Fig. 6. rVEGF rescues the Mmp9^-/- fracture repair defect. (A) Tissue sections from PBS- and rVEGF-injected Mmp9^-/- calluses were stained with SO-FG at 10 and 14 days post-fracture to illustrate that, during the maturation phase of fracture healing (10 days), there is no difference in either the amount of cartilage (red) or the size of the callus (indicated by dotted black line). However, by 14 days post-fracture, the amount of cartilage was substantially reduced in Mmp9^-/- mice that received rVEGF compared with those that received PBS. These histological observations were confirmed by histomorphometric measurements of the total callus volume and the cartilage volume (B). By 14 days post-fracture, there was a statistically significant decrease in both the total callus volume (left graft, asterisks) and in the cartilage volume (right graft, asterisks) of Mmp9^-/- mice that received rVEGF (n=10) versus PBS (n=10), as assessed by ANOVA (P=0.03 and P=0.02, respectively; bars represent mean±s.e.m.). (C) By 14 days post-fracture, substantially less hypertrophic cartilage was detected in the Mmp9^-/- calluses that had been treated with rVEGF compared with their PBS counterparts. Left panels show the yellow ColX hybridization signal superimposed upon a tissue section stained with SO-FG. Higher magnification of area boxed in red illustrates that ossification was also accelerated by rVEGF when compared with PBS controls [Analine Blue, (AB) stain superimposed upon an adjacent tissue section stained with SOFG]. Higher magnification of the area boxed in black illustrates that rVEGF induced substantially greater osteoclast-mediated degradation of the callus compared with PBS controls (arrowheads in upper and lower panels indicate TRAP immunostaining, red dotted line demarcates the boundary between the remaining hypertrophic cartilage and the newly forming bone). Higher magnification of this same region demonstrates that rVEGF resulted in an increased vascular invasion of the callus, as shown by the presence of PECAM-positive endothelial cells penetrating the Mmp9^-/- hypertrophic cartilage callus (arrows, bottom right panel). Conversely, endothelial cells remain at the periphery of the PBS-injected Mmp9^-/- callus. Scale bars: in A,C (SO/ColX panel), 1 mm; in C (TRAP and PECAM panels), 200 µm.