Endocrine Abstracts

The discovery of the fundamental involvement of receptor activator for nuclear factor-_KB ligand (RANKL) in osteoclast (OC) formation, has enabled the routine generation of human OCs in vitro from human peripheral blood mononuclear cells (PBMCs). Beta C-terminal telopeptide (beta-CTx) is a specific resorption marker for degradation of bone type I collagen by OCs. We have evaluated the correlation between beta-CTx released from dentine wafers on which OC were cultured as above, and area of resorption lacunae excavated by these cells. Venous blood from patients with Paget's disease of bone and a healthy volunteer, was used to isolate PBMCs. PBMCs from Paget's subjects were cultured in medium containing either 10% FCS or 1% or 10% human serum derived from the same individual, 30 nano-grams per milli-litre RANKL, and 25 nano-grams per milli-litre macrophage-colony-stimulating factor (M-CSF). PBMCs from normal subjects were cultured in the presence of 30, or 60 nano-grams per milli-litre RANKL, 25 nano-grams per milli-litre M-CSF, and 10%FCS. Cell culture medium was harvested at specific time points (7, 14, and 21 days) during a 3 week incubation for measurement of beta-CTx by electrochemiluminescence immunoassay (ROCHE, Lewes UK). End-point area of resorption was determined by point counting using reflected light microscopy. Incubation of Pagetic PBMCs with 10% Pagetic serum or 10% FCS resulted in significantly higher release of beta-CTx and a greater area of resorption than from cells cultured in 1% Pagetic serum. Beta-CTx was significantly correlated with the area of resorption (r=0.92, n=62). Beta-CTx release from cultures of 'healthy' PBMCs was also significantly correlated with the increasing area of resorption produced from OC formed in the presence of increasing concentrations of RANKL (r=0.97, n=13). Measurement of beta-CTx can replace the laborious and time-consuming microscopic evaluation of resorption lacunae formed by osteoclasts generated in cultures of PBMCs supplemented with recombinant RANKL.