*3.3. Immunological Assessment*

#### 3.3.1. Cell Proliferation Detected by the MTT Assay

The proliferation of MG-63 osteoblasts, L929 fibroblasts, and RAW 264.7 macrophages on the surface of the tested specimens was evaluated with the MTT assay (Figure 2A). MG-63 cells proliferated on all tested specimens, except for the TNT5/Ag samples. It was also noted that only TNT5 specimens promoted proliferation when referring to the reference samples (Ti6Al4V foil) after 72 and 120 h. On the other hand, the slowest cell proliferation was observed on TNT5 coatings enriched with silver nanoparticles. TNT15 specimens both with and without silver nanograins inhibited cell proliferation compared with the reference samples. Among all the investigated coatings, only TNT5 increased L929 fibroblast proliferation after 24, 72, and 120 h (Figure 2B). Moreover, TNT15 nanolayers also induced L929 cell proliferation after 120 h. Importantly, in contrast to MG-63 osteoblasts, with an increase in the incubation time, more L929 cells proliferated on all tested specimens, and none of the tested coatings caused a decrease in the level of L929 cell proliferation. The RAW 264.7 cell proliferation results after 24 and 48 h of incubation are plotted in Figure 2C. Macrophages were cultured in the pro-inflammatory environment created by adding LPS to the cell growth media or in the absence of LPS. As can be seen, with an increase of the incubation period, more cells proliferated on all tested substrates. Importantly, LPS did not affect the level of cell proliferation. After 24 h, macrophages that grew on Ti6Al4V/Ag, TNT5, and TNT5/Ag showed a greater proliferation rate than cells growing on Ti6Al4V reference alloys. After 48 h, all modified implant surfaces showed an increased proliferation rate apart from Ti6Al4V/Ag.

**Figure 2.** Proliferation of human osteoblast-like MG 63 cells (**A**), L929 murine fibroblast cells (**B**), and murine macrophage cell line RAW 264.7 (**C**) on the surface of TiO2 nanotube coatings analyzed by the MTT assay (a colorimetric assay for assessing cell metabolic activity). MG-63 osteoblasts and L929 fibroblasts were cultured on the specimens for 24, 72, and 120 h, whereas RAW 264.7 macrophages were cultivated for 24 and 48 h in the presence or absence of LPS (Lipopolysaccharide). The absorbance values are expressed as means ±SEM of five independent experiments. Asterisks indicate significant differences comparing to the reference Ti6Al4V alloy foils (Ti6Al4V) (\*\*\* *p* < 0.001, \* *p* < 0.05). Hash marks denote significant differences when the level of cell proliferation was lower in comparison with the reference Ti6Al4V alloy foils (### *p* < 0.001, ## *p* < 0.01, # *p* <0.05).

3.3.2. Morphology and Proliferation Rate of MG-63 Osteoblasts Observed by Scanning Electron Microscopy

Biointegration of the TiO2 nanotube coating was also evaluated with SEM micrographs. Comparative SEM images show the morphology and proliferation level of the MG-63 osteoblasts in Figure 3. These data support the MTT results and clearly demonstrate that the highest biocompatibility was observed for TNT5 samples, which is mainly related to the increase in the cell proliferation level over time (compare the micrographs presented in Figure 3c,i,o). Importantly, as can be seen in Figure 3o, MG-63 osteoblasts started to grow in layers on top of each other, which was observed after 120 h of incubation. This phenomenon was not noticed for the TNT5 samples enriched with silver nanoparticles (Figure 3p). SEM images also showed that MG-63 osteoblasts have an elongated shape and form numerous filopodia, which strongly attach the cells to the nanocoatings' surface (arrows in Figure 3g–r). These thin actin-rich plasma membrane protrusions were also generated between the cells (arrows in Figure 3l). Finally, SEM micrographs were also used to evaluate the biointegration level of Ti6Al4V orthopedic implants, which were produced using selective laser sintering 3D technology. As can be seen in Figure 3m,n, MG-63 osteoblasts effectively attached to the implant's surface. Moreover, with an increase of the incubation time, the number of cells and their density increased.

**Figure 3.** Scanning electron microscopy (SEM) images showing human osteoblast-like MG-63 cells that grow on the surface of the tested titania coatings and the reference Ti6Al4V alloy foils enriched or not with silver nanograins. Micrographs (**<sup>m</sup>**,**<sup>n</sup>**) present the cells grown on the surface of Ti6Al4V orthopedic implants, which were produced using selective laser sintering 3D technology. Arrows in image (**l**) indicate filopodia spread between cells and those in image (**q**,**<sup>r</sup>**) present filopodia penetrating deep into the samples and attaching the cells to the surface. The type of sample, cell incubation time, and scale of the images are shown in the figures as indicated.

#### 3.3.3. Alkaline Phosphatase Activity of MG-63 Cells

Osteoblastic cell differentiation was assessed by measuring ALP activity, normalized to the total protein content after 24, 72, and 120 h of culture. Figure 4 shows the comparison of the ALP activity of MG-63 cells cultured on the tested specimens with reference Ti6Al4V alloy foils. The ALP activity of MG-63 cells grown on the all tested specimens increased over time. However, MG-63 cells cultured on the substrates enriched with silver nanograins had significantly lower ALP activity than those cultured on the reference Ti6Al4V alloy foils at the respective incubation time. In contrast, among all of the tested samples, only the TNT5 specimens induced higher ALP activity in comparison with the reference Ti6Al4V samples at a given incubation time. This phenomenon was also observed for TNT15 substrates but only after 24 h of culture.

**Figure 4.** Alkaline phosphatase activity (ALP) of MG-63 osteoblasts growing on TiO2 nanotube coatings produced by electrochemical anodic oxidation at potentials of 5 (TNT5) or 15 V (TNT15) and enriched with silver nanoparticles in comparison with the reference Ti6Al4V alloy foils and enriched or not with silver nanograins. The cells were cultured on the surface of the tested specimens for 24, 72, and 120 h. ALP activity [units] was calculated per μg of protein and it is expressed as the means ± SEM of five independent experiments. Asterisks indicate significant differences at the appropriate incubation time when the ALP activity of the cells growing on the tested specimens was higher compared to the reference Ti6Al4V alloy foils (Ti6Al4V) (\*\*\* *p* < 0.001, \* *p* < 0.05). Hash marks denote significant differences at the appropriate incubation time when the ALP activity of osteoblasts cultivated on the tested samples was lower in comparison with the reference Ti6Al4V alloy foils (### *p* < 0.001, ## *p* < 0.01, # *p* < 0.05).

#### 3.3.4. Secretion of Cytokines and Nitric Oxide by RAW 264.7 Macrophages

The time-course of the protein release of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α), anti-inflammatory cytokines (IL-10), and NO (nitric oxide) was assessed in 24 to 48 h of incubation by performing ELISA assays. Data show that RAW 264.7 macrophages stimulated with LPS released higher amounts of cytokines and NO over time for all tested substrates (Figure 5). However, TiO2 nanotube coatings produced by electrochemical anodic oxidation at potentials of 5 (TNT5) and 15 V (TNT15), enriched or not with silver nanoparticles, displayed a different production of cytokines and NO. Generally, the TNT5 and TNT5/Ag samples inhibited the LPS-induced release of pro-inflammatory cytokines and NO in comparison with the reference Ti6Al4V alloy foils, whereas TNT15 and TNT15/Ag specimens enhanced the production of IL-1β, IL-6, TNF-<sup>α</sup>, and NO. Moreover, cells that grew on the surface of TNT15 substrates released significant amounts of these cytokines and NO without LPS stimulation (Figure 5E). In contrast, in the absence of LPS, the amounts of IL-1β and IL-6 measured from cells cultured on TNT5, TNT5/Ag, and Ti6Al4V/Ag specimens were below the assay detection limits, at both analyzed time points. Importantly, the presence of silver nanoparticles on the surface of all tested coatings (Ti6Al4V, TNT5, and TNT15) inhibited pro-inflammatory cytokine production in comparison with the same respective layers not enriched with silver nanograins. The level of anti-inflammatory cytokine (IL-10) was also measured. As can be seen in Figure 5D, the biggest amount of IL-10 was released by the LPS-stimulated cells growing on the surface of TNT5 and TNT5/Ag samples. On the other hand, the levels of IL-10 from cells growing on the TNT15 and TNT15/Ag specimens were lower in comparison with the reference Ti6Al4V alloy foils.

**Figure 5.** Secretion of pro-inflammatory (**A**–**C**) and anti-inflammatory (**D**) cytokines or total nitric oxide (**E**) by RAW 264.7 macrophages in the standard and LPS-stimulated conditions. The cells were cultured on the tested specimens for 24 and 48 h. Cytokine and nitric oxide (NO) production was normalized to a number of 105 cells. Data are expressed as mean ± SE (*n* = 3). Asterisks indicate significant differences at the appropriate incubation time when the amounts of cytokines and NO produced by the cells growing on the tested specimens were higher in comparison with the reference Ti6Al4V alloy foils (Ti6Al4V) (\*\*\* *p* < 0.001, \*\* *p* < 0.01, \* *p* < 0.05). Hash marks denote significant differences at the appropriate incubation time when the levels of cytokines and NO secreted by the cells cultivated on the tested samples were lower in comparison with the reference Ti6Al4V alloy foils (### *p* < 0.001, ## *p* < 0.01, # *p* < 0.05).
