Skyscan 1276

The left femurs were soaked in 4% paraformaldehyde over 24 h and then analyzed immediately by a micro-CT system (SkyScan1276, Bruker microCT company, Kontich, Belgium) using 60 kV voltage, 140 μA current, and 9 μm resolution ratio in the distal growth plate of femurs. After the scan was completed, the 3D images were reconstructed. Dataviewer software 1.5.6.2 was used to adjust the direction and other parameters of the scanned sample images to ensure all samples were processed under the same conditions to generate VOI images. Meanwhile, the bone density formula was constructed in CTAn software 1.17.7.2 using standard product parameters. Then, the single VOI image of each sample was imported to select 100–200 layers below the femoral growth plate to obtain bone morphometric parameters, including bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular space (Tb.Sp) and trabecular number (Tb.N). BV/TV is the ratio of the total volume of voxels representing bone structures in the ROI to the total volume of all voxels in the region. Tb.Th is the average thickness of the trabecular bone. Tb.N is the number of intersections between bone tissue and non-bone tissue in a given length of bone. Tb.Sp is the average width of the pulp cavity between the trabeculae, indicating the porosity of the trabecular bone.

X-ray micro-computed tomography (Skyscan 1,276, Bruker, Belgium) was used. The mouse maxilla was scanned at a voltage of 60 kV and a current of 200 μA with a scan resolution of 6.5 μm. As the filament was placed on the second molar, the region of interest (ROI) for 3D reconstruction was defined as the area above the root tip below the crown of the second molar, with a volume of 0.5 mm × 0.25 mm × 0.4 mm. Three-dimensional image reconstruction was performed using N-Recon software (V 1.7.4.6). Three-dimensional analysis was performed using CT-AN software (V 1.19.11.1+).

Tibiae were carefully excised and fixed in 4% paraformaldehyde for 24 h, rinsed in PBS and stored in PBS. Tibiae were scanned using the Skyscan 1276 (micro-computed tomography imager, Bruker, Kontich, Belgium) at 9 μm resolution, 0.25 mm aluminium filter, 56 kV voltage, 200 μA, 560 ms exposure time, and 0.4° step rotation with frame averaging of 2. Images were reconstructed and analyzed using NRecon (v1.7.4.6, Bruker), Dataviewer (v1.5.6.2, Bruker), CT Analyzer (CTAn; v1.18.8.0, Bruker) and CTVox (v3.3.0 r1403, Bruker). Tibial lengths were determined after scanning. Regions of interest were determined as described previously (Chan et al., 2021 (link)). The trabecular bone was assessed in the proximal region commencing at 3% of bone length from the growth plate and extended distally for a total of 13.5% (equivalent to 0.5-3 mm of the growth plate). For cortical bone, a region of interest beginning at 50% of bone length and extending distally for 2% (approximately 0.5 mm) was used for three-dimensional cortical bone analyses using CT Analyzer. Representative images were taken at the mid-diaphysis (50% of bone length) using a pseudodensity filter in CTVox. The length of the tibial crest was measured using a custom script written in FIJI/ImageJ using individual cross-sectional images spanning 15 to 40% of the tibial length, as described previously (Chan et al., 2023 (link)).

The specimens were subjected to micro-CT scanning (Bruker SkyScan 1276, Billerica, MA, USA; 85 kV, 200 mA, 10 μm resolution). The three-dimensional reconstruction of the sagittal and axial sections was performed using the DataViewer software (Version 1.5.2.4; Bruker, Billerica, MA, USA). The new bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular spacing (Tb.Sp) values were calculated using CTAn (Version 1.17; Bruker, Billerica, MA, USA).

Extracted canine teeth were numbered in a manner to identify the cadaveric specimen they came from and whether they were maxillary or mandibular. Radiolucent hydro-plastic beads were used to fashion trays that could hold up to 6 teeth. Two to three trays of teeth were stacked at once in the micro-CT tube with the plane of the tray orientated parallel to the axis of rotation of the CT. This ensured that the long axes of the teeth were aligned parallel to the axis of rotation, resulting in a superior image quality in comparison to other orientations. The trays were secured in the micro-CT tube to ensure no movement for the duration of the scan. The teeth were scanned using a high-resolution micro-CT (Skyscan 1,276, Bruker, Germany) and scanned at an x-ray energy of 100 kV, with an aluminum and copper filter, using a slice thickness of 10 μm. Image data was exported in TIFF (Tagged Image File) format and then converted to AM (AmiraMesh) format for use in a dedicated digital imaging visualization and analysis software (Avizo v2022.2) for viewing by a dentistry and oral surgery resident in training (EC). Multiplanar reconstructions (MPR) in the transverse, sagittal and dorsal planes were utilized for analysis.