3.3.1. Comparison of the Effect of Nb and Mo on Titanium Biocompatibility

Figure 5 shows the number of cells on each gradient after three and seven days for the same donor (other donors generating the same trends). This allowed us to rapidly compare the effects of chemistry, roughness and hyperdeformation. Figure 5a shows that pure titanium and Ti6Al4V have the same trends on adhesion and proliferation after three and seven days. Ti6Al4V appeared to have a slightly more beneficial effect than pure titanium on adhesion. The number of cells on polished samples after seven days remained almost unchanged which confirms the results obtained by Johansson et al. [49]. The S + P samples for both materials had the same behavior as the polished ones. The Ti SMATed samples were unlike the Ti6Al4V SMATed samples which had a sharp decrease in the number of cells after seven days. Nevertheless, whatever the material, the SMAT had a positive effect on the adhesion because of the increase of the roughness, which is consistent with previous studies from the literature [46–48]. Figure 5b shows that the biocompatibility of these two materials does not seem to be affected by the presence of low amount of alloying elements (≤25%).

When the percentage of alloying element is increased (50 and 75%), the behavior of the cells was strongly modified. Niobium was found to have an excellent biocompatibility, which greatly increased the proliferation of the cells (Figure 5c). This effect of the chemistry was more important after seven days if the samples were SMATed. This is because of the increase in the number of adhering cells, which further increased subsequently after seven days, due to the cell division process in an excellent biocompatible environment. As shown in Figure 5d, the harmfulness of molybdenum began to have a very important effect from 75%. Even for the polished sample where the adhesion was best after three days, the cells then died quickly avec seven days. The toxicity of this alloy was even greatly increased by the microstructure modification induced by the SMAT. Indeed, on the samples, S and S + P, no cell survived after seven days. These results validated the differences in biocompatibility between Nb and Mo observed by Eisenbarth et al. [50].

In the case of pure elements, as no cells adhered or survived for all conditions of pure Mo, Figure 5e demonstrates the toxicity of pure Mo and the better biocompatibility of Nb.

All these results mainly demonstrated that the best biocompatibility was obtained for the SMATed TiNb alloys when the concentration in niobium was between 50 and 75%. In addition, it was clear that SMAT has a beneficial effect only during the adhesion stage by increasing the roughness and that the toxicity of molybdenum was increased by the microstructure modification induced by SMAT.

**Figure 5.** Comparison of the effect of Nb and Mo on cells adhesion and subsequent proliferation for different amount of alloying element. (**a**) 0%; (**b**) 25%; (**c**) 50%; (**d**) 75%; (**e**) 100%.

As the molybdenum has a poor biocompatibility compare to niobium, only the results on TiNb are showed in the rest of this paper.
