Fig. 2. Mmp9^−/− and myeloid-specific Mmp14 conditional knockout mice display normal osteoclast activity in vitro and in vivo.

(A) BMDMs were isolated from wild-type (WT) or Mmp9^−/− mice, cultured on plastic substrata with M-CSF and RANKL for 5 days, and stained with TRAP. Scale bar, 500 μm. (B) Wild-type or Mmp9^−/− BMDMs were cultured atop bone slices and induced into osteoclasts for 6 days. After cell removal, resorption pits were visualized by WGA-DAB staining. Scale bar, 100 μm. (C) Quantification of bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) as determined by μCT of 5-month-old wild-type and Mmp9^−/− male mice (n = 9). (D) BMDMs were isolated from wild-type or Mmp14^ΔM/ΔM mice, cultured atop plastic substrata with M-CSF and RANKL for 5 days, and stained with TRAP. Scale bar, 500 μm. (E) Wild-type or Mmp14^ΔM/ΔM BMDMs were cultured atop bovine bone slices and induced into osteoclasts for 6 days. Cells were removed and resorption pits visualized by WGA-DAB staining. Scale bar, 100 μm. (F) Quantification of BV/TV, Tb.Th, Tb.N, and Tb.Sp as determined by μCT of 5-month-old wild-type and Mmp14^ΔM/ΔM male mice (n = 9). (G) Relative mRNA expression of Mmp14 or Mmp9 in osteoclasts differentiated from Mmp9^−/− or Mmp14^ΔM/ΔM BMDMs (n = 3). (H) Western blot and quantification of Mmp9 and Mmp14 expression in osteoclasts differentiated from Mmp9^−/− or Mmp14^ΔM/ΔM BMDMs (n = 3). All results are representative of data generated in at least three independent experiments. ns, not significant. **P < 0.01. Error bars are means ± SEM. All data were analyzed using unpaired Student’s t test. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.