Purification of Osteoclast Differentiation Factor produced by Epiphyseal Cartilage

软骨内成骨是由软骨组织形成开始的。软骨模具形成后不久，模具中心部分的软骨细胞变得肥大和钙化。在钙化软骨区新生血管形成的同时，骨细胞前体被招募到骨化中心，分化的破骨细胞逐渐取代软骨成为骨并挖掘骨髓腔。因此，钙化软骨一定是胚胎发育过程中分化破骨细胞第一次出现的地方。在这里，我们研究了钙化软骨可能在分化破骨细胞的形成中发挥功能作用的可能性。我们从幼兔身上分离生长板软骨细胞，并进行培养。培养基分别在第6天（融合期）、第22天（成熟期）、第42天（肥厚期）、第48天（早期钙化期）和第57天（重度钙化期）进行2天的培养。通过坑形成试验（Bone Min.17: 347-359,1992）和TRAP试验（Blood 74: 1295-1302,1989）评估培养基对破骨细胞分化的刺激活性。结果表明钙化软骨细胞分泌一种促进破骨细胞分化的因子。然后我们在胎牛骨骺软骨的胍苷提取物中发现了类似的破骨细胞分化因子。经纯化，发现其活性原理与软骨调节素- ii （ChM-II）相同。ChM-II在3 ~ 10 ng/ml剂量范围内明显刺激破骨细胞分化，但不需要维生素D_3的存在才能起作用。

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.