Microview software

Three-dimensional images of the implants and surrounding tissue were produced by Micro-CT and used for quantification of the newly formed bone. Ex vivo acquisitions at 15 µm resolution were performed from 900 X-ray radiographs obtained on an Explore Locus SP X-Ray µCT device (General Electric, Milwaukee/WI, USA) with a source voltage of 80 kV, a current of 80 µA, and an exposure time of 3000 ms. Reconstruction of the region of interest was performed after correction of the center of rotation and calibration of mineral density. After calibration, three dimensional analyses were performed using Microview software (GE Healthcare Inc., Princeton/NJ, USA). The threshold for newly formed bone was determined for each scan individually. Quantification included Bone Mineral density (BMD), and Bone Mineral Content (BMC) as defined previously [51] (link). BMC values were analyzed from regions of interest (ROI) of a fixed volume. Six ROI throughout the defect were determined for each sample. The means of all ROI per sample were analyzed further to evaluate the means with standard deviation of six samples per condition at each time point. For the analysis of BMD, the entire defect site of six samples per condition was taken into account and the results of six samples per condition are shown as the means with standard deviation.

Tomographic scans were performed in vivo on anesthetised mouse using a ExploreLocus 80 Micro-CT (GE Healthcare), with the following parameters : X-ray tube (80 kV), current (450 μA), exposure time (400ms), angular rotation (360°), angular increment (0.5°), voxel (45 μm isotropically). Datasets were reconstructed using reconstruction algorithm provided with the scanner. Analysis was performed with GE Healthcare Microview software.

Formalin-fixed bones were imaged using a Faxitron MX-20 digital x-ray system with a 24kV, 4-s exposure time for 2D analysis. For whole-body micro-CT imaging, formalin-fixed mouse skeletons were placed in a GE Locus Ultra Micro-CT (GE Medical Systems) and subjected to a 16-s Anatomical Scan Protocol (total of 680 images) at 80 kV, 70 mA, using a 0.15-mm Cu Filter, to achieve ∼150-µm resolution. The same machine was also used for live-mouse imaging at acquisition parameters 80 kV, 50 mA; 16-s anatomical scan; 154-µm isotropic voxels (total of 680 slices) and 3D rendered using Siemens Inveon. For high-resolution micro-CT imaging, fixed mouse legs were immobilized on 1.25% agarose. Specimens were scanned in 360° rotation using a Siemens Inveon Micro-CT high-resolution scanner (Siemens Medical Systems) with the x-ray source at 80 kVp and 0.5 μA. 3D micro-CT data were reconstructed at 13.5-µm resolution. Raw data processing was performed using ImageJ software (National Institutes of Health), and 3D isosurfaces were rendered using Microview software (GE Healthcare). Bone length was measured digitally with ImageJ.

Rat femora were examined by 3D micro-CT (GE eXplore Locus micro-CT system for small animals, RS-9, GE Healthcare, Ontario, Canada) under anesthesia with 10% chloral hydrate (0.3 mL/100 g). Femora were scanned at an isotropic voxel size of 0.046 mm. A fixed length of bone of 100 image layers, located between the proximal and distal boundaries of the callus, was analyzed. The outer boundary of the callus and the area enclosed by the periosteal surface of the preexisting cortical bone were defined by automated segmentation; the volume enclosed by these two surfaces was the callus volume of interest. The MicroView software (GE Healthcare, Ontario, Canada) was used to reconstruct and process the 3D images and calculate the following bone morphometric parameters: total callus volume (TCV) and tissue mineralized density (TMD) [21 (link)]. Mineralized tissue was distinguished from unmineralized and poorly mineralized tissue using a fixed global threshold of 25% of the maximum gray value, corresponding to a mineral density of 641.9 mg HA/cm³ and approximately 45% of the attenuation of mature cortical bone in the specimens examined. This value was based on visual inspection of the tomograms and qualitative comparison with paired, decalcified histological sections from additional specimens.

Specimens were immobilized using cotton gauze and scanned using a GE Locus SP microCT scanner to produce 14 μm voxel size volumes, using X-ray settings of 80 kVp, 118 uA and a 0.02 mm aluminium filter to attenuate harder X-rays. Three-dimensional isosurfaces were generated and measured using the dvanced region of interest and isosurface tools of the Microview software package (GE). Images were rendered using Drishti30 (link).