Dynamic histomorphometry analysis

Tetracycline and calcein labelling was performed to analyse dynamic histomorphometry. Briefly, rabbits were injected intramuscularly with 50 mg/kg of tetracycline 10 days before sacrifice and 5 mg/kg of calcein (Sigma) 3 days before sacrifice. Then, the thighbones were dissected out, fixed in 70% ethanol, dehydrated, and embedded undecalcified in methylmethacrylate. Then, 10-μm sections were cut, and the unstained sections were viewed using a fluorescence microscope. The mineral appositional rate (MAR, μm/day) was measured. MAR was defined as the mean distance between two fluorescent labels divided by the number of days between labels.