Figure 3. Effects of rhSCL on TRAP⁺ multinucleated cell formation.

The effect of rhSCL on osteoclastogenesis was tested in co-cultures of MLO-Y4 cells with A) mouse splenocytes and B) human PBMC. In both cases MLO-Y4 cells were seeded into type I collagen coated culture wells and cultured for 72 h in the absence or presence of rhSCL, as indicated, prior to the addition of either splenocytes or PBMC. All cultures received rhM-CSF at 25 ng/ml. RhOPG was added to some cultures at 100 ng/ml and rhRANKL (100 ng/ml) added to monocultures of either splenocytes or PBMC to confirm the osteoclast-forming potential of these populations (not shown). Media were replenished every 3 days. Cultures were fixed and stained for TRAP at day 6. TRAP-positive MNC, defined as cells with >3 nuclei, were counted from quadruplicate wells. Asterisks denote significant differences to the no SCL control (** p<0.01, *** p<0.001) and the effect of OPG relative to the corresponding SCL-only treatment is indicated by Φ (p<0.001). C) In the case of MLO-Y4/PBMC co-cultures and cells formed in PBMC monocultures treated with rhRANKL, the relative size (in pixels) of TRAP⁺ MNC formed (in each case >50 cells) was measured by Image J analysis. Difference to the no SCL control is indicated by ** p<0.01; † indicates difference of rhRANKL control to rhSCL at 100 ng/ml (p<0.01). Data shown are representative of at least 3 independent experiments.