Characterization of circulating human osteoclast progenitors: development of in vitro resorption assay

Abstract

Several cell surface markers were used to isolate monocytes as osteoclast progenitors with an immunomagnetic cell separation system. Use of this system with specific monocyte antibodies produced 99% pure monocytes. When purified monocytes were cultured on bovine bone slices in the presence of receptor activator of nuclear factor-kappaB (RANKL), macrophage-colony stimulating factor (M-CSF), tumor necrosis factor alpha (TNF-alpha), and dexamethasone for 14 days, CD14(+) CD11b(+), and CD61(+) monocytes had approximately 90-, 30- and 20-fold higher osteoclast formation capacities/plated cells compared to the control culture. CD15(+) monocytes generated few tartrate-resistant acid phosphatase-positive multinucleated cells (TRACP+ MNC), and CD169(+) monocytes generated no TRACP+ MNC. This suggests, that there are various subsets of monocytes in the blood circulation and that they have different capacities in osteoclast formation. These results show that circulating human osteoclast progenitors can be efficiently purified by immunomagnetic cell separation system using anti-CD14, -CD11b, and -CD61 antibodies. These purified monocyte fractions had different ability to give rise to osteoclasts. CD169 was not found to be suitable for osteoclast progenitor isolation. Optimal concentration of dexamethasone for osteoclast formation and bone resorption was 10 nM. To develop a human resorption assay, osteoclasts were first induced for 7 days, whole media were replaced, cultures were continued for additional 3 days and C-terminal telopeptide of type I collagen was determined from culture media. This assay was shown to be functional, since two well-known resorption inhibitors, bafilomycin A(1) and calcitonin, dose-dependently inhibited the resorption activity of osteoclasts.