Effect of soluble TWEAK on osteoclast formation and function. Human unfractionated PBMC were cultured for nine days in medium containing rhM-CSF only (25 ng/ml) (A), M-CSF and rhTWEAK at 100 ng/ml (B), 400 ng/ml (C) or 800 ng/ml (D), M-CSF and rhRANKL (50 ng/ml) (E), or all of M-CSF (25 ng/ml), RANKL (50 ng/ml) and TWEAK (100 ng/ml) (F). Cultures were then fixed and stained for TRAP. (G) Multinucleated cells (MNC), containing >3 nuclei, positive for TRAP, were counted from quadruplicate wells. Data are expressed as means ± standard deviation. Significant differences were determined by one way analysis of variance (ANOVA) with Tukey post-hoc test: a indicates difference to M-CSF only control (P < 0.001) and b denotes difference to M-CSF+RANKL treatment (P < 0.05). (H) PBMC were seeded onto dentine slices in the presence of rhM-CSF (25 ng/ml) with the addition of rhRANKL (50 ng/ml) and/or rhTWEAK as indicated. Resorption was assessed after 14 days of culture by SEM and is expressed as the mean ± SD resorption expressed as a percentage of that measured for the RANKL/M-CSF control. Data shown are pooled from two independent experiments with resorption assessed for four dentine slices/treatment/donor. No significant differences were observed between RANKL treatments.