Methodology

A novel assay for analysis of the regulation of the function of human osteoclasts

Barrie Kirstein¹ , Urszula Grabowska² , Bertil Samuelsson² , Masahiro Shiroo² , Timothy J Chambers¹ and Karen Fuller¹

¹  Department of Cellular Pathology, St George's, University of London, London SW17 0RE, UK

²  Medivir UK, Little Chesterford, Essex CB10 1XL, UK

author email corresponding author email

Journal of Translational Medicine 2006, 4:45doi:10.1186/1479-5876-4-45

Published:	7 November 2006

Abstract

Background

Very little is known of the regulation of the function of human osteoclasts, largely due to the virtual impossibility of obtaining human osteoclasts ex vivo. It has recently become possible to generate human osteoclasts in vitro, by incubation of peripheral blood mononuclear cells (PBMCs) in macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). However, the assays at present available do not distinguish clearly between the distinct effects of agents on differentiation and function.

Materials and methods

We developed a novel assay for resorptive function of human osteoclasts that minimizes inter-assay variability by using each culture as its own baseline, and that minimizes the confounding effects of agents on differentiation by assessing resorptive function over a short test period. In this assay, the development of resorptive activity is monitored in sample cultures. When resorption is underway, bone resorption (measured as the release of the C-terminal telopeptide degradation product of type I collagen (CTX-I) into the supernatant) is compared before vs after incubation for 1–24 h in test agent.

Results

Using this assay, we found that changes in bone resorption could be detected using substantially fewer cultures per variable. Moreover, we could detect effects of agents on resorption within 1 h of addition, a time sufficiently short that a change in release is likely to reflect an effect on function rather than on differentiation.

Conclusion

The assay makes it possible to distinguish the effects of agents on osteoclastic function, independent of their effects on differentiation.