Fig. 2
Direct contact with mOBs inhibits the bone-resorbing activity of mOCs. a A representative MIP image of bone-resorptive activity in skull bone tissue of a Col2.3-ECFP/TRAP-tdTomato mouse injected with a pH-sensing chemical probe, pHocas-3. Green, pHocas-3; cyan, mOBs expressing Col2.3-ECFP; red, mOCs expressing TRAP-tdTomato; blue, bone tissues (SHG). Scale bar, 50 µm. b, c Assessment of mOC bone-resorbing activity. b Areas containing mOCs were automatically binarized from the original images. Red, mOC areas; gray, regions outside the mOC areas. c Mean pHocas-3 fluorescence intensities were measured inside (pHocas-3 signal) and outside the mOC areas (pHocas-3 noise). The bone resorbing index (BRI) was the ratio of the pHocas-3 signal to the pHocas-3 noise. d, e Images processed for BRI calculations (d for the mOC indicated with white asterisk in the outlined region of a, and e for the mOC indicated with black asterisk in the region delineated with a dotted line in a). Scale bar, 20 µm. f, g Magnified MIP images from the region outlined in a and f, and the region delineated by the dotted line in a and g, captured at 0, 160, and 320 min (upper panels). The 3D images yielded by colocalization analysis (bottom panels). Scale bar, 20 µm. The contact areas were those where mOBs and mOCs colocalized and are shown in yellow. The filled arrowheads show areas of mOB–mOC contact. The open arrowheads indicate separated mOBs and mOCs. The actual BRI values are shown to the right of the images. h BRI of mOCs in contact, or not, with mOBs. Snapshot MIP images were collected from 14 independent experiments; n = 34 (mOCs in contact with mOBs), n = 67 (mOCs not in such contact). Data are presented as means ± SDs. ****p < 0.0001 (Mann–Whitney test)
