A SEQUENTIAL CULTURE APPROACH TO STUDY OSTEOCLAST DIFFERENTIATION FROM NONADHERENT PORCINE BONE-MARROW CELLS

A ''sequential culture step'' system was devised to study osteoclast d ifferentiation from newborn porcine bone marrow cells. Nonadherent cel ls were collected from cultures of bone marrow cells, and subsequently precultured at a low cell density in low-serum medium supplemented wi th L929-conditioned medium (L9-CM) derived M-CSF/CSF-1. After 4 d, adh erent cells mainly composed of M-CSF-dependent macrophage/osteoclast p rogenitors, but devoid of stromal-like cells, were further cultured in medium supplemented with L9-CM and CM derived from serum-free culture s of fetal rat calvarial bones. This phase was characterized by a rapi d induction of mono- and multinucleated (pre)osteoclast-like cells, po sitive for cytochemical TRAP activity, but negative for nonspecific es terase (NSE) staining. The presence of 1,25-dihydroxyvitamin D3 [1,25( OH)(2)D-3] stimulated osteoclast generation, whereas calcitonin treatm ent significantly inhibited this process. The osteoclastic nature of t he cells was confirmed by the occurrence of extensive, characteristic bone resorption on dentin slices, which was associated with release of type I collagen N-telopeptides from the bone matrix into the culture medium. The presence of a DNA synthesis inhibitor (HU) during the firs t 3 d of culture completely inhibited osteoclast formation, whereas HU treatment during the last phase did not affect production of multinuc leated osteoclast-like cells. Likewise, a specific antibody directed a gainst M-CSF during the first preculture period, completely abolished osteoclast formation. Adding the antibody during the last phase of the culture, however, strongly inhibited multinucleated osteoclast format ion, accompanied by a significant increase in a mononuclear TRAP-posit ive, NSE-positive (osteoclast precursor) cell fraction. These results indicate that M-CSF is essential for progenitor proliferation as well as for (pre)osteoclast maturation and/ or fusion into multinucleated c ells, but also suggest that additional soluble (bone-derived) factors are involved as cofactors in the differentiation process to committed mononuclear osteoclast precursors. The porcine marrow culture approach provides a suitable model system to investigate specific soluble oste oclast-inducing factors affecting different stages of osteoclast devel opment.