2.3.1. Cell Culture

Human osteoblast-like MG 63 cells (European Collection of Cell Cultures, Salisbury, UK, cat. no. 86051601) were cultured at 310 K in 5% CO2 and 95% humidity in Eagle's minimum essential medium (EMEM) containing 2 mM L-glutamine, 1 mM sodium pyruvate, MEM non-essential amino acid, heat-inactivated 10% fetal bovine serum (FBS), 100 μg/mL streptomycin, and 100 IU/mL penicillin (all compounds from Sigma-Aldrich, Darmstadt, Germany). The culture medium was changed every 2–3 days. The cells were passaged using 0.25% trypsin- ethylenediaminetetraacetic acid (EDTA) solution (Sigma-Aldrich Darmstadt, Germany). The murine macrophage cell line RAW 264.7 was obtained from European Collection of Cell Cultures (Salisbury, UK, cat. no. 91062702). The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% Fetal Bovine *Serum* (FBS), 100 μg/mL streptomycin, and 100 IU/mL penicillin (all compounds from Sigma-Aldrich). Macrophages were maintained at 310 K in a 5% CO2/95% humidified atmosphere, subjected to no more than 15 cell passages and utilized for experimentation at approximately 70%–80% confluency. L929 murine fibroblast cells (American Type Culture Collection, Manassas, VA, USA) were cultured at 310 K in a humidified atmosphere with 5% CO2. The culture medium consisted of RPMI 1640 medium containing 2 mM l-glutamine (Sigma-Aldrich, Darmstadt, Germany), 10% heat-inactivated fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 μg/mL streptomycin (PAA Laboratories GmbH, Cölbe, Germany). L929 cells were passaged using a cell scraper.

#### 2.3.2. Cell Proliferation Assays

The effect of the tested specimens on the cell proliferation (measured after 24, 72, and 120 h) was studied using the MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide; Sigma Aldrich, Darmstadt, Germany) assay. MG-63 osteoblasts and L929 fibroblasts were seeded onto the autoclaved tested nanolayers placed in a 24-well culture plate (Corning, NY, USA) at a density of 1 x 10<sup>4</sup> cells/well and cultured for 24, 72, and 120 h. RAW 264.7 macrophages were seeded onto the substrates at a density of 25 × 10<sup>4</sup> cells/well and cultivated for 24 and 48 h. Moreover, the proliferation rate of the RAW 264.7 cell line was assessed for the cells stimulated with lipopolysaccharide (LPS; derived from *Escherichia coli*; 0111:B4, Sigma Chemicals, St. Louis, MO, USA) at a dose of 10 ng/mL, which was added to the cell growth medium to create the pro-inflammatory environment. The control cells were incubated on the test samples without the presence of LPS. After the respective incubation time, the substrates were rinsed with phosphate-buffered saline (PBS, pH 7.4; 1 × working concentration, contains 155.2 mM NaCl, 2.97 mM Na2HPO4 × 7H20 and 1.06 mM KH2PO4) and transferred to a new 24-well culture plate. The MTT (5 mg/mL; Sigma-Aldrich) solution in a respective culture medium without phenol red was added to each well and the plates were incubated for 3 h. Then, the MTT solution was aspirated and 500 μL of dimethyl sulfoxide (DMSO; 100% *v*/*v*; Sigma Aldrich, Darmstadt, Germany) was added to each well. Finally, the plates were shaken for 10 min. The absorbance was measured at the wavelength of 570 nm with the subtraction of the 630 nm background, using a microplate reader (Synergy HT; BioTek, Winooski, VT, USA). The blank groups (the plates incubated without the cells) were treated with the same procedures as the experimental groups. All measurements were done in duplicate in five independent experiments.

#### 2.3.3. MG-63 Osteoblasts Morphology Observed by SEM

The analysis of the morphology changes and number of MG-63 osteoblasts growing on the surface of TNT coatings and Ti6Al4V orthopedic implants, which were produced using selective laser sintering 3D technology, was performed using scanning electron microscopy (SEM; Quanta 3D FEG; Carl Zeiss, Göttingen, Germany). In the case of the TNT coatings, the cells were seeded onto the specimens placed in the 24-well plate at a density of 1 × 10<sup>4</sup> cells/well, whereas the osteoblasts growing on the surface of the Ti6Al4V orthopedic implant placed in the 6-well plates were seeded at a density of 1 × 10<sup>4</sup> cells/cm2. After the selected incubation time, the nanolayers were rinsed with PBS to remove non-adherent cells and fixed in 2.5% *v*/*v* glutaraldehyde (Sigma Aldrich, Darmstadt, Germany) for a minimum of 4 h (maximum 1 week). Then, the samples were washed again with PBS and dehydrated in a graded series of ethanol concentration (50%, 75%, 90%, and 100%) for 10 min. Finally, the specimens were dried in vacuum-assisted desiccators overnight and stored at room temperature until the SEM analysis was performed.

#### 2.3.4. Alkaline Phosphatase Activity Assay

MG-63 osteoblasts were seeded onto the tested nanolayers placed in a 24-well culture plate at a density of 1 × 10<sup>4</sup> cells/well and cultured for 24, 72, and 120 h. Then, the samples were washed with PBS and lysed in 0.2% (*v*/*v*) Triton X-100 (Sigma Aldrich, Darmstadt, Germany), with the lysate centrifuged at 14.000× *g* for 5 min. The clear supernatants were used to measure the alkaline phosphatase (ALP) activity, which was determined using the ALP assay kit from Abcam (London, UK, cat. no. ab83369) according to the manufacturer's instructions. The intracellular total nuclear protein concentration in the final supernatants was determined using the Pierce ™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) and the ALP activity was normalized to it.

#### 2.3.5. ELISA Quantification of Cytokines and Nitric Oxide

Murine macrophage cell line RAW 264.7 were seeded in triplicate onto the tested specimens placed in 24-well tissue culture plates (Corning, NY, USA) at a density of 25 × 10<sup>4</sup> cells/well and cultured for 24 and 48 h. The pro-inflammatory environment was created by adding 10 ng/mL of LPS to the cell growth media. The control cells were incubated on the tested substrates without the presence of LPS. Protein levels of the pro-inflammatory cytokines, interleukin (IL) 1β, IL-6, and tumor necrosis factor (TNF) α; anti-inflammatory cytokine, IL-10; and total nitric oxide, secreted into the cell culture media were measured with sandwich enzyme-linked immunosorbent assays (ELISA) kits from R & D Systems (Minneapolis, MN, USA; cat. no. MLB00C, M6000B, MTA00B, M1000B and KGE001, respectively), according to the manufacturer's instructions. Colorimetric changes in the assays were detected using a Synergy HT Multi-Mode Microplate Reader. The sensitivity of the 1β, IL-6, TNFα, IL-10, and total NO (nitric oxide) kits were less than 4.8, 1.8, 7.21, 5.22, and 0.78 μmol/L, respectively. To eliminate variation due to di fferences in the cell density among the samples, the cytokines and NO production were normalized to a number of 10<sup>5</sup> cells.
