*3.4. Wettability*

Wettability plays an important role in biological performances of materials. Hydrophilicity of the titania-based coatings on M30NW biomedical alloy samples was evaluated by measuring their water contact angles Θ (Figure 7).

**Figure 7.** Contact angles Θ of the titania-based coatings on M30NW alloy samples depending on doped ions.

The TiO2 and Ca\_TiO2 coatings have similar Θ values of 49◦ and 46◦, respectively. The addition of silver ions causes an increase in the contact angle. As the content of silver ions in the coating increases, the value of the contact angle increases up to 61◦ for the highest Ag content. On the basis of wetting measurements, it was found that the surface free energy decreases with increasing concentration of silver ions in the coatings (Figure 8).

**Figure 8.** Surface free energy γ values (with distinction between dispersive and polar components) of the titania-based coatings doped with Ca and Ag ions in different molar ratios.

As it is known, the surface free energy (γ) is a sum of the dispersive (γLW) and acid-base (γAB) components (Equation (4)), which both determine this value.

$$
\gamma = \gamma^{LW} + \gamma^{AB}.\tag{4}
$$

Figure 8 gives the values of the γ, with distinction between polar (γAB) and dispersive (γLW) components. In our study, the surface free energy value is mainly influenced by the γAB component, since the value of the γLW component is similar for all surfaces and amounts ca. 40 mJ/m2. It can be clearly seen that both surface free energy γ and its polar component γAB decrease with increasing Ag concentration in the coatings. It is related to the formation of silver oxide particles on the surface of the coatings. Silver atoms present on the coatings' surface are exposed to the atmosphere and are free to bond with other atoms, especially with oxygen and water [46]. Therefore, as the concentration of silver in the coating increases, the number of Ag-O bonds increases, which, in consequence, changes the properties of surface.

It can be concluded that all investigated TiO2-based coatings are hydrophilic regardless of the type and molar ratio of the dopants. The higher surface wettability results in better adhesion and proliferation of the eukaryotic cells [47]. That can be beneficial for biological applications, especially for use in the circulatory system. According to the literature [48], the hydrophilic titania coating reduces adsorption of proteins and minimizes adherence of blood platelets on the surface.

#### *3.5. Biological Evaluation: Cell Viability and Proliferation Ability Assays*

Figure 9a presents the results obtained from the live/dead test and the determined amounts of cells viability after direct contact with the examined surfaces. The highest amount of live cells (i.e., above 98%) was observed for the sample 50Ca50Ag\_TiO2. The biggest percentage of dead cells was noted for the sample 25Ca75Ag\_TiO2 (~22% of all collected cells). Statistical significance on the level of *p* < 0.05 was noted between sample 25Ca75Ag\_TiO2 and TiO2, Ag\_TiO2, 75Ca25Ag\_TiO2 for live cells as well as for the dead ones. Nevertheless, none of the examined materials is cytotoxic. All the samples fulfill the requirements defined in the ISO 10993-5—the viability is above 70%. That states that prepared sol-gel coatings, regardless of doped element—Ca, Ag or their mixture—are biocompatible materials. Taking into consideration the average amount of cells, in the case of a cells' proliferation (Figure 9b), it was observed that the trend of obtained results is similar to the trend for results obtained for contact angle measurement. It can be stated also that cells' proliferation slightly increases with decreasing surface free energy—the highest is for Ag\_TiO2 sample, which has the lowest γ values. Calcium addition does not influence the cells, most proliferate onto the samples doped only with Ag (Ag\_TiO2) and their amount is higher than for uncoated basic sample. Nevertheless there are no statistically significant differences between all evaluated coatings. It can state that, in the case of doped sol-gel coatings, their biological response depends mainly on surface topography and wettability of the samples, and to a very small extent is the effect of the content of individual elements included in the coating.

**Figure 9.** The results of (**a**) the live/dead test and (**b**) the cells' proliferation evaluation for all the examined coatings after 48 h of direct contact (conducted according to the protocol of ISO 10993-5: Tests for Cytotoxicity—In Vitro Methods).

The biocompatibility is usually defined as "the ability of a material to perform with an appropriate host response in a specific application" [49]. Interactions between biological system and biomaterial surface run in the following order: In the first few nanoseconds, the water molecules and proteins reach the surface, being followed by the cells [50]. The interaction of proteins and cells with the surface is driven by the specific surface features: Surface chemistry, topography, roughness, wettability, and crystallinity. Cells can sense the chemistry and topography of the surface to which they adhere. Cell behavior is different on different nanosurfaces, because nanomorphology of the material may significantly influence protein and cell adhesion. In general, cells show good spreading, proliferation, and differentiation on hydrophilic surfaces. Nevertheless, the major factor determining the nature of the cells' interaction with biomaterials is the composition and conformation of the proteins adsorbed on the surface. The adhesion and behavior of cells are affected by adsorption of serum and extracellular matrix proteins [51]. Therefore, the observed difference in the proliferation and viability of osteoblast cells may be caused by the difference in the absorption of proteins responsible for the cell colonization process. The adsorption of proteins responsible for the cell colonization and their activity may be affected by one or more interactions between proteins and surfaces, including van der Waal's interactions, electrostatic interactions, hydrogen bonding, and hydrophobic interactions [52–56]. According to the literature, generally higher surface wettability results in better adhesion and proliferation of the eukaryotic cells [57]. Although, when the surface wettability is very high, water adsorbs preferentially on the surface [58] and thus can reduce adsorption of the proteins. It has been shown by the study of Xu et al. that surfaces with θ > ∼60◦–65◦ show stronger adhesion forces for proteins than the surfaces with θ < 60◦ [57]. Generally, hydrophobic surfaces are considered to be more protein adsorbent than hydrophilic surfaces, due to strong hydrophobic interactions occurring at these surfaces [59] in direct contrast to the repulsive solvation forces arising from strongly bound water at the hydrophilic surface [53]. As proteins determine the cell proliferation results, for the sample with the highest amount of Ag having the highest contact angle (above 61◦), the average number of proliferated cells is the highest—almost at the same level as a control. For the other doped samples, with the changing molar ratios of calcium ions Ca2+ and silver ions Ag+ (Ca/Ag 3:1, 1:1, 1:3) in TiO2 sol, contact angle is on the level of <sup>∼</sup>50◦–55◦. The surface wettability is on a very similar level for all doped TiO2 coatings, so the results obtained from the live/dead assay confirmed this dependence—osteoblast proliferation for all doubly-doped coatings is at the comparable level. However, it should be noted that the differences between proliferation results for all coatings are not significant and are in the range of experimental error. Therefore, it could be concluded that, in general, osteoblast cells growth is promoted on all coated surfaces, regardless of the increase of particular component elements as Ag and Ca, nor differences in nanotopography. Similarly, no morphological differences for the osteoblast-like cells were reported in the literature for the Ca-Ag coexisting nano-structured titania layer on Ti metal surface [34], as well as for Ag-Sr co-doped hydroxyapatite/TiO2 nanotube bilayer coatings [33]. These reports prove that incorporating a secondary bioactive compound (e.g., Ca or Sr ions) not only improves bioactivity of the coating, but it is also effective in lessening Ag cytotoxicity and optimally preserving its antibacterial properties.

According to T.T Liao el at. [60], less ordered phases in a coating results in a lower adsorption of proteins and cells on the surface, while increasing crystallinity of the coating improves cell colonization. For the coatings examined in this work, the XRD results showed that the crystallinity of the anatase in TiO2 coating increases with increasing amount of Ag and the highest was obtained for the Ag\_TiO2 sample. At the same time, for this sample, the highest value of Rq = 7.88 nm was observed. Surface topography plays an important role in providing three-dimensionality of cells [61]. For instance, the topography of the collagen fibers, with repeated 66 nm binding, has shown to affect cell shape [62]. Focal adhesion interacting with the surface is established by cell filopodia (which are 0.25–0.5 μm wide and 2–10 μm long) [63]. Filopodia can interact with the surface due to surface features, which are either arranged randomly or in some geometrical order and have dimensions from the micro to the nanometer range [61]. Beyond micrometers, it has been shown that nanometric (1–500 nm) features

can elicit specific cell responses [61,62]. In case of our experiment, as the surfaces of the coatings show different nanostructures, we noted that osteoblast cells react differently on the surface revealing dissimilar morphology (Figure 10). The results we go<sup>t</sup> are consistent with the observations made by S. Lee et al. [64]. In their results they noticed that as micropore size increases, cell number is reduced and cell differentiation and matrix production is increased. Their study demonstrated that the surface topography plays an important role for phenotypic expression of the MG63 osteoblast-like cells. In our case, we observed that cell shape and proliferation level are different as the coatings' topography in nanoscale is different. For most porous surfaces (Ca\_TiO2, 75Ca25Ag\_TiO2) we observed fewer cells and increased matrix production, although their number is still relatively high. For surfaces without pores, osteoblasts are more elongated (more natural morphology), although other factors decrease their number compared to control.

**Figure 10.** The osteoblast cells images (fluorescent stained, live cells—green colour, bar 200 μm) after 48 h of growing on samples (direct contact) modified by coatings with different composition and nanostructure.
