The L3–5 vertebrae and femur were harvested, fixed in 4% paraformaldehyde (PFA) for 1 day at 4 °C, and washed with PBS. The fixed samples were placed in centrifuge tubes containing styrofoam spacers, placed in a μ-CT scanner (vivaCT 80, Scanco Medical, Wayne, PA). Samples were scanned at 55 keV with a pixel resolution of 10.4 μm. Reconstruction was made of the images using μ-CT Evaluation Program V6.6 (Scanco Medical). Bone mineral density was quantified and expressed as percentage of bone volume (BV) per total volume (TV) (BV/TV) using the phantom as a reference from 100 contiguous slices. Trabecular number (Tb.N), trabecular spacing (Tb. Sp), connectivity density (Conn. D), and trabecular thickness (Tb. Th) were quantified through μ-CT Evaluation Program V6.6 (Scanco Medical) software. Vertebral-3D model images were acquired using CTAn software (n = 4–5 per group).
μct evaluation program v6
Manufactured by Scanco
Sourced in Switzerland
The μCT Evaluation Program V6.6 is a software tool designed for the analysis and visualization of micro-computed tomography (μCT) data. It provides a comprehensive platform for viewing, processing, and evaluating μCT scans.
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13 protocols using μct evaluation program v6
1
Microstructural Analysis of Vertebral and Femoral Bone
2
Microstructural Analysis of Knockout Dentin
3
Quantifying Intramuscular Adipose Tissue via μCT
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The specimens were scanned at a resolution of 8.4 μm isotropic voxel size at a beam energy of 70 kVp, current of 114 μA, and 200 ms of integration time (μCT40, Scanco Medical, Brüttisellen, Switzerland). A stack of sixty slices covering 0.480 mm were scanned at the middle of each gastrocnemius muscle. The images were analyzed using the manufacturer’s evaluation program (μCT Evaluation Program v6.0, Scanco) according to a custom analysis protocol, determining the IMAT volume based on segmentation. Analyses of muscle tissues and IMAT were performed at the region of interest (ROI) consisting of a contour covering each muscle, spanning all slices of the scan. A Gaussian filter was applied to all images to reduce noise (sigma = 2.5, support = 5) as noise is known to influence the quantification of IMAT [30 (link)]. “Intra”-muscular IMAT was segmented using a lower threshold of −600 HU (Hounsfield units), which was supported by Erlandson et al. for the segmentation of fat for the evaluation of muscle and myotendinous tissue [23 (link)], and an upper threshold of 100 HU, which produced the best correlation between MD and IMAT in our preliminary study. To avoid border artefacts, the defined ROI was peeled with 3 voxels. The ROI, MD, and percentage of IMAT volume over the total ROI volume (IMAT%) were determined.
4
Alveolar Bone Microstructure Analysis
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Scans for volume, density, and bone mass of mandible alveolar bone were performed with a microtomograph imaging system (μCT 50, Scanco Medical AG, Brüttisellen, Switzerland). The scanning parameters were X-ray bulb voltage 90 kV, current 200 μA, resolution 9 μm, and exposure time 500 ms. The images were processed by three-dimensional reconstruction software (μCT Evaluation Program v6.0, Scanco Medical) and analyzed for determination of bone mass and density.
Bone mass analysis was performed on the alveolar bone of the first molar root bifurcation. The VOI (volume of interest) of the analysis region was selected as a cube with a length of 270 μm below the alveolar crest of the left molar mandibular root bifurcation. The middle 30 layers were selected for trabecular bone parameter analysis, including bone mineral density (BMD), bone volume fraction (bone volume per total volume, BV/TV) (%), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular bone number (the mean trabecular number, Tb.N). Each sample was analyzed three times to calculate the average value.
Bone mass analysis was performed on the alveolar bone of the first molar root bifurcation. The VOI (volume of interest) of the analysis region was selected as a cube with a length of 270 μm below the alveolar crest of the left molar mandibular root bifurcation. The middle 30 layers were selected for trabecular bone parameter analysis, including bone mineral density (BMD), bone volume fraction (bone volume per total volume, BV/TV) (%), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular bone number (the mean trabecular number, Tb.N). Each sample was analyzed three times to calculate the average value.
5
Quantifying Femoral Cortical Bone Microarchitecture
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Femurs were harvested from 8-week and 12-month old mice, cleaned of soft tissue and placed in a 20.5 mm tube. Samples were scanned horizontally, approximately 300 slices per sample, with a desktop cone-beam μCT scanner (SCANCO Model μCT40; SCANCO Medical). Femurs were then rotated to transverse slices and analyzed. Cortical bone was measured at the midshaft (50 % of the length of the femur), and then 50 slices were included on either side (100 slices total). Contours for cortical bone were drawn using the midshaft contour script (#16), and each was inspected & modified for best fit before analysis. Cortical bone was analyzed using μCT Evaluation Program V6.0 (SCANCO Medical) with the midshaft cortical bone script (#23) with multiple thresholds to gauge best fit, and a threshold of 350 was selected to be the most appropriate.
6
Alveolar Bone Density and Eruption Rate
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Mice were sacrificed using an excess of isoflurane anesthesia at 0 and 12 days after surgery. The mandibular alveolar bone was harvested, and bone specimens were scanned with a micro-CT scanner equipped with a custom software package (Micro-CTCH-8306, Scanco Medical, Basserdorf, Switzerland). Specimens were scanned at 70 kVp and 114 μA at high resolution (37 μm). The images were processed by three-dimensional reconstruction software (μCT Evaluation Program v6.0, Scanco Medical). Taking the line of the outermost tangent point above the bilateral mandibular foramen of mice as the reference line, the distance between the two parallel lines at 12 day after tooth extraction was measured from the highest point of the right mandibular first molar as the parallel line with the reference line, and the distance between the two parallel lines at 0 days after tooth extraction was subtracted as the eruption rate. Each sample was analyzed by two different investigators independently. The analysis software Mimics Medical 17.0 was used to measure bone mineral density (BMD).
7
Micro-CT Analysis of Knee Samples
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Knee samples were fixed in 3.5% formafix for 24 h. Afterwards, micro-CT scanning was carried out using Scanco vivaCT 80 (SCANCO Medical, Brüttisellen, Switzerland) with a voxel size of 15.6 µm at 70 kVp, 113 µA and 400 ms integration time. µCT Ray V4.0-4 (Scanco Medical AG, Brüttisellen, Switzerland) and imageJ were used to generate the representative images. For subchondral trabecular bone and osteophyte evaluation, the volume-of-interest (VOI) was defined by manual contouring and calculated using the μCT Evaluation Program V6.6 (Scanco Medical AG, Brüttisellen, Switzerland). Data were reported according to the guidelines for tissue imaging by the American Society of Bone and Mineral Research45 (link).
8
Calvaria Bone Micro-CT Analysis
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The calvaria bones were scanned using high-resolution micro-CT (μCT-50, SCANCO Medical AG). The scanning parameters were as follows: 70 kV, 114 μA, and 7 μm. The μCT Evaluation Program V6.6 (SCANCO, Switzerland) was used to generate three-dimensional (3D) images and analyze the bone volume/tissue volume (BV/TV).
9
Micro-CT Analysis of Ankle Bone Volume
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Right ankle joints were fixed in 10% formalin for 48 h, washed in phosphate-buffered saline (PBS) for 2 h and then soaked in 75% ethanol, scanned by micro-CT system (Scanco VIVA CT80, SCANCO Medical AG, Switzerland). The scanning parameters were as follows: pixel size 15.6 μm, tube voltage 55 kV, tube current 72 μA, integration time 200 ms. The cross-section images were then reconstructed and realigned in 3D, the bone volume (BV) of astragalus were measured and a density threshold was set from 370 to 1000 as Bone by μCT Evaluation program V6.6 (Scanco Medical AG, Switzerland). A stack of 340–441 cross-sections was reconstructed, with an inter slice distance of 1 pixel (15.6 μm), corresponding to a reconstructed height of 5.3–6.9 mm, recreating the ankle joints.
10
Micro-CT Analysis of Arthritis Joints
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The knee and ankle joints of each experimental mouse were scanned using micro-CT system (Scanco VIVA CT80; SCANCO Medical AG, Brüttisellen, Switzerland). Images were acquired at 55 kV, 72 μA and 300 ms/frame, with 360 views. The 3-dimensional structures of knee joint and ankle joint were reconstructed and evaluated by μCT Evaluation Program V6.6 (SCANCO Medical AG). Three-dimensional bone volume of patellas and astragalus was measured using Aquarius software (T Ray V4.04) to determine the volumetric change of arthritis joints, and a density threshold was set higher than 11,000 AU as “bone”. Analysis typically took around 15 min per joint for an experienced operator.
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