X-ray Microscopic Examination of Murine Femora

Figure 1: Rendered cut view along the length of a mouse femur showing both trabecular and cortical bone

Rodent models are widely used in many types of biomedical research because they can mimic aspects of a biological process or disease found in humans. For this research, micro-CT imaging is highly effective for the assessment of trabecular and cortical bone morphology, allowing researchers to measure bone microarchitecture without relying on stereologic models. Researchers can non-destructively acquire the three-dimensional architecture of bone from any site within a small animal. The sample remains intact, allowing researchers to follow up with genetic and histological analysis.

We imaged a mouse femur at high resolution using the SkyScan 2214 nano-CT, obtaining a highly detailed view using an isotropic voxel size of 2 µm. For most studies involving murine bones, the nano-resolution possible on the SkyScan 2214 would not be required for a quantitative analysis. However, some studies do explore the substructure of bone beyond the standard quantitation of volume and density. For these types of applications, the SkyScan 2214 allows a deeper view within the structure of the mouse limbs.

X-Ray Microscopic Imaging of Murine Femora

Figure 2: Planar 2D views within the mouse femur

As shown in Figure 2, the SkyScan 2214 nano-CT captured a highly detailed view through the full length of the bone using an isotropic voxel size of 2 µm.  The proximal and distal growth plates are clear and visible along with the trabecular mesh that extends through the length of the bone.

The growth plate is clearly visible within the distal end of the bone and the trabecular structures grow from this point up towards the proximal end of the bone. Historically, identification of the distal growth plate of a very proximal-distal oriented bone is often the first step in defining a region of interest to examine quantitative differences in trabecular bone growth among samples.

 

Figure 3: Volumetric 3D rendering of the mouse femur

When looking through the 3D view of the full bone, we can easily identify the growth plate, trabecular bone, cortical bone, and other key features examined in most bone imaging studies (Figure 3). We also begin to see some additional porosity within the cortical bone not typically observed at more standard imaging resolutions (8-15 µm).

Figure 4: Planar 2D views of femoral cortical bone

For an even deeper look into the structure of the bone, a second acquisition was completed on the femur, focusing on a region of interest within the cortical bone of the femur around the middiaphysis of the bone at an isotropic voxel size of 500nm (Figure 4). Fine pores and blood vessel pathways are even more pronounced when stepping up to the increased resolution available in this dataset.

Figure 5: Volumetric 3D rendering of the mouse cortical ROI acquired at 500nm voxel size

When looking through the 3D view of the cortical region of interest, the presence of blood vessels and fine pores appear (Figure 5). It may not be common knowledge to all, but despite the general idea of bone as being dense and solid, most types of bone contain many blood vessels and pores.


Figure 6: Volumetric 3D renderings calculated pore diameters overlaid onto the original cortical ROI dataset

Within CTAnalyzer software, we isolated and quantified the diameters of the pores within the cortical region of interest. We then utilized CTVox software to present both the original dataset and the colorized pore diameters simultaneously (Figure 6). While we see fine orange pores throughout the sample, tubular pathways which carry blood through the bone are also visible at larger diameters.

 

 

Figure 7: Quantitative views of all identified pores (top) and the pores isolated by size to highlight the vessels (bottom) within the cortical ROI
Figure 7: Quantitative views of all identified pores (top) and the pores isolated by size to highlight the vessels (bottom) within the cortical ROI

Figure 7 presents two views of our quantitative assessment of the pores within the cortical region of interest, showing either all pores identified or using CTVox to isolate only the largest pores within view to highlight the channels present within the sample. For this region of interest, we determined the bone had a porosity of 4.7%, with about 3% being closed porosity (orange spheres) and about 1.8% being open porosity accessible to the outside or inside of the bone (channels and surface pores). The average pore size was 6.91 ± 4.87 µm throughout the cortical region of interest.

Figure 8: Calculated size distribution of pores within the cortical ROI

We also can present the pore distribution as a function of total pore volume within the region of interest graphically as shown in Figure 8.

Conclusion

The extreme resolution of the SkyScan 2214 nano-CT was a great match in capturing a detailed view of the entire mouse femur as well as capturing a higher resolution view focusing on a region of interest within the sample.

We hope you found this Image of the Month informative and encourage you to subscribe to our newsletter and social media channels in preparation for the continuation of our Image of the Month series next month.

Scan Specifications

Sample Full Bone Cortical ROI
Voltage (kV) 60 60
Current (µA) 178 180
Filter 0.5 mm Aluminum 0.5 mm Aluminum
Pixel Size (µm) 2 0.5
Rotation Step 0.3 0.2
Exposure Time (ms) 1170 6500
Rotation Extent (deg.) 360 180
Scan Time (HH:MM:SS) 17:46:08 18:32:18

 

These scans were completed on our SkyScan 2214 nano-CT system at the Micro Photonics Imaging Laboratory in Allentown, PA. Reconstructions were completed using NRecon while visualization and volumetric inspection of the 2D and 3D results were completed using DataViewer and CTVox. CTAn was utilized to isolate and quantify the pores within the bone.

Would you like your work to be featured in our monthly newsletter? If so, please contact us by calling Seth Hogg at 610-366-7103 or e-mailing seth.hogg@microphotonics.com.

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