*2.3. Cytotoxicity*

Human osteoblast-like MG-63 cell lines (ATCC: CRL-1427) were cultured in DMEM (Dulbecco's Modified Eagle's Medium) medium (Immuniq, Zory, Poland) without phenol red with the addition of ˙ 10% fetal bovine serum (FBS) and 1% streptomycin/penicillin (Gibco-BRL, Life Technologies, Karlsruhe, Germany) in standard cell conditions (37 ◦C, 5% CO2). Cytotoxicity assay was conducted for both pristine polyethylene and modified samples. The samples were sterilized overnight in ethanol vapor. Then, the samples were placed in a sterile 24-well culture plate and a cell suspension with a density of 50 × 10<sup>4</sup> cells/0.2 mL was added. Cells without the tested samples were treated as a reference sample. Treated and untreated cells were incubated for 72 h. Every 24 h, the medium was changed

for a fresh one. After that time, cytotoxicity was determined by Alamar Blue assay (Sigma-Aldrich, Bornem, Belgium) according to the well-described protocol in [24]. In brief, cells were washed with phosphate-buffered saline (PBS) and incubated with resazurin sodium salt solution (25 μM in PBS) for 4 h at 37 ◦C in the dark. The fluorescence caused by the cellular metabolic activity was measured at 605 nm (excitation wavelength 560 nm) with a multimode microplate reader (Infinite 200M PRO NanoQuant, Tecan, Männedorf, Switzerland). Cytotoxicity was expressed as a percentage of viable cells after treatment with coated polyethylene samples in reference to untreated cells (control). The experiment was repeated three times. Cell morphology was examined using a fluorescence microscope (Olympus IX51, Olympus, Tokyo, Japan) with an excitation filter of 470/20 nm. After staining with acridine orange (viable cells) and propidium iodide (dead cells), cells were observed for morphological changes. At least five viewing fields containing ca. 100 cells each were analyzed. Photographs of CT26 cells after treatment with the tested alloys were taken using an inverted microscope equipped with a reflected fluorescence system (Olympus IX51, Olympus, Tokyo, Japan).
