Sodium bicarbonate

All of the in vitro analyses were performed in an experimental model using a commercially available monocyte/macrophage peripheral blood cell line—SC (ATCC CRL-9855) (ATCC; Manassas, VA, USA). Cell cultures were kept under standard conditions (37 °C; 5% pCO₂; 95% humidity) according to the guidelines provided by the manufacturer. Cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) with 4-mML-glutamine adjusted to contain 1.5 g/L sodium bicarbonate (ATCC; Manassas, VA, USA) and supplemented with 0.05-mM 2-mercaptoethanol (Sigma-Aldrich Corp., St. Louis, MO, USA), 0.1-mM hypoxanthine and 0.016-mM thymidine (90%) (ATCC; Manassas, VA, USA), fetal bovine serum (10%) (ATCC; Manassas, VA, USA) and 1% penicillin/streptomycin solution (P/S) (ScienCell Research Laboratories, San Diego ad, CA, USA). Each cell culture was split when it reached 90–95% confluency. All of the tested cements were mixed according to manufacturer’s instructions and then cured in sterile hemi-sphere molds, r = 3.75 mm (surface area 1.33 cm², volume of 140 μL). Immediately after reaching the setting time as provided by the manufacturer, the specimens were placed in Eppendorf tubes containing 1 mL of cell culture medium and were incubated for 24 and 48 h at 37 °C. The eluates were centrifugated for 5 min (2000 rpm) and then used for further analysis [33 (link)].

The materials were sterilized by UV light for 0.5 h from both sides. The samples were inserted into polystyrene 24-well cell culture plates (TPP, Trasadingen, Switzerland) and were seeded with endothelial cells originating from bovine pulmonary artery (line CPAE ATCC CCL-209, Rockville, MA, USA) in a minimum essential Eagle medium (E-MEM) supplemented with 2 mM l-glutamine, 1.0 mM sodium pyruvate, 0.1 mM non-essential aminoacids and 1.5 g/L sodium bicarbonate (all chemicals from Sigma). In order to avoid potential adsorption of serum-derived proteins and masking the oligopeptidic ligands, fetal bovine serum (Sebak GmbH, Aidenbach, Germany) was added to the medium 5 h after cell seeding to a final concentration of 20 % in each well [23 (link)]. Each well contained 30,000 cells and 1.5 ml of the medium. The cells were cultured in a humidified atmosphere containing 5 % CO₂ in the air.

All in vitro analyses were performed with a commercially available monocyte/macrophage peripheral blood cell line—SC (ATCC CRL-9855) (ATCC; Manassas, VA, USA). Cells were maintained under standard conditions (37 °C; 5% pCO₂; 95% humidity) according to the manufacturer’s guidelines. Cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) with 4-mM l-glutamine adjusted to contain 1.5 g/L sodium bicarbonate (ATCC; Manassas, VA, USA) and supplemented with 0.05-mM 2-mercaptoethanol (Sigma-Aldrich Corp., St. Louis, MO, USA), 0.1-mM hypoxanthine and 0.016-mM thymidine (90%) (ATCC; Manassas, VA, USA), fetal bovine serum (10%) (ATCC; Manassas, VA, USA) and 1% penicillin/streptomycin solution (P/S) (ScienCell Research Laboratories, San Diego ad, CA, USA). Cells were split every 2–3 days, when the cell culture reached 90–95% confluency. A total of 50 µL of each investigated UDA was placed in Eppendorf tubes and polymerized according to the manufacturer’s instructions (LED lamp intensity over 1000 mw/cm², The CURE-TC-01, Spring Health Products, PA, USA). Afterwards, 1 mL of cell culture medium was added and the Eppendorfs were incubated for 24 h at 37 °C. The eluates obtained after centrifugation for 5 min at the speed of 2000 rpm were used for further experiments.