Osteoblast differentiation and mineralization

On day 21, calcium mineralization was measured via alizarin red staining assay—this time period was chosen based on past published studies using melatonin to induce differentiation of hMSCs into osteoblasts [33]. Human PBMCs were added to osteoblastic cultures on day 13 because past studies using osteogenic medium have shown that pre-osteoblasts proliferate and start to differentiate into mature osteoblasts between 14 to 21 days [33]; and RANKL, M-CSF and/or OPG levels would be high enough to modulate osteoclastogenesis [41, 90]. Alizarin red staining was performed on the bottom chamber of the transwell co-culture and directly on 6-well plates (both hMSCs and hPBMCs) of the layered co-culture using the commercially available osteogenesis quantification kit (EMD Millipore, MA, USA) per manufacturer's instructions. Microscopic evaluation of osteogenesis was performed using a Vistavision microscope (VWR International) with a progress C3 camera (Zenoptik; Data not shown). Osteogenic differen-tiation was quantified by spectrophotometry at 405 nm using the Perkin Elmer Victor 1420 Multilabel plate reader (Waltham, MA, USA). Concentrations of alizarin red reflecting osteoblast mineralization activity were calculated, normalized against Os-/Veh and compared between groups.