Quantitative analysis of bone and soft tissue by micro-computed tomography: applications to ex vivo and in vivo studies

Abstract

Micro-computed tomography (micro-CT) is a high-resolution imaging modality that is capable of analysing bone structure with a voxel size on the order of 10 μm. With the development of in vivo micro-CT, where disease progression and treatment can be monitored in a living animal over a period of time, this modality has become a standard tool for preclinical assessment of bone architecture during disease progression and treatment. For meaningful comparison between micro-CT studies, it is essential that the same parameters for data acquisition and analysis methods be used. This protocol outlines the common procedures that are currently used for sample preparation, scanning, reconstruction and analysis in micro-CT studies. Scan and analysis methods for trabecular and cortical bone are covered for the femur, tibia, vertebra and the full neonate body of small rodents. The analysis procedures using the software provided by ScancoMedical and Bruker are discussed, and the routinely used bone architectural parameters are outlined. This protocol also provides a section dedicated to in vivo scanning and analysis, which covers the topics of anaesthesia, radiation dose and image registration. Because of the expanding research using micro-CT to study other skeletal sites, as well as soft tissues, we also provide a review of current techniques to examine the skull and mandible, adipose tissue, vasculature, tumour severity and cartilage. Lists of recommended further reading and literature references are included to provide the reader with more detail on the methods described.

Figure 1

Scout scan of sample holders with specimen. (a) Stack of entire tibiae; (b) stack of entire femora; (c) lumbar spine; (d) entire 2-day-old mouse neonate.

Figure 2

Scanned area for trabecular bone analysis at the proximal tibial metaphysis (a), distal femoral metaphysis (b) and fifth lumbar (L5) vertebra of the spine (c).

Figure 3

Area scanned for cortical bone analysis in tibial (a) and femoral (b) midpoints.

Figure 4

Scanned area for whole skeletal analysis of a 2-day-old mouse neonate.

Figure 5

Region of interest for trabecular bone analysis in tibial metaphysis (a), femoral metaphysis (b) and fifth lumbar (L5) vertebra of the spine (c).

Figure 6

Region of interest for cortical bone analysis in tibial (a) and femoral (b) midshaft.

Figure 7

Region of interest for the skeletal analysis of the entire 2-day-old mouse neonate specified by a regular circle. (a) A single frame showing spine and ribs. (b) A single frame showing the skull.

Figure 8

Greyscale (left) and binarised (middle) slices from a micro-CT image of the distal rat femur. The binarised images are stacked to form a 3D volume (right) for structural analysis.

Figure 9

3D images of 2-day-old mouse neonate (a), tibial trabecular bone (b), femoral trabecular bone (c), vertebral trabecular bone (d), tibial cortical bone (e) and femoral cortical bone (f).

Figure 10

Procedure to transfer baseline scans onto follow-up. The VOI including cortical and trabecular bone of the follow-up image is registered to baseline to generate the transformation matrix [T] (top). The transformation matrix is then inverted and used to apply the baseline VOI of a specific region (that is, trabecular bone) to the follow-up scan for analysis (bottom).

Figure 11

Subtraction images obtained by image registration of micro-CT images at the proximal tibia and lumbar vertebra in a mouse at baseline and 14 days postovariectomy. Localised bone formation (orange) and resorption (blue) can be observed. This technique can be used to determine the amount and rate of bone formation and resorption in vivo over a given time period.

Figure 12

Segmentation of visceral and subcutaneous adipose tissue in a mouse. Regions of lean, visceral fat and subcutaneous fat tissue are shown in a tomographic slice (a), and the corresponding visualisation of the segmentation is shown (b) with the inner visceral fat depicted in red and the outer subcutaneous fat depicted in semitransparent yellow.

Figure 13

Time-lapsed micro-CT image of the development of an osteolytic lesion in the distal femur of a mouse. Baseline (a) and follow-up (b) micro-CT image show the change in bone structure over time. The fusion image (c) obtained after image registration depicts the natural bone growth in orange alongside the osteolytic bone loss in blue.