Compression fractures and bone resorption in Chi/+ and Chi/+LP vertebral bodies. (A) Toluidine blue stained medio-sagittal section of a compression fracture from three months old mutant zebrafish (representative for both Chi/+ and Chi/+LP) observed with polarised light. The compression fracture is characterised by several fractures in the central region of the vertebral body (black arrowheads); a bone callus is present on the outside of the vertebral centrum (white arrowheads). The fracture also disrupts the notochord tissue and induces condensation of chordocytes into a fibrous tissue (a known reaction of notochord tissue to injuries) (red asterisks). (B) High magnification of the fractured bone (black arrowheads) inside the notochord. The reaction of the notochord tissue can be seen (red asterisks). (C) A fibrocartilaginous callus is present around the fractured central part of the vertebral body, the typical appearance for fracture callus at initial stages of repair (white asterisks). Polarised light (insert) shows collagen fibres (green) within the cartilaginous callus. Red asterisk indicates the notochord tissue condensation. (D) Sagittal section of a healed compression fracture. Fracture repair and remodelling processes replaced the fibrocartilaginous tissue by a bone callus (white arrowhead). Remnants of the fibrocartilaginous tissue are visible (black arrow). (E) Tartrate-resistant acid phosphatase (TRAP) staining confirms compression fracture repair. TRAP activity (red staining, red arrowhead) indicates resorption of the fibrous tissue (white asterisks) that is being replaced by a hard bone callus. The fractured bone fragments (black arrowheads) in the lumen of the notochord do not show resorption, which is consistent with the absence of blood vessels, lymphatic vessels and innervation inside the notochord. (F) TRAP activity is detected also in the trabecular bone (white arrowheads) and vertebral endplate (white arrow) of a vertebra showing a compression fracture in mutant zebrafish (representative for both Chi/+ and Chi/+LP). White asterisk indicates the fibrocartilaginous callus. (G, H) WT display TRAP activity (red) at sites of bone remodelling linked to bone growth, i.e. the endosteal surfaces of the neural (black arrowheads) and haemal arches (black arrow). NT, neural tube. (I, J)Chi/+ animals (representative also for Chi/+LP) show TRAP activity at the same locations as in WT, however mutants exhibit expanded TRAP activity at all endosteal and periosteal bone surfaces, i.e. arches (black arrowheads) and bone trabeculae connecting the endplates (white arrowheads). NT, neural tube.
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