Figure 6.

The effects of heparin on osteoclastogenesis induced by MLO-Y4 conditioned medium. (A) MLO-Y4 conditioned medium preparation flowchart. MLO-Y4 cells were pre-treated with heparin for 6 h, then cultured in fresh medium. The supernatant were then collected after 2 h (2h Hpt-Y4) and 24 h (24h Hpt-Y4). The preparation process of Hin-Y4 conditioned medium were displayed on the right flow line. MLO-Y4 cells were cultured for 48 h and the supernatant were collected as “empty Y4 supernatant”. Empty Y4 supernatant was then incubated with various concentrations of heparin to obtain heparin-incubated MLO-Y4-conditioned medium (Hin-Y4). Each conditioned medium was added with recombinant RANKL to make sure the osteoclast can form; (B,C) RAW264.7 cells were cultured in 2h Hpt-Y4 and 24h Hpt-Y4, and the osteoclast number were counted eight days later. (n = 3). No significance among each groups (p > 0.05); (D) RAW 264.7 were cultured in Hin-Y4 for eight days, and osteoclasts in each group were counted. (n = 3). osteoclastogenesis induced by 5 ng/mL recombinant RANKL was served as a control. MNC: multinuclear cell; TRAP: tartrate-resistant acid phosphatase; RANKL: receptor activator of NFκB ligand; OPG: osteoprotegerin; each of the above values is expressed as the mean ± S.D. * p < 0.05 represent significant differences from the relevant group 0.