Fig. 1. MMP9 expression during non-stable fracture healing. (Left column) Sagittal sections through the wild-type callus at 3, 6, 10, 14 and 21 days post- fracture, stained with Safranin-O/Fast Green (SO-FG), illustrates the abundant proteoglycan-containing cartilage (red, c) that appears during the soft callus phase of healing (4-10 days post-fracture) and is resorbed during the hard callus phase of healing (10-21 days post-fracture). The borders of the fracture callus are delimited with a dotted line. The proximal half of the broken tibia is on the left side, showing the growth plate, and the distal part of the tibia is on the right side. (Right column) In situ hybridization and immunostaining analyses at 3, 6, 10, 14 and 21 days post-fracture. At 3 days, analyses show Mmp9 mRNA (green) and MMP9 protein (brown) in inflammatory cells around the fracture site. At 6 days, MMP9 protein is detected in TRAP-positive cells at the fracture site. At 10 days, Mmp9 mRNA is localized to cells at the boundary between the cartilage callus (c) and newly forming bone (b). This cartilage-bone boundary, demarcated by a dotted line, is visualized with SO-FG staining on an adjacent section. At 14 days, Mmp9-expressing cells are detected at the site of hypertrophic cartilage degradation. At 21 days, MMP9- and TRAP- positive osteoclasts are localized in the callus at the site of new bone remodeling. Higher magnifications in the right column correspond to the boxed areas in the left column. gp, growth plate; bm, bone marrow; b, bone. Scale bars: SO-FG staining (low magnification), 2 mm; SO-FG staining (high magnification) and Mmp9 in situ hybridization, 250 µm; MMP9 immunostaining and TRAP-MMP9 double staining, 10 µm.

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