Ctvox 3

Bones for micro-CT were fixed in 4% paraformaldehyde (PFA) and scanned using Bruker’s Skyscan 1272 (Bruker, Belgium) by rotating over 360° in 0.8° rotational steps. The X-ray settings were standardised to 70 kV and 142 μA with an exposure time of 1450 ms and the X-ray filter used was a 0.5 mm aluminium. Projections were acquired with nominal resolutions of 21.5 and each resulting image contained 1144 x 1144 pixels. All X-ray projections were reconstructed using NRecon 1.7.3.1 software (Bruker) to create cross-sectional images and viewed using CTvox 3.3.0 (Bruker).

The maxilla of the mice from the experimental and control group were scanned using an animal micro-computed tomography (μCT) system (SkyScan1172; Bruker‐microCT, Belgium) at Tufts Medical Center. The 3D models were then reconstructed from the raw images with Bruker NRecon and analyzed with software from Bruker, including CT Analyser 1.17.7.2, DataViewer 1.5.4.0 and CT Vox 3.3.0. The alveolar bone loss was described as the distance from the cementoenamel junction to the alveolar bone crest (CEJ‐ABC), and the values were measured at six periodontal sites (mesiobuccal, midbuccal, distobuccal, mesiopalatal, midpalatal, and distopalatal) of the second molars.

Left-hind limbs (LHL) were fixed in 4% PFA and subsequently transferred into PBS for high-resolution micro-CT scanning using Bruker's Skyscan 1272. X-ray settings were standardised to 70 kV and 142 μA and used a 0.5-mm aluminium X-ray filter. Each entire femur was scanned over 360° rotation in 0.8° rotational steps and the exposure time was set to 470 ms. Projections were acquired with nominal resolutions of 10 μm and each slice contained 1224×820 pixels. All X-ray projections were reconstructed using a modified back-projection reconstruction algorithm (NRecon 1.7.3.1 software-SkyScan, Bruker) to create cross-sectional images. Reconstruction parameters included ring artefact correction (2-6), beam hardening correction (40-50%) and misalignment correction. Reconstruction was performed in a blinded manner. 3D reconstructions were viewed using CTvox 3.3.0 (Bruker). Reconstructed images were analysed through CTAn 1.19 software (Bruker), which has inherent 2D and 3D analysis tools. All analyses were performed as per the updated guidelines for the assessment of bone density and microarchitecture in vivo using high-resolution peripheral quantitative CT (Whittier et al., 2020 (link)).