Bone Abstracts

Site-specific osteoclasts (OC) and OC-like cells from different pathologies exhibit heterogeneity e.g. in the resorption machinery, influence of anti-resorptive drugs and response to local or systemic hormones e.g. PTH. The explanation for this is not known but different precursors, local regulation through osteoblasts (OB), osteocytes and extracellular matrix (ECM) could be involved. To study these differences a simple and reproducible cell model is an advantage in pre-clinical studies. For that purpose, RAW264.7, a cell line used for its capacity to form OC-like cells was evaluated for its suitability as a model for OC heterogeneity.

RAW 264.7 subclones were acquired by single cell cloning. Cells were seeded on bone or coated hydroxyapatite wells (Corning Osteo-Assay Surface 96 wells) at 3×10⁴ cells/cm², then stimulated with RANKL or RANKL+M-CSF at 10 ng/ml for 14 and 7 days respectively, and evaluated by TRAP staining, immunohistochemistry, morphological analysis, gene expression profiling as well as functional assays e.g. capacity to dissolve mineral matrix through acidification.

Gene expression profiling revealed three subclones groups; (i) TRAP^low. (ii) TRAP^high where TRAP and Cathepsin K were closely correlated. (iii) TRAP^moderate/Cathepsin K^low but MMP9^high. RANKL differentiation of group (i) resulted in a homogenous population of smaller TRAP+ multinucleated cells (MNC) dissolving hydroxyapatite, thus resembling OC. On the other hand, group (ii) resulted in a heterogeneous population of partly larger MNC with lower TRAP expression exhibiting minimal dissolvent of hydroxyapatite, thus resembling multinucleated giant cells. RANKL +M-CSF differentiation of subclones from group (i) and (ii) resulted in inhibition of osteoclast differentiation with no TRAP positive staining, no multinucleated cells and less expression of osteoclast genes markers.

In summary, subclones of the RAW264.7 cell line can be isolated representing macrophage and osteoclast precursors with different phenotypic and functional properties.