In vivo x-rays were obtained using an Xtreme scanner (Bruker, Billerica, MA) at 45 kilovolt peak (kVp), 0.4 mA, and 2-s exposure time. A binary bridging score was assigned by two independent, blinded observers and determined as mineralized tissue fully traversing the defect. In vivo microCT scans were performed at 4, 8, and 12 weeks to assess longitudinal defect healing. For initial studies, animals were scanned using an Inveon microPET/CT system (Siemens Medical Solutions, Malvern, PA) at 45 kVp, 0.2 mA, and 35-μm isotropic voxels. Data were reconstructed using system default parameters for analyzing bone and accounting for the metal in the fixation plates. DICOM-exported files were processed for three-dimensional (3D) analysis (CTAn software, SKYSCAN/Bruker) using a gauss filter at 1.0 pixel radius and a global threshold range of 28 to 255 for all samples. Bone volume was determined in a standard region of interest (ROI) spanning the length of the defect. For the main study, animals were scanned using an Albira PET/SPECT/CT system (Bruker) at 45 kVp, 0.4 mA, and 125-μm voxel size. A global threshold was applied for each dataset, and bone volume was determined in a standard ROI spanning the length of the defect.

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