Differentiation and function of osteoclasts cultured on bone and cartilage

We examined the differentiation and resorptive function of osteoclasts (OC) cultured on the slices of calcified bone, decalcified bone and hyaline cartilage, and found that OC differentiation depends on the co-cultured substratum, as well as osteoblast-derived factors. Bone marrow-derived macrophages (BMM) were formed from marrow cells of 5 week old ddY mice and cultured for 3 days on freeze-dried slices of calcified bone, decalcified bone or cartilage, all prepared from rabbit costal bone. BMM cultured on calcified bone slices exhibited tartrate-resistant acid phosphatase (TRAP) activity and were structurally characterized by multinucleation and ruffled border development. However, on decalcified bone slices, BMM seldom became multinucleated and exhibited weak TRAP activity. BMM cultured on cartilage slices were mononuclear, devoid of TRAP activity and structurally resembled mononuclear phagocytes. In SEM observations of co-cultured slices, resorption lacunae were formed only on calcified bone slices, and not on slices of decalcified bone and cartilage. Our results, therefore, indicated that BMM could differentiate into functional OC only on calcified bone slices, suggesting a key role of calcified components in the bone matrix for the terminal OC differentiation. Then, we cultured BMM on the same slices with yeast particles. In cultures with yeast particles, BMM exhibited intense TRAP activity, developed a ruffled border-like structure and formed resorption lacunae even on decalcified bone and cartilage slices. Vacuolar-type H⁺-ATPase was strongly expressed along the ruffled border membranes of these OC. Only the BMM that had not incorporated yeast particles developed a ruffled border, whereas the BMM that had incorporated yeast particles did not become multinucleated and lacked a ruffled border structure. Thus, our results further suggest that, even on uncalcified substrata, the terminal differentiation of BMM into functional OC is induced by an unidentified external stimulus, which may be contained in the cell membrane of yeast particles.