Rrankl

Bone marrow cells were recovered by flushing Dulbecco’s modified Eagle medium (DMEM) (Invitrogen) supplemented with 0.0001% of gentamycin (Invitrogen) and 0.002% Fungizone amphotericin B (Invitrogen) into the femur and tibia marrow space of BALB/c mice [15 (link)]. The culture was washed twice with DMEM (Invitrogen) and separated from blood cells and lymphocytes by Histopac 1083 (Sigma). The culture was washed twice in alpha-MEM medium (Sigma) supplemented as above. Cells (2.5×10⁶ cells.mL⁻¹) were cultured with 100 ng.mL⁻¹ of rRANKL (Peprotech), 10 ng.mL⁻¹ of rM-CSF (Peprotech) and capillaries with or without MTA, per 6 days. Half of the medium was changed on day 3 by replacing with half of a new medium and new rM-CSF (Peprotech) and rRANKL (Peprotech).

RAW cells were cultured as described above with 100 ng.mL⁻¹ of rRANKL (Peprotech) in a plate coated with type I rat tail collagen (BD Biosciences Bedford, MA, USA) and 0.02 N acetic acid (Fisher, Trenton, NJ, USA) until osteoclasts were differentiated. Cells were harvested with trypsin-EDTA (Invitrogen) and the cellular concentration adjusted to 4×10⁵ cells/well. Cells were applied onto dentin slices (Immunodiagnostic systems, Boldon, UK), with 100 ng.mL⁻¹ of rRANKL (Peprotech) and capillaries with or without MTA. After osteoclast differentiation, dentin slices were stained with toluidine blue 1% (Fisher). Four pictures were taken from each group, and the reabsorbed pit area was counted in these pictures using the NIH image software analyzer at a 100-fold magnification.

Non-confluent culture of RAW 264.7 osteoclast precursor (RAW) cell line (from ATCC) was harvested and cultured with minimum essential medium eagle alpha modification (alpha-MEM) (Sigma, St. Louis, MO, USA) supplemented with 2.2 G.L⁻¹ NaHCO₃ (Sigma), 15% fetal calf serum (Altana, Lawrenceville, GA, USA), 1% penicillin/streptomycin (1000 U/mL) (Invitrogen, Grand Island, NY, USA), 1% MEM amino acids solution (Invitrogen), 1% L-glutamine (Invitrogen) and 0.1% gentamycin (Invitrogen). Cells (2×10⁵) were cultured in a 24-well plate (Corning, New York, NY, USA) with capillaries with or without MTA, for 24 hours for cell viability assay. Another culture was performed, including 2-capillaries/well with or without MTA, for 24 hours. In order to observe osteoclast differentiation, 100ng.mL⁻¹ of recombinant (r) RANKL (Peprotech, Rock Hill, NJ, USA) was added to this culture, and every three days, half of medium and rRANKL (Peprotech) were changed, and the cultures were harvested on days 5, 6 and 8. RNA extractions were performed after 48h of cell incubation. The evaluation of the effect of aluminum and calcium on osteoclastogenesis was performed through RAW cells incubation with or without 40μg.mL⁻¹ of aluminum oxide, 99.99% (Sigma) and calcium oxide Reagent Plus, 99.99% (Sigma), with/without rRANKL for 7 days.