The current study was undertaken to adapt Equilibrium Partitioning of an Ionic Contrast agent via microcomputed tomography (EPIC-μCT) to mouse articular cartilage (AC), which presents a particular challenge because it is thin (30 μm) and has a small volume (0.2-0.4 mm(3)), meaning there is only approximately 2-4 μg of chondroitin sulfate (CS) glycosaminoglycan per joint surface cartilage.
Using 6 μm isotropic voxels and the negatively charged contrast agent ioxaglate (Hexabrix), we optimized contrast agent concentration and incubation time, assessed two methods of tissue preservation (formalin fixation and freezing), examined the effect of ex vivo chondroitinase ABC digestion on X-ray attenuation, assessed accuracy and precision, compared young and skeletally mature cartilage, and determined patterns of degradation in a murine cartilage damage model induced by treadmill running.
The optimal concentration of the contrast agent was 15%, formalin fixation was preferred to freezing, and 2 h of incubation was needed to reach contrast agent equilibrium with formalin-fixed specimens. There was good agreement with histologic measurements of cartilage thickness, although μCT over-estimated thickness by 13% (5 μm) in 6-week-old mice. Enzymatic release of 0.8 μg of chondrotin sulfate (about 40% of the total) increased X-ray attenuation by 17%. There was a 15% increase in X-ray attenuation in 14-week-old mice compared to 6-week-old mice (P < 0.001) and this corresponded to 65% decrease in CS content at 14 weeks. The older mice also had reductions of 33% in cartilage thickness and 44% in cartilage volume (P < 0.001). Treadmill running induced a 16% decrease in cartilage thickness (P = 0.012) and a 12% increase in X-ray attenuation (P = 0.006) in 14-week-old mice.
This technique enables non-destructive visualization and quantification of murine femoral AC in three dimensions with anatomic specificity and should prove to be a useful new tool in studying degeneration of cartilage in mouse models.