Fig. 5. Mmp9^-/- fracture healing is hindered by the reduction in chondroclasts/osteoclasts and a delay in vascular invasion. 10 days, left half; 14 days right half. (A) Top row, left panels show that in wild-type mice at 10 days post-fracture, Mmp9-expressing chondroclasts/osteoclasts (inset shows cells with characteristic ruffled borders) begin to accumulate at the wild-type cartilage/bone boundary (dotted red line). MMP9 is not expressed in null-mutant mice. In an adjacent section (middle row, left panels), TRAP activity highlights the location of osteoclasts in the wild-type callus (arrowheads), which are largely absent from the Mmp9^-/- callus. b, bone; c, cartilage. In near-adjacent sections (bottom row, left panels), PECAM-expressing endothelial cells (arrow) have accumulated at the border between wild-type hypertrophic cartilage (c) and newly forming bone (b; arrow). Few PECAM-positive cells are detected in the Mmp9^-/- callus (arrow). By 14 days post-fracture, abundant MMP9 protein (upper right panels, brown) and TRAP activity (middle right panels, arrowheads) indicate that osteoclasts-mediated degradation of the cartilage callus is well underway (dotted red line indicates the cartilage-bone junction). This degradation activity is associated with increased vascular invasion as illustrated by PECAM-expressing endothelial cells (arrows). Although more PECAM-positive cells are present at 14 days in the Mmp9^-/- callus (arrow), they are restricted to the edge of the cartilage-bone junction (dotted red line). (B) Tissue sections adjacent to those analyzed in A were examined for the expression of Vegf (top, fuchsia) and one of its receptors, Flk1 (bottom, green). Vegf transcripts were detected in late hypertrophic chondrocytes in both wild-type and Mmp9^-/- calluses at 10 and 14 days post-fracture. Transcripts for Flk1 were detected in endothelial cells surrounding the cartilage callus of both wild-type and Mmp9^-/- calluses. b, bone; c, cartilage. The dotted lines in B delimit the boundary of the cartilage in the callus. Scale bars: in A (low magnification) and in B, 200 µm; in A (high magnification), 20 µm.