post-hoc test or unpaired T-test. Statistical significance was set at P,0.05. Treatment groups with annotations containing the same letter are not significantly different. (For example: 3 groups annotated with ‘‘a’’, ‘‘b’’ and ‘‘c’’, are all different to each other. In the case of 3 groups annotated with ‘‘a’’, ‘‘ab’’ and ‘‘b’’, ‘‘a’’ is different to ‘‘b’’, whereas ‘‘a’’ and ‘‘ab’’ are not different, likewise ‘‘ab’’ and ‘‘b’’.) Results M-CSF acutely augments RANKL-induced resorption in mature human OCs We assessed the survival and resorbing activity of mature human OCs by seeding freshly harvested cells onto dentine slices and culturing them for 72 h. Survival of OCs cultured in media/ 10% FBS alone was enhanced (62% increase) by the addition of M-CSF (25 ng/mL) but more effectively so (165% increase) by M-CSF Augments Resorption by Human Osteoclasts RANKL (125 ng/mL). Neither M-CSF nor RANKL had any substantial effect on OC plan area (size). In the absence of RANKL, resorption was negligible. Activation of RANKL- induced resorption per OC was markedly enhanced (300% increase) by the addition of M-CSF (25 ng/mL) (Fig. 1A). In six independent experiments, the addition of M-CSF (25 ng/mL) resulted in a 220612% (mean6SEM) increase in resorption compared to RANKL (125 ng/mL) alone (p = 0.004, not shown). This effect was concentration dependent (Fig. 1B). At M-CSF concentrations between 50 and 200 ng/mL, a concentration- dependent, although non-significant, trend to decreased resorption was apparent. In cultures treated with RANKL only, co-treatment with a neutralising antibody to M-CSF had no significant effect on survival or resorption, indicating that endogenous M-CSF [11] is not necessary for RANKL-induced survival or resorption activation (Fig. 1B). A 130% stimulation of resorption in the presence of M-CSF (37.5 ng/mL) was accompanied by an Figure 1. M-CSF augments RANKL-induced resorption and F-actin sealing zone formation by harvested OCs. Mature OCs were formed from CFU-GM precursors cultured on plastic in the presence of RANKL (125 ng/mL) and M-CSF (25 ng/mL) for 21 d. Mature OCs were then detached with non-enzymatic dissociation buffer, re-adhered to dentine slices and cultured for a further 72 h. (A) Survival and activity of harvested OCs cultured in media alone or combinations of RANKL (125 ng/mL) and M-CSF (25 ng/mL); data representative of 6 independent experiments. (B) Concentration-dependent stimulation of RANKL-induced resorption by M-CSF; data representative of 3 independent experiments. RANKL (125 ng/ml) added to all cultures. (C) M-CSF (25 ng/mL) potentiates RANKL-induced (125 ng/mL) sealing zone formation. (D) Photomicrographs show OCs stained for F-actin. Results expressed as mean 6 SEM (n = 6 dentine slices/group). Groups with different annotations are significantly different; Panel A&B p = 0.001; one-way ANOVA; Fishers multiple comparison test; Panel C p = 0.001; Two-sample T-test. doi:10.1371/journal.pone.0021462.g001 PLoS ONE | www.plosone.org 3 June 2011 | Volume 6 | Issue 6 | e21462