Quantifying Alveolar Bone Loss via μCT

To quantify alveolar bone loss, we used microcomputed tomography (MILabs, The Netherlands) on live animals. The measurement was carried out with the following settings: voltage, 50 kV; tube current, 0.21 mA; exposure time, 75 ms; and step angle, 0.1°. Briefly, the linear measurements of the distances between cementoenamel junction (CEJ) and alveolar bone crest (ABC) were taken at two sites of second molars in the right and left dental arches (50 (link)). Data were quantified using PMOD software (PMOD Technologies Ltd., Switzerland). Additionally, bone loss was visualized by methylene blue and eosin staining after euthanasia, as described previously (51 (link)). Soft tissue was removed from bones, and the jaws were immersed overnight in 3% hydrogen peroxide. The next day, jaws were incubated in 1% bleach and then washed and air dried. Alveolar bone and teeth were stained with 0.5% eosin for 5 min and 1% methylene blue (Merck Millipore) for 1 min, followed by several washes in distilled water. Bone loss was visualized with a dissecting microscope (SZX-9; Olympus) connected to a digital camera (EOS 1000D; Canon).