equivalent increase in sealing zone formation when compared to treatment with RANKL alone (Figs. 1C and 1D). In our 72 h cultures, we could not exclude the possibility that the observed resorption-stimulating effect was due to ongoing OC differentiation in the presence of M-CSF. In CFU-GM osteoclas- togenesis cultures we previously found a linear increase in OC plan area over three weeks due to progressive cell fusion [19] yet in the current study, no substantive increase in OC plan area was observed with M-CSF, suggesting an absence of significant cell fusion. Nevertheless, to exclude this possibility, we conducted experiments over a shorter time of 4–24 h. Since the small amount of dentine resorption present in these short-term assays could not be accurately quantified using whole-slice image analysis, we counted resorption pits at higher magnification and individually measured their plan area and diameter or, in the case of non- circular ‘‘trail or trench’’ pits, trail pit linear length (trail length). In these experiments, OCs were treated with RANKL (125 ng/mL) alone or together with M-CSF (25 ng/mL) (Figs. 2A and 2B). No significant effect on OC number was seen over this period. In the presence of RANKL only, the resorption pit number, size and trail length increased with time in a linear manner. Co-treatment with M-CSF increased the pit number and pits per OC at each time point (200% at 24 h) but had no substantive effect on the pit size or trail length (Fig. 2A). The effect of M-CSF at 6 h in an independent experiment is shown in Fig. 2B. The addition of M-CSF Augments Resorption by Human Osteoclasts M-CSF to RANKL had no effect on OC characteristics but increased the number of pits and pits per OC by 66% and 62%, respectively. M-CSF pre-treatment facilitates RANKL-induced activation of c-fos but not of NFkB In mature human OCs, NFATc1 is not activated by either RANKL or M-CSF. Acute treatment with RANKL of OCs that had been serum-starved for 18 h produced a 320% activation of NFkB at 30 min (Fig. 3A). Pre-treatment with M-CSF (37.5 ng/ mL) for 18 h had no effect on basal or RANKL-induced activation of NFkB (Fig. 3A). In contrast, in control OCs, RANKL did not activate c-fos, whereas pre-treatment with M-CSF increased basal activation by 70% and RANKL-induced activation by 190% (Fig. 3A). We also found that M-CSF pre-treatment increased basal c-fos protein expression by 41.5% (Fig. 3B). No effect on NFATc1 activation was seen with M-CSF pre-treatment or acute RANKL treatment (Fig. 3A). Phosphorylation of ERK1/2 by M-CSF is augmented by co-treatment with RANKL in mature human OCs Since MEK/ERK pathways are known to be involved in both RANK and c-fms signalling [22–24], we hypothesised that they may be involved in the observed effect of M-CSF on RANKL- Figure 2. M-CSF rapidly potentiates RANKL-induced resorption by harvested OCs. Mature OCs were re-adhered to dentine slices and cultured for up to 24 h in RANKL (125 ng/mL) alone or RANKL (125 ng/mL) + M-CSF (25 ng/mL). A. Time course of OC survival and resorption. Results expressed as mean 6 SEM (n = 6 dentine slices/group). Groups with different annotations (a,b,c) are significantly different for treatment; P,0.05 ANOVA GLM Tukey pairwise comparisons. Groups with different annotations (x,y,z) are significantly different for time; P,0.003 ANOVA GLM Tukey pairwise comparisons; data from one experiment. B. Potentiation of OC activity by M-CSF (25 ng/mL) at 6 h of treatment. Results expressed as mean 6 SEM (n = 6 dentine slices/group). P,0.05 Two-sample T-test; data from one experiment. doi:10.1371/journal.pone.0021462.g002 PLoS ONE | www.plosone.org 4 June 2011 | Volume 6 | Issue 6 | e21462