Heat-treated osteoblastic cell (ROS17/2.8)-conditioned medium induces the formation of osteoclast-like cells

To study the effects of osteoblast products on osteoclast formation, we added the conditioned medium (CM) of rat osteoblastic cell line ROS17/2.8 to rat bone marrow cultures, in which tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like multinucleate cells (MNCs) formed in the presence of 10(-8) M 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). The formation of 1,25(OH)2D3-dependent TRAP-positive MNC at day 7 of culture was strongly inhibited by the > 10 kDa fraction of ROS17/2.8 cell-CM (ROSCM), but heat treated ROSCM (htROSCM) expressed marked stimulation in the formation of the MNCs. The expression of several osteoclastic phenotypes of the MNCs induced by htROSCM and 1,25(OH)2D3 was more enhanced compared with that of the MNCs induced by 1,25(OH)2D3 alone. The MNCs induced by htROSCM and 1,25(OH)2D3 were highly motile, were sensitive to calcitonin (CT), and had high bone resorbing activity. These data suggest that htROSCM promotes the osteoclast differentiation in the presence of 1,25(OH)2D3 in a rat bone marrow culture system. The stimulatory activity of TRAP-positive MNC formation in htROSCM is derived from heat-stable protein(s) that is (are) thought to be different from colony-stimulating factors (CSFs) such as macrophage-CSF (M-CSF) or granulocyte-macrophage-CSF (GM-CSF).