Implant disks

Titanium disks made up with the same materials and surface characteristics as those with the original implants were obtained from the respective manufacturers. Two types of disks were used; the small disks represented 3M™ESPE™ MDI implants, while the large disks represented Ankylos®, Dentsply Friadent implants. A total of 10 disks of each brand were used for the study.

Cell culture and in vitro mineralization

Disks were sterilized and coated with 2% gelatin solution to facilitate the attachment of cells. MC3T3-E1 and C2C12 cells were purchased from ATCC (Manassas, VA, USA). Recombinant human bone morphogenetic protein 2 BMP-2 was purchased from GenScript (Piscataway, NJ, USA). MC3T3-E1 and C2C12 cells were cultured in alpha-MEM (Invitrogen, Carlsbad, CA, USA) and DMEM (Invitrogen, Carlsbad, CA, USA), respectively. Culture media were supplemented with 10% FBS (PAA, Etobocoke, Ontario, Canada) and 100 U/ml penicillin–streptomycin (Invitrogen, Carlsbad, CA, USA). Cells were grown at 37 °C under 5% CO2 in a humidified incubator. Mineralization of MC3T3-E1 cultures was induced by addition of ascorbic acid (50 µg/ml) and sodium phosphate (4 mM) to the culture medium for 12 days.

Calcein staining

Cells were fixed with 4% paraformaldehyde. 0.25% calcein (Sigma-Aldrich, Saint Louis, MO, USA), and 2% NaHCO₃ solution prepared in 0.15 M NaCl was added to the fixed cells and incubated for 5 min at room temperature. After washing once in PBS, H33258 nuclear staining was performed, washed twice in PBS and images were taken using an inverted fluorescence microscope (EVOS FL, Thermo Fisher Scientific).

Alamar blue

In order to examine cellular viability/metabolic activity, Alamar blue solution (Resazurin sodium salt, Sigma-Aldrich, Saint Louis, MO, USA) was directly added to the medium to 100 μM final concentration.