Purification of Osteoclast Differentiation Factor produced by Epiphyseal Cartilage

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Endochondral bone formation is initiated by the formation of cartilage tissue. Soon after the cartilaginous mold is formed, chondrocytes in the central part of the mold become hypertrophic and calcified. Concomitantly with neovascularization in the calcified cartilage zone, the bone-cell precursors are recruited to the ossification center where the differentiated osteoclasts gradually replace cartilage into bone and excavate the bone marrow cavity. Therefore, calcified cartilage must be the place where the differentiated osteoclasts appear for the fist time during embryonic development. Here we examined a possibility that calcified cartilage may play a functional role for formation of the differentiated osteoclasts. We isolated grwoth-plate chondrocytes from young rabbits, and cultured. The culture media were conditioned for two days each on Day 6 (at the confluent stage), Day 22 (at the maturing stage), Day 42 (at the hypertrophic stage), Day 48 (at the early calcified stage), and Day 57 (at the heavily calcified stage), respectively. Stimulatory activity of the medium on osteoclastic differentiation was assessed by pit formation assay (Bone Min.17 : 347-359,1992) and TRAP assay (Blood 74 : 1295-1302,1989). The results clearly indicated that calcified chondrocytes secreted a factor stimulatory for osteoclastic differentiation. Then we found the similar osteoclast differentiation factor in the guanidine extracts of fetal bovine epiphyseal cartilage of developing bone. The active principle was purified, and found identical with chondromodulin-II (ChM-II). ChM-II markedly stimulated osteoclastic differentiation at the dose rage of 3-10 ng/ml, but it did not required the presence of vitamin D_3 for action.