Fig. 4. Mmp9^-/- mice exhibit an ossification defect during stabilized fracture repair. In A and B, wild-type is left, Mmp9^-/- is right. (A) SO-FG stained sections through the stabilized fracture site at 10 days confirm the presence of abundant new bone (arrows indicate Oc expression) and the absence of cartilage in the wild-type callus (no ColIIa signal is evident despite hybridization with this RNA probe). By sharp contrast, abundant cartilage forms in the Mmp9^-/- callus regardless of stabilization of the bone segments, as shown by the expression of ColIIa (pink). Oc expression (black) is also detected in the periosteum, adjacent to the fracture site (arrows) and in the endosteum. (B) SO-FG staining of sections at a pin implant (*) at 10 days shows that although no cartilage is detected in wild-type animals, abundant cartilage (red) is present in Mmp9^-/- animals. This cartilage is restricted to the periosteal surface (po). Oc expression (black) is localized both at the periosteal and endosteal (en) surfaces (arrows) in wild-type and Mmp9^-/- animals. (C) SO-FG stained sections through the wild-type callus illustrate that if fractures are left unstable for 24 hours (left, 24h) and subsequently stabilized, they heal without evidence of cartilage (10 days; arrow indicates the healing fracture). However, fractures that are unstable for 48 hours (right, 48h) and subsequently stabilized tend to heal with abundant cartilage (red). (D) MMP9 immunostaining and double-staining with TRAP illustrate that MMP9 protein (brown) is detected by day 3 in the endosteal matrix (box 1), in inflammatory cells (box 2), in mesenchymal cells within the fracture gap and surrounding soft tissues (box 3), and in the periosteum (box 4). These MMP9-positive cells are TRAP-negative. TRAP-positive osteoclasts/chondroclasts are present at the epiphyseal growth plates of the fractured bone (not shown), and in the cortical bone. bm, bone marrow; c, cortex. Scale bars: in A,C, 1 mm; in B,D (low magnification), 500 µm; in D (high magnification), 10 µm.