Fig. 2. Effects of AGEs on osteoclast and osteoblast differentiation. (A) TRAP-positive MNCs in RANKL-induced marrow macrophage cell cultures. Bone marrow mononuclear cells were incubated for three days with 30 ng/mL M-CSF, 70 ng/mL RANKL, 25 mM glucose, 25 mM mannitol, and 1, 10, or 100 ug/mL of AGEs (^*P < 0.05). (B) ALP activities in MC3T3-E1 cell cultures. MC3TC-E1 cells were incubated for eight days with 50 ug/mL ascorbic acid, 10 mM β-GP, 25 mM glucose, 25 mM mannitol, and 1, 10, or 100 ug/mL of AGEs (^*P < 0.05, ^**P < 0.01, ^***P < 0.001). (C) Bone nodule formations in MC3T3-E1 cell cultures. MC3TC-E1 cells were incubated in 24-well plates (5,000 cells/well) for 14 days with 50 ug/mL ascorbic acid, 10 mM β-GP, 25 mM glucose, 25 mM mannitol, and 1, 10, or 100 ug/mL of AGEs (^*P < 0.05, ^**P < 0.01).

TRAP = tartrate-resistant acid phosphatase, MNC = multinucleated cell, M-CSF = macrophage colony-stimulating factor, RANKL = receptor activator of nuclear factor-κB ligand, AGE = advanced glycation end product, ALP = alkaline phosphatase.