Mct 35

Tissues from the original defect of each group of mice were fixed in 10% formalin at 4°C for 48 hrs and then scanned by micro‐CT (mCT‐35, Scanco Medical AG, Bassersdorf, Switzerland) with a resolution of 7 μm. The samples were then decalcified in 10% ethylenediaminetetraacetic acid for 2 weeks, embedded in paraffin and cut into serial buccal and lingual sagittal cross sections. The sections were stained with haematoxylin and eosin (H&E) according to standard methods. Newly formed bone in the H&E‐stained sections was quantified in four sections with three different defects per treatment. H&E stains were examined and photographed with a Nikon Eclipse E600 microscope and Spot Advanced software (Diagnostic Instruments, Sterling Heights, MI, USA). Then, six images of three slices of each specimen were randomly collected and were randomly collected under the same exposure value at high magnification (×200). And the inner field of the defect area of each H&E staining slice was determined under low magnification (×40). The area (mm²) percentages of the new bone to the area of the defect were calculated using Image‐Pro Plus bioimage professional analysis software. Furthermore, the boundary of the defects around the tooth root was defined, and the new alveolar bone, PDL and cementum were observed.

Micro-CT imaging and analysis were performed using a benchtop, cone-beam animal scanner (mCT 35; SCANCO Medical, Switzerland). The parameters were 70 kVp and 141 μA; we used a 0.5-mm-thick aluminum filter. The pixel size was 8.0 μm and the rotation step 0.4°C. Cross-sectional images were reconstructed using a filtered back-projection algorithm (NRecon software, Bruker Micro CT, Belgium). For each scan, a stack of 286 cross-sections was reconstructed at 2,000 × 1,335 pixels. Bone volume and surface were analyzed at the femur.

Dissected mouse knee joints were fixed in 4% paraformaldehyde overnight at 4 °C and replaced with PBS before micro-CT analysis. Knee joints were scanned at an isotropic voxel size of 7 μm, with a peak tube voltage of 70 kV and current of 0.114 mA (mCT 35; Scanco Medical AG, Bassersdorf, Switzerland). A three-dimensional Gaussian filter (s = 0.8) with a limited, finite filter support of one was used to suppress noise in the images, and a threshold of 220–1 000 was used to segment mineralized bone from the air and soft tissues. The region of interest was selected for calcified osteophyte tissues, and the bone volume was calculated to measure the osteophyte size.

Micro-computed tomography imaging was performed using a bench-top cone-beam-type in vivo animal scanner (mCT 35; Scanco Medical, Brüttisellen, Switzerland). Mice were euthanized; their spines were dissected and immediately placed in 10% neutral buffered formalin, where they were incubated for 2 weeks. Samples were imaged at settings of 60 kVp and 166 μA using a 0.25-mm-thick aluminum filter. The pixel size was 16 μm and the rotation step was 0.6°. Cross-sectional images were reconstructed using a filtered back-projection algorithm (NRecon software; Bruker microCT, Kontich, Belgium). For each scan, a stack of 286 cross-sections was reconstructed at 2,000 × 1,335 pixels.