2.3.1. Cell Culture

Human osteoblast-like MG-63 cells (European Collection of Cell Cultures, Salisbury, UK) were plated in a 25 cm<sup>2</sup> cell culture flask (Corning, Corning, NY, USA) and culture with Eagle's Minimum Essential Medium containing 2 mM L-glutamine, 1 mM sodium pyruvate, Minimum Essential Medium (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). Cultures were maintained at 37 ◦C in a 95% humidified atmosphere of 5% CO2. 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) when reaching 70–80% of confluence.

Murine fibroblast cell line L929 (American Type Culture Collection, Manassas, VA, USA) were cultured at 37 ◦C in 5% CO2 and 95% humidity in a complete Roswell Park Memorial Institute (RPMI) 1640 medium containing 2 mM L-glutamine, heat-inactivated 10% FBS, 100 μg/mL streptomycin and 100 IU/mL penicillin. L929 cells culture conditions were the same as we have described previously [32].

The cells belong to both cell lines, in a volume of 1 mL of appropriate culture medium, were seeded onto the autoclaved tested specimens placed in a 24-well culture plate (Corning, Corning, NY, USA) at a density of 1 × 10<sup>4</sup> cells/well for 24 h, 72 h or 120 h, respectively. The cells incubated with the tested plates in the above incubation time were also analyzed for the observation of cell morphology. Cell morphology and proliferation was investigated for the Ti6Al4V/TNH samples as well as for Ti6Al4V/TNT specimens.

#### 2.3.2. Proliferation of L929 Fibroblasts and MG-63 Osteoblasts Detected by MTT Assay

The effect of the tested specimens on the cell proliferation (after 24-, 72- and 120 h, respectively) was studied by the MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide; Sigma Aldrich, Darmstadt, Germany) assay using the same method as it was reported in Lewandowska et al. [28]. Briefly, after the respective incubation time, the plates were washed with phosphate buffered saline (PBS) and transferred to a new 24-well culture plate. The MTT (5 mg/mL; Sigma-Aldrich, Darmstadt, Germany) solution in a culture medium without phenol red (RPMI 1640 medium for L929 fibroblasts or Eagle's Minimum Essential Medium for MG-63 osteoblasts; both from Sigma-Aldrich, Darmstadt, Germany) was added to each well. After 3 h of incubation at 37 ◦C in a humidified atmosphere of 5% CO2, the solution was aspirated, 500 μL of dimethyl sulfoxide (DMSO; 100% *v/v*; Sigma Aldrich, Darmstadt, Germany) was added to each well and 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 cells) were treated with the same procedures as the experimental groups. All measurements were done in duplicate in five independent experiments.
