A sequential culture approach to study osteoclast differentiation from nonadherent porcine bone marrow cells |
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Authors: | Ben A A Scheven John S Milne Simon P Robins |
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Institution: | (1) Skeletal Research Unit, Rowett Research Institute, AB21 9SB Bucksburn, Aberdeen, Scotland, United Kingdom |
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Abstract: | Summary A “sequential culture step” system was devised to study osteoclast differentiation from newborn porcine bone marrow cells.
Nonadherent cells were collected from cultures of bone marrow cells, and subsequently precultured at a low cell density in
low-serum medium supplemented with L929-conditioned medium (L9-CM) derived M-CSF/CSF-1. After 4 d, adherent cells mainly composed
of M-CSF-dependent macrophage/osteoclast progenitors, but devoid of stromal-like cells, were further cultured in medium supplemented
with L9-CM and CM derived from serum-free cultures of fetal rat calvarial bones. This phase was characterized by a rapid induction
of mono- and multinucleated (pre)osteoclast-like cells, positive for cytochemical TRAP activity, but negative for nonspecific
esterase (NSE) staining. The presence of 1,25-dihydroxyvitamin D3 1,25(OH)2D3] stimulated osteoclast generation, whereas calcitonin treatment significantly inhibited this process.
The osteoclastic nature of the 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 first 3 d of culture completely inhibited osteoclast formation,
whereas HU treatment during the last phase did not affect production of multinucleated osteoclast-like cells. Likewise, a
specific antibody directed against 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 formation, accompanied
by a significant increase in a mononuclear TRAP-positive, 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 cells, 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 osteoclast-inducing factors affecting different stages of osteoclast
development. |
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Keywords: | bone resorption M-CSF macrophage in vitro acid phosphatase |
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