Dentine disks

The functional resorption activity of the differentiated osteoclasts was evaluated by resorption pit formation, as described below. Cells were cultured for 30 days on top of dentine disks (Immunodiagnostic Systems, Boldon, UK) in 96-well culture plates in the presence of M-CSF (20 ng/mL), RANKL (50 ng/mL), or IL-7 (2 ng/mL) to differentiate osteoclasts. Then, the dentine slices were washed three times and immersed in 70% sodium hypochlorite to remove adherent cells. The resorption lacunae were counterstained with 1% (w/v) toluidine blue in 0.5% sodium borate for 60 s (Sigma-Aldrich). Photographs were taken through an LSM 5 PASCAL confocal microscope (Carl Zeiss, Jena, Germany) to analyze the surface topography and the area of the resorption pits was measured in four randomly selected areas for each dentine slice with the LSM 5 Image Browser (Carl Zeiss). Roughness parameters obtained were:roughness average (Ra), which is the main height calculated over the entire measured length or area, Rq, statistical moments of peak distribution (symmetry), Rz, mean roughness depth, and Rv, maximum profile valley depth, which are the distances from the mean line/surface to the highest/lowest point in the evaluation length/area (26 (link)).