Identification of inhibitor of osteoclastogenesis

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The purpose of this research was to purify and identify inhibitors of osteoclast formation released from mouse stromal cells (pre-osteoblasts, TMS-12). To quantify the activity of inhibition of osteoclast formation, we developed the assay system for osteoclast formation, and to purify the inhibitors we determined the cultured condition of TMS-14 without serum. Using this system, we carried out the purification of inhibitors from the 120 liter of the conditioned medium of TMS-14 with ion-exchangers, molecular cutting, dialysis and so on. Finally after applying Superdex 200HR10/30 it was determined that the molecular of the inhibitors was 200K and 22K. Now, we carry out the cloning of this substances. Next, we characterized the inhibitors. As a result, the inhibitors were characterized as follows ; 1) It was determined the inhibitors was not IL-4, IL-10, TNFα, known-inhibitors of osteoclast formation by the experiments of antibody neutrization, 2) The substances inhibited osteoclast differentiation, but not proliferation of osteoclast progenitor cells, 3) The substances also changed the morphology of mature osteoclasts and inhibited the pit formation.These data indicated that the characterization of the inhibitors of osteoclast formation was quietly different from other inhibitors such as osteoprotegrin/osteoclastogenesis inhibitory factor.