 50 μg/mL streptomycin (Biological Industries). Cells were cultured in standard conditions (37 ◦C, humidified atmosphere of 5% CO2 in air) and medium was replaced every 2–3 days (75% confluence). Cells were used between passages 5 and 8.

For the evaluation of proliferation and cytotoxicity marking method, a "live/dead" test (Viability/Cytotoxicity Kit, Molecular Probes) was applied. Cells were seeded at 6 × 10<sup>4</sup> cells/mL/well/sample in 2 mL of McCoy's 5A medium (ATCC, Manassas, VA, USA) and cultured for 48 h. After that time, a mixture of two fluorescent dyes was used. One of the fluorescent dyes within the live cells produces an intense uniform green fluorescence and the second one, when the membranes are damaged, penetrates the cells and binds to nucleic acids, thereby producing a bright red fluorescence in dead cells. The samples were examined in a fluorescence microscope Olympus GX 71 equipped with a digital camera (DP70).

The obtained results were analyzed by one-way ANOVA analysis with a significance level of *p* < 0.05. Statistical analysis was performed using OriginPro 9 software.

#### **3. Results and Discussion**
