Micro-Scale Distribution of CA4+ in Ex vivo Human Articular Cartilage Detected with Contrast-Enhanced Micro-Computed Tomography Imaging

Abstract

Contrast-enhanced micro-computed tomography (CEµCT) with cationic and anionic contrast agents reveals glycosaminoglycan (GAG) content and distribution in articular cartilage (AC). The advantage of using cationic stains (e.g., CA4+) compared to anionic stains (e.g., Hexabrix ® ), is that it distributes proportionally with GAGs, while anionic stain distribution in AC is inversely proportional to the GAG content. To date, studies using cationic stains have been conducted with sufficient resolution to study its distributions on the macro-scale, but with insufficient resolution to study its distributions on the micro-scale. Therefore, it is not known whether the cationic contrast agents accumulate in extra/pericellular matrix and if they interact with chondrocytes. The insufficient resolution has also prevented to answer the question whether CA4+ accumulation in chondrons could lead to an erroneous quantification of GAG distribution with low-resolution µCT setups. In this study, we use high-resolution µCT to investigate whether CA4+ accumulates in chondrocytes, and further, to determine whether it affects the low-resolution ex vivo µCT studies of CA4+ stained human AC with varying degree of osteoarthritis. Human osteochondral samples were immersed in three different concentrations of CA4+ (3 mgI/ml, 6 mgI/ml, and 24 mgI/ml) and imaged with high-resolution µCT at several timepoints. Different uptake diffusion profiles of CA4+ were observed between the segmented chondrons and the rest of the tissue. While the X-ray -detected CA4+ concentration in chondrons was greater than in the rest of the AC, its contribution to the uptake into the whole tissue was negligible and in line with macro-scale GAG Karhula et al. CA4+ Accumulation in Human Cartilagecontent detected from histology. The efficient uptake of CA4+ into chondrons and surrounding territorial matrix can be explained by the micro-scale distribution of GAG content. CA4+ uptake in chondrons occurred regardless of the progression stage of osteoarthritis in the samples and the relative difference between the interterritorial matrix and segmented chondron area was less than 4%. To conclude, our results suggest that GAG quantification with CEµCT is not affected by the chondron uptake of CA4+. This further confirms the use of CA4+ for macro-scale assessment of GAG throughout the AC, and highlight the capability of studying chondron properties in 3D at the micro scale.

Triple Contrast CT Method Enables Simultaneous Evaluation of Articular Cartilage Composition and Segmentation

“…2,24,25 Despite this, the widespread use of CA4+ is hindered due to suboptimal performance at imaging times during early diffusion (0-2 h from contrast agent administration). 18,22 The distribution of cationic contrast agents with articular cartilage is proportional to PG content as the negative fixed charge carried by PG molecules attracts the positively charged contrast agents. However, especially in the early stage of diffusion, the diffusion of cationic contrast agent is also controlled by degeneration-related factors having opposite effects; the loss of PGs decreases the diffusion of cationic contrast agent while increase in water content and decrease in steric hindrance (i.e., physical diffusion barrier of the tissue caused by collagen network architecture and PGs in the matrix) increase the diffusion.…”

Synchrotron MicroCT Reveals the Potential of the Dual Contrast Technique for Quantitative Assessment of Human Articular Cartilage Composition

“…Cationic contrast agents are more sensitive to cartilage PG content at diffusion equilibrium than anionic agents due to electrostatic attraction between cationic molecules and negatively charged PGs. However, cationic contrast agents have limitations in assessing PG content of cartilage at clinically feasible early diffusion time points, that is, 1–2 h after contrast agent administration . At early diffusion time points, contrast agent diffusion into cartilage is affected by water content and surface permeability as well as by the PG content, diminishing the sensitivity of cationic contrast agents to variations in PG content.…”

“…However, cationic contrast agents have limitations in assessing PG content of cartilage at clinically feasible early diffusion time points, that is, 1-2 h after contrast agent administration. 18 At early diffusion time points, contrast agent diffusion into cartilage is affected by water content and surface permeability as well as by the PG content, diminishing the sensitivity of cationic contrast agents to variations in PG content. As a solution, we introduced a dual contrast agent (a mixture of two contrast agents) and a dual energy CT technique (two X-ray energies) for imaging at diffusion equilibrium.…”

“…The contrast agent series for (A) CA4+ (iodine, I) with3,6,12,18,24,30,36, and 42 mg I/ml and (B) gadoteridol (gadolinium, Gd) with 6, 9, 12, 15, and 18 mg Gd/ml were imaged with energies of 32 and 34 keV separately to determine the iodine and gadolinium mass attenuation coefficients. The figures show the X-ray attenuation as a function of the true concentration of the contrast agents.…”

Deep-Learning for Tidemark Segmentation in Human Osteochondral Tissues Imaged with Micro-computed Tomography

“…Thus, direct µCT imaging of soft tissues, such us AC, is not possible. To mitigate this limitation of X-ray imaging, several contrast agents have been introduced to provide X-ray attenuation contrast for the AC, such as phosphotungstic acid (PTA), CA4+ and others [14,10,6].…”