Skyscan 1172

The excised right femurs were scanned using a high-resolution micro-CT scanner (SkyScan1172, Bruker, USA) at a resolution of 15 μm, with a voltage of 80 kV, a current of 100 μA, a 0.5 mm aluminum filter, a rotation step of 0.6° and an exposure time of 360 ms. Images were then reconstructed using bundled software Nrecon1.7. Another bundled software, DataViewer1.4.3, was employed to orient the cross-sectional images parallel to the transaxial plane. A cylindrical volume of interest (VOI) with a diameter of 1 mm and height of 1 mm was placed randomly in the distal femoral metaphysis. See an example shown in Figures 1A–C.
Morphometric analysis of the VOI trabecular bone was performed using CTAn1.16 (SkyScan1172, Bruker, USA). A cohort of global threshold lower limits ranging from 65 to 100 at an interval of 5 was chosen to segment out trabeculae under the same VOI region. Different global thresholds will lead to microstructural models exhibiting various degrees of osteoporosis. The higher the global threshold chosen, the higher the degree of simulated osteoporosis (Figure 2A). The trabecular parameters BV/TV, Tb. Th, Tb. Sp, were measured for each VOI. The VOI trabecular bones were also saved in “stl” file format for subsequent 3D model optimization and 3D printing.

Femurs, calvariae, vertebrae, and jaws were dissected from WT or Il1rn^−/− mice and fixed with 4% paraformaldehyde for 48 h, then stored in 70% ethanol at 4 °C. Scans were obtained with a SCANCO Medical machine and ex vivo µCT analyses were performed with Skyscan 1172 Software (Bruker, Germany). The region of interest (ROI) was set using a manually determined global threshold. Three-dimensional microstructural bone properties, including the bone volume fraction (BV/TV), specific bone surface (BS/BV), trabecular thickness (Tb.Th.), and trabecular separation (Tb.Sp.), were calculated according to the manufacturer’s software.

The mandibles of Mmp13^−/− and WT mice were fixed in 70% ethanol and prepared for high-resolution µCT (SkyScan 1172, Bruker, Kontich, Belgium) of incisor and first molar teeth. A three-dimensional analysis was carried out to determine total volume, enamel volume, dentin volume, pulp volume, and total mineral density (TMD). The samples were scanned using a 10-MP digital detector, 10W of energy (70 kV and 142 mA), and a pixel size of 7.5microns, exposure 850 ms/frame rotation step 0.3° with ×10 frame averaging, 0.5 mm aluminum filter, and scan rotation of 180°. After scanning, the radiographs were reconstructed using NRecon software (version 1.7.3.0; Bruker). Reconstruction was conducted with NRecon using GPU acceleration. Gaussian smoothing was applied with a 2-voxel radius, ring artifact and beam hardening corrections were applied in reconstruction. Ring artefact reduction set to 7 pixels. Beam hardening correction was set to 40%. CTAn software (CTAn Micro-CT software, Bruker) was used to generate 2-D images for color density and 3-D images for CT volume.

After 60 days, SB and SBlyo were collected, washed with PBS to remove the remaining medium, and fixed in a neutral buffer containing 4% v/v formaldehyde. Samples were analyzed using high-resolution X-ray microtomography (SkyScan 1172, Bruker, Billerica, MA, USA) to study the structure and quantify the newly formed tissue after SVF colonization [27 (link)]. Acquisitions were performed at 80 kV using a 0.5 mm Al filter at a resolution of 6 m, 0.4° of rotation step, 360° scan, and 4× frame averaging. Datasets were reconstructed with NRecon software (Bruker, Billerica, MA, USA). A color contrast mask was used to identify the new formed mineralized tissue. Two expert operators performed quantification on axial slices, measuring the mineralized tissue length using DataViewer.