Continuous Compressive Force Induces Differentiation of Osteoclasts with High Levels of Inorganic Dissolution

Abstract

BACKGROUND Osteoclast precursor cells are constitutively differentiated into mature osteoclasts on bone tissues. We previously reported that the continuous stimulation of RAW264.7 precursor cells with compressive force induces the formation of multinucleated giant cells via receptor activator of nuclear factor kappaB (RANK)-RANK ligand (RANKL) signaling. Here, we examined the bone resorptive function of multinucleated osteoclasts induced by continuous compressive force. MATERIAL AND METHODS Cells were continuously stimulated with 0.3, 0.6, and 1.1 g/cm² compressive force created by increasing the amount of the culture solution in the presence of RANKL. Actin ring organization was evaluated by fluorescence microscopy. mRNA expression of genes encoding osteoclastic bone resorption-related enzymes was examined by quantitative real-time reverse transcription-polymerase chain reaction. Mineral resorption was evaluated using calcium phosphate-coated plates. RESULTS Multinucleated osteoclast-like cells with actin rings were observed for all three magnitudes of compressive force, and the area of actin rings increased as a function of the applied force. Carbonic anhydrase II expression as well as calcium elution from the calcium phosphate plate was markedly higher after stimulation with 0.6 and 1.1 g/cm² force than 0.3 g/cm². Matrix metalloproteinase-9 expression decreased and cathepsin K expression increased slightly by the continuous application of compressive force. CONCLUSIONS Our study demonstrated that multinucleated osteoclast-like cells induced by the stimulation of RAW264.7 cells with continuous compressive force exhibit high dissolution of the inorganic phase of bone by upregulating carbonic anhydrase II expression and actin ring formation. These findings improve our understanding of the role of mechanical load in bone remodeling.

Figure 1

Schema of compressive force generated by increasing the volume of culture medium and effect of compressive force on actin ring organization and TRAP staining. White arrow indicates compressive force (A). Cells were continuously stimulated with 0.3, 0.6, or 1.1 g/cm² compressive force in the presence of 5 ng of RANKL for 4 days. Actin labeled with fluorescently tagged phalloidin (green) and nuclei labeled with DAPI (blue) were observed with a fluorescence microscope (B). The cells were stimulated with 0.3, 0.6, or 1.1 g/cm² compressive force for 4 days, and then stained with TRAP and observed by light microscopy (C). Scale bar=100 μm.

Figure 2

Effect of compressive force on mRNA levels of osteoclastic bone resorption-related enzymes in RAW264.7 cells. The mRNA expression levels of carbonic anhydrase II (A), matrix metalloproteinase-9 (B), and cathepsin K (C) were determined by real-time RT-PCR on days 3 and 4 of culture. Bars indicate the means ± standard deviation of 4 independent experiments. * P<0.05, ** P<0.01, *** P<0.001 (vs. 0.3 g/cm²), ^## P<0.01 (0.6 vs. 1.1 g/cm²).

Figure 3

Effect of compressive force on protein levels of osteoclastic bone resorption-related enzymes in RAW264.7 cells. The protein levels of carbonic anhydrase II, matrix metalloproteinase-9, and cathepsin K were determined by western blotting on day 4 of culture. The expression of β-tubulin was used as an internal reference.

Figure 4

Effect of compressive force on mineral resorption activity. Cells on the calcium phosphate-coated plate were continuously stimulated with, 0.3, 0.6, or 1.1 g/cm² compressive force in the presence of 5 ng of RANKL for 4 days. (A) Resorption pits were observed by light microscopy. Scale bar=100 μm. (B) Calcium phosphate released from the plate was quantified by fluorescence intensity. Bars indicate the means ± standard deviation of four independent experiments. * P<0.05, *** P<0.01 (vs. 0.3 g/cm²), ^## P<0.01 (0.6 vs. 1.1 g/cm²).