Development of new assay systems for examining the relation between osteoblasts and osteoclasts, and identification of new factors controlling bone metabolism

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The aim of the present study was to develop new assay systems for examining the relation between osteoblasts and osteoclasts and to identify new factors controlling bone metabolism. In order to address these problems, we performed following experiments: (1) isolation and characterization of osteotropic factors produced by osteoblasts, (2) development of new assay systems for examining osteoclast formation in vitro, and (3) clarification of the role of osteoblasts in osteoclast formation.1. We previously reported that osteoblastic MC3T3-E1 cells produced differentiation inducing factor (DIF) which promoted differentiation of mouse myeloid leukemia cells (M1) into macrophage-like cells. The DIF was partially purified from the conditioned meda of MC3T3-E1 cells and its biological activity in bone resorption was examined. The DIF exhibited a marked bone resorbing activity in a Raisz's assay system and it stimulated osteoclast-like cell formation in a mouse marrow culture system.2. We devel … More oped a mouse bone marrow culture system to examine osteoclast formation in vitro. Bone resorption-stimulating hormones and factors such as 1alpha,25(OH)_2D_3, PTH, PGE_2, IL-1, TNFalpha, and TGF markedly stimulated the formation of osteoclast-like multinucleated cells (MNCs), whereas calcitnin and IFN<@2Y<@D2 strongly inhibited the formation induced by those hormones and factors.3. In order to examine the role of osteoblasts in osteoclast formation, we developed a co-culture system of mouse spleen cells and osteoblastic cells freshly isolated from fetal mouse calvariae. When mouse spleen cells were co-cultured with osteoblastic cells in the presence of 1alpha, 25(OH)_2D_3, osteoclast-like MNCs were formed within 8 days. Neither the same co-culture without the vitamin nor separate cultures of either spleen cells or osteoblastic cells with the vitamin produced osteoclast-like MNCs. These results indicate that osteoblasts are required for differentiation of osteoclast progenitors into multinucleated osteoclasts. Less