Fluorescent labelling analysis

Fluorescent calcium-binding dyes were injected subcutaneously into all animals to identify new bone formation and bone remodelling over time. Calcein green (10 mg/kg; Sigma, St. Louis, MO, USA) was injected after 4 weeks, and xylenol orange (90 mg/kg; Sigma) was administered 7 weeks after defect creation. Unstained ground sections were used to analyse fluorescent labelling, which was visualised using a fluorescence microscope (Olympus Optical) and photographed with a built-in digital camera.