MMP-9 is important for stimulation of bone angiogenesis by osteoclasts in vivo. (A) Supra-calvarial RANKL increases angiogenesis and osteoclast formation to a greater extent in calvaria of WT than Mmp9^−/− mice. Images of the outer table of calvarial sections stained for TRAP (red) and CD31 (brown) and lightly counterstained with hematoxylin. Images are WT vehicle-treated (i), WT RANKL-treated (ii), Mmp9^−/− vehicle-treated (iii), or Mmp9^−/− RANKL-treated (iv). Images were acquired as in Figure 4. indicates CD31⁺ vessel in remodeling bone; 2-headed arrow, thickness of RANKL-induced bone remodeling. Scale bars represent 50 μm. Outer surface is at the top of the image. Auto contrast and auto color adjustment were performed with Adobe Photoshop software. (B) Quantification of RANKL-induced vessel response by calculation of CD31⁺ vessel density or area in the outer table of 10× objective images taken at the calvarial midline. (C) Total percentage TRAP⁺ area of the calvarial outer table or resorptive surface of the outer table subperiosteal surface. (D) Vessels per osteoclast were calculated by dividing vessel density by TRAP⁺ area for each RANKL-treated animal. (E) In vitro osteoclast formation from bone marrow from WT and Mmp9^−/− C57BL/6 mice was quantified by counting TRAP⁺ multinucleated cells. Data are mean ± SEM. *P < .05. NS indicates not significant.