Contrast-enhanced CT using a cationic contrast agent enables non-destructive assessment of the biochemical and biomechanical properties of mouse tibial plateau cartilage

Abstract

Mouse models of osteoarthritis (OA) are commonly used to study the disease's pathogenesis and efficacy of potential treatments. However, measuring the biochemical and mechanical properties of articular cartilage in these models currently requires destructive and time-consuming histology and mechanical testing. Therefore, we examined the feasibility of using contrast-enhanced CT (CECT) to rapidly and non-destructively image and assess the glycosaminoglycan (GAG) content. Using three ex vivo C57BL/6 mouse tibial plateaus, we determined the time required for the cationic contrast agent CA4+ to equilibrate in the cartilage. The whole-joint coefficient of friction (μ) of 10 mouse knees (some digested with Chondroitenase ABC to introduce variation in GAG) was evaluated using a modified Stanton pendulum. For both the medial and lateral tibial plateau cartilage of these knees, linear regression was used to compare the equilibrium CECT attenuations to μ, as well as each side's indentation equilibrium modulus (E) and Safranin-O determined GAG content. CA4+ equilibrated in the cartilage in 30.9 ± 0.95 min (mean ± SD, tau value of 6.17 ± 0.19 min). The mean medial and lateral CECT attenuation was correlated with μ (R(2) = 0.69, p < 0.05), and the individual medial and lateral CECT attenuations correlated with their respective GAG contents (R(2) ≥ 0.63, p < 0.05) and E (R(2) ≥ 0.63, p < 0.05). In conclusion, CECT using CA4+ is a simple, non-destructive technique for three-dimensional imaging of ex vivo mouse cartilage, and significant correlations between CECT attenuation and GAG, E, and μ are observed. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1130-1138, 2016.

Keywords: coefficient of friction; compressive modulus; glycosaminoglycan; micro-computed tomography; osteoarthritis.

Figure 1

Diffusion-in data for CA4+ into the cartilage of three mouse tibial plateaus. Data is plotted as the mean CECT attenuation of both the medial and lateral tibial plateau cartilage normalized by the mean baseline (not contrast-enhanced) attenuations (lines are for visualization purposes only). By fitting the data for each sample with an equation of the form CECT Atten. = a*e^−b(time) + c, tau values (calculated as 1/b) can be computed for each sample, and the mean tau value was found to be 6.17 ± 0.19 min (mean ± SD).

Figure 2

Standard curve correlating mean Ip Value (a measure of the red intensity from Safranin-O stained histology sections) to GAG content determined via the DMMB assay for 42 depth-wise layers obtained from six neighboring bovine cartilage plug pairs. The correlation was R² = 0.93, and statistically significant (p < 0.05), indicating that quantifying the red content from Safranin-O stained histology sections reflects the GAG content of the cartilage tissue.

Figure 3

Correlations for mouse medial ( ) and lateral ( ) tibial plateau surfaces between: a) mean CECT attenuation and GAG content, b) indentation equilibrium modulus (E) and GAG content, and c) mean CECT attenuation and E. All correlations had R² > 0.6 and were statistically significant (p < 0.05) for both surfaces.

Figure 4

Correlations between a) pendulum coefficient of friction (μ) and average tibial plateau GAG content and b) mean tibial plateau cartilage CECT attenuation and μ for mouse knees. Both correlations had coefficients of variation greater than or equal to 0.59, and were statistically significant (p<0.05).

Figure 5

Using contrast-enhanced CT, color maps can be generated in any orientation for visualization of depth-wise cartilage attenuation with the underlying bone morphology and attenuation also visible. For example, traditional sagittal CECT color maps can be generated for the medial tibial plateau samples with a) the highest GAG content and b) the lowest GAG content found in this study, which are analogous to their corresponding Safranin-O stained histological sections (c & d, respectively). CECT also enables 3D color map generation that can be visually sectioned in any view independently or up to three planes simultaneously, as demonstrated by the 3D color maps for the same e) highest GAG and f) lowest GAG samples. By zooming into the regions encompassed in the red squares, it is easier to distinguish the difference in depth-wise attenuation patterns for the same g) high GAG and h) low GAG samples. Note that the dotted lines in g and h separating the articular cartilage surface (Cart. Surface), cartilage (Cart.), and bone are manually drawn for visualization purposes only, and the actual cartilage-bone and cartilage-air boundaries were found quantitatively using the region-growing algorithm in the Analyze software “Volume Edit” module. The color scale bar indicates the corresponding CECT attenuation in Hounsfield Units (HU) for all CECT color maps. The distribution of the CA4+ contrast agent matches the typical distribution of Safranin-O stain in histological sections, with a greater attenuation in the middle and deep zone for the sample with high GAG and a fairly uniform distribution for the sample with low GAG.