3.3.3. Adhesion Properties

In Table 2 the results of adhesion tests of the Ti6Al4V/TNT20-60 and Ti6Al4V/TNH20-60 systems are presented.


**Table 2.** Results of nano scratch-tests of Ti6Al4V/TNH20-60 and Ti6Al4V/TNT20-60 systems.

The critical load was assumed as the maximum force at which the nanotubular or nanostructural layers were delaminated from titanium alloy substrate, and critical friction as the maximum friction force during layers' delamination. Scratch-tests showed lower values of critical load and critical friction for Ti6Al4V/TNH20-60 systems comparing to Ti6Al4V/TNT20-60 ones. It means that the adhesion of non-tubular coatings is lower than adequate tubular ones. And that additional drying of nanotubular samples before autoclaving increases the adhesion to titanium alloy substrate. The biggest difference in the critical force (delamination force) between Ti6Al4V/TNT20-60 and Ti6Al4V/TNH20-60 systems is visible for samples TNT30 and TNH30 and is equal 25%, while the smallest difference is visible for TNT60 and TNH60 (12%).

#### *3.4. Cell Proliferation Detected by MTT Assay*

Biointegration of Ti6Al4V/TNH20-60 and Ti6Al4V/TNT20-60 systems were evaluated based on the results of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assays made for the two different cell lines: murine L929 fibroblasts and human osteoblast-like MG 63 cells. The level of proliferation (assessed after 24-, 72- and 120 h) of the cells growing on the Ti6Al4V/TNH20-60 surface was compared to that observed for the cells cultured on the Ti6Al4V/TNT20-60 e.g., TNH20 *vs.* TNT20. As it can be seen in Figure 7, with an increase of incubation time more L929 fibroblasts (Figure 7A), as well as MG-63 osteoblasts (Figure 7B), proliferated on the surface of the all tested biomaterials (*p* < 0.001).

**Figure 7.** L929 murine fibroblasts (**A**) and human osteoblasts MG-63 (**B**) proliferation (after 24-, 72- and 120 h) on the surface of Ti6Al4V/TNH20-TNH60 and Ti6Al4V/TNT20-TNT60 samples, detected by MTT assay. The absorbance values are expressed as means ± SEM of five independent experiments. Asterisk and hash mark indicate significant differences between the cells incubated with the alloy references samples (Ti6Al4V) compared to the TNH and TNT specimens after 72 h (\* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001) or 120 h (# *p* < 0.05, ## *p* < 0.01, ### *p* < 0.001) of incubation time, respectively. Tables below the graphs A and B present the statistical differences in the proliferation of the cells, between TNH and TNT coatings produced by the electrochemical anodization of the Ti6Al4V foil at the same selected potential (\$ *p* < 0.05, \$\$ *p* < 0.01, \$\$\$ *p* < 0.001).

Analysis of these data revealed also that there were no differences in the cells proliferation after 24 h between the all tested samples and titanium alloy references sample (Ti6Al4V). In contrast, Ti6Al4V/TNH coatings, as well as Ti6Al4V/TNT nanolayers, provoked a significant increase in cells proliferation compared to the Ti6Al4V reference alloy. This phenomenon was the most noticeable after 120 h of incubation both in the culture of fibroblasts (with the exception of TNH60 sample) and osteoblasts (*p* < 0.001). In the tables below the graphs in Figure 7, the statistical differences in the cells

biointegration between TNH and TNT coatings produced by the electrochemical anodization of the Ti6Al4V foil at the same selected potential were presented. Analysis of these tables shows that there were no differences in cells proliferation measured after 24 h for both tested cells lines. In contrast, TNT nanolayers caused a greater increase in MG-63 osteoblasts proliferation than TNH coatings, which was noticed after 72 h as well as 120 h of incubation time (Table in Figure 7B). On the other hand, L929 fibroblasts cultured on the surface of TNT samples showed a higher rate of proliferation than the cells that grew on the TNH specimens only after 72 h of incubation time, except for the comparison of TNH40 and TNT40 layers, when the differences were also observed after 120 h (Table in Figure 7A). However, it should be clearly emphasized that all the tested TNH coatings showed a much higher level of biocompatibility than titanium alloy references sample (Ti6Al4V) and TNT coatings even higher than TNH ones.

#### *3.5. Cell Morphology Observed by Scanning Electron Microscopy*

SEM images micrographs present L929 murine fibroblasts (Figure 8) and MG-63 human osteoblasts (Figure 9) morphology, and proliferation. The cells showed of both figures were cultured on the surface of Ti6Al4V references sample, Ti6Al4V/TNH40, and Ti6Al4V/TNT40 nanocomposites. These data support the MTT results and clearly demonstrate the high biocompatibility properties of both types of tested nanomaterials, which are mainly related to the increase in cells proliferation level over time (compare micrographs (a–c), (d–f) and (g–i) in Figures 8 and 9, respectively).

**Figure 8.** *Cont.*

**Figure 8.** Scanning electron microscopy (SEM) images presenting proliferation of the murine L929 fibroblasts growing on the surface of the titanium alloy references sample (Ti6Al4V; (**<sup>a</sup>**–**<sup>c</sup>**) in comparison with Ti6Al4V/TNH40 sample; (**d**–**f**) and Ti6Al4V/TNT40 sample; (**g**–**i**). Arrows in the image (**j**) show the cells growing in layers on top of each other. Arrows in micrographs (**k**) and (**l**) indicate filopodia spread between cells or penetrating deep into the TNT40 nanolayers, respectively

**Figure 9.** Scanning electron microscopy (SEM) micrographs showing the human osteoblast-like MG-63 cell proliferation on the surface of the references sample (Ti6Al4V; (**<sup>a</sup>**–**<sup>c</sup>**) compared to Ti6Al4V/TNH40 sample; (**d**–**f**) and the Ti6Al4V/TNT40 sample; (**g**–**i**). Micrograph (**j**) presents the multilayer growth of cells. Arrows indicate numerous filopodia spreading between cells (**k**) or filopodia, which attached osteoblasts to the nanocoatings surface (**l**).

Importantly, as it can be seen in Figure 8j, L929 fibroblasts also start to grow in layers on top of each other and this phenomenon is much more noticeable during the MG-63 osteoblasts incubation (see, e.g., Figure 9j), when the entire surface of TNH40 as well as TNT40 coatings is overgrown with multilayer structure of growing cells after 120 h of incubation time (Figure 9f,i, respectively). Finally, SEM images show that L929 fibroblasts as well as MG-63 osteoblasts form numerous filopodia, which strongly attach the cells to the nanocoatings surface by penetrating deep into nanolayers (arrows in Figures 8l and 9l, respectively) or the cells generate filopodia between themselves (arrows in Figures 8k and 9k, respectively).
