The ligations were maintained for 2 weeks, and after which the mice were euthanized by CO₂ inhalation and the maxilla were harvested. The gingival tissues of half group of mice were isolated and collected for mRNA expression study. The skulls left were defleshed by beetles for 1 week. Briefly, in beetle’s chamber, freshly dissected skull was put in a paper cup with 0.5 cm diameter holes at the bottom, so beetles can move in with relatively controlled numbers. After that, the skulls were bleached by H₂O₂ (3%) for 4 h. Bone resorption was measured by microscope imaging analysis and μCT scan analysis. The skulls of the other half of the group were fixed in formalin overnight at 4 °C followed by EDTA decalcification for 3 weeks with agitation. After complete demineralization, implants were removed manually by rotating counterclockwise. All the decalcification samples were embedded into paraffin and cut in 5 μm sections along the mesial-distal plane and then subjected to H&E staining and TRAP staining.

The two dimension (2D) bone resorption measurements were assessed under a microscope (Nikon SMZ745T, Nikon Instruments Inc.) and analyzed by software ImageJ (NIH) on buccal and palatal surfaces for each segment, and a standard calibrator was used for calibration at the same magnification as previously described [36]. The bone resorption area was enclosed coronally by the CEJ of the molars, laterally by the exposed distal root of the first molar and the exposed mesial root of the third molar, and apically by the alveolar crest. The results are presented in square millimeters.

Mice maxillae were scanned with a high-resolution scanner (mCT-40, Scanco Medical). Samples were exposed to polychromatic X-rays on a rotating stage at a steep angle of 0.18° over 360°. Measurements were taken at an operating voltage of 70 kVp and 114 mA current and 6 mm isotropic voxel resolution, with an exposure time of 200 ms and five frames averaged per view. Quantitative three dimension (3D) measurements of the bone resorption were performed using Seg3D software as previously described [5].