Skyscan1175

Calvaria were dissected free and evaluated using a SkyScan1175 (Bruker, MA, USA) high-resolution microCT imaging system. Each calvaria was scanned separately at 65 kV and 153 µA with a 1.0-mm aluminum filter to obtain a 10 µm voxel size. Scan slices were acquired in the coronal plane by placing the bone parallel to the z-axis of the scanner. NRecon (Bruker) was used to reconstruct images using the following settings: smoothing 1, ring artifact reduction of 5% and beam hardening correction of 20%, and quantitative analysis was performed using CTAn (Bruker) in accordance with the recommendations of the American Society for Bone and Mineral Research.^{38 (link)} Volumes of interest were disc shaped to encompass the circular bone defect and were 4.3 mm in diameter. All analysis was performed in a blinded fashion.

For all micro-CT analysis, mice knee joints were fixed overnight in 10% formalin at 4°C, and then scanned at a voltage of 55 kVp, a current of 181 μA, and a resolution of 9.0 μm per pixel by high-resolution micro-CT (Bruker MicroCT, Skyscan 1175) (Hu et al., 2020 (link)). We used NRecon image reconstruction software, version 1.6 (Bruker MicroCT), CTAn data-analysis software, version 1.9 (Bruker MicroCT) to reconstruct and analyze the parameters of the tibia subchondral bone. 10 coronal images of the medial suchondral bone comartment were selected and used for 3D reconstruction using CTVol v2.0 (Bruker MicroCT).

For micro-CT analysis, the mice femur was fixed overnight in 10% formalin at 4°C and then scanned and analyzed by a high-resolution micro-CT (Bruker MicroCT, Skyscan 1175, Belgium). The X-ray was set at 65 kV, 153 μA, and a resolution of 11.0 μm/pixel. We used NRecon image reconstruction software (version 1.6, Bruker MicroCT) and CTAn data-analysis software (version 1.9, Bruker MicroCT) to reconstruct and analyze the subchondral trabecular bone at the injury site. To select the region of interest (ROI) in analysis of the trabecular bone, we first identified the subchondral bone and drew the regions of interest from below the injured articular surface (with a diameter of 1mm) and extended toward the distal direction for proximally 0.7mm in length. The trabecular bone was analyzed to determine trabecular BV/TV, Tb. Th, Tb. N, and Tb. Sp.