Contrast-Enhanced Micro–Computed Tomography for 3D Visualization and Quantification of Glycosaminoglycans in Different Cartilage Types

Abstract

Objective To compare CA4+-enhanced micro–computed tomography (microCT) of bovine articular, meniscal, nasal, and auricular cartilage, each of which possesses a different extracellular matrix (ECM) composition and structure. Design The diffusion kinetics of CA4+ in different native cartilage types were assessed over 20 hours. The feasibility of CA4+-enhanced microCT to visualize and quantify glycosaminoglycans (GAGs) in these different tissues was tested using safranin-O staining and 1,9-dimethylmethylene blue assay. Results The diffusion kinetics of CA4+ in auricular cartilage are significantly slower compared with all other cartilage types. Total GAG content per volume correlates to microCT attenuation with an R2 value of 0.79 for all cartilage types. Three-dimensional contrast-enhanced microCT images of spatial GAG distribution reflect safranin-O staining and highlight the differences in ECM structure, with heterogeneous regions with higher GAG concentrations highlighted by the contrast agent. Conclusions CA4+-enhanced microCT enables assessment of 3-dimensiona distribution and GAG content in different types of cartilage and has promise as an ex vivo diagnostic technique to monitor matrix development in different tissues over time as well as tissue-engineered constructs.

Anisotropic Articular Cartilage Biofabrication based on Decellularized Extracellular Matrix

“…The GAGs/DNA ratio in the weight-bearing regions of human articular cartilage is approximately 0.2 µg ng -1 . [91] Our engineered cartilage grafts surpassed this value, indicating that cells were very metabolically active during this period. Chondrogenic hypertrophy of adult hMSCs, marking the terminal stage of chondrocyte differentiation, is undesirable in cartilage regeneration approaches.…”

Non‐enzymatic glycation reduces glucose transport in the human cartilage endplate independently of matrix porosity or proteoglycan content

“…Contrast‐enhanced micro‐CT via equilibrium partitioning of an ionic contrast agent is a nondestructive technique used to estimate sGAG content in cartilaginous tissues. Owing to the net negative charge of the sGAGs, immersion of cartilage and disc tissues in the cationic contrast agent CA 4+ results in uptake of the positively charged contrast agent, and inter‐sample differences in the measured micro‐CT attenuation are positively correlated with inter‐sample differences in the mean sGAG content 29 . Here, we used the iodinated cationic contrast agent CA 4+ , 19,20,29,30 which was synthesized as described previously 19,20 .…”

“…Owing to the net negative charge of the sGAGs, immersion of cartilage and disc tissues in the cationic contrast agent CA 4+ results in uptake of the positively charged contrast agent, and inter‐sample differences in the measured micro‐CT attenuation are positively correlated with inter‐sample differences in the mean sGAG content. 29 Here, we used the iodinated cationic contrast agent CA 4+ , 19 , 20 , 29 , 30 which was synthesized as described previously. 19 , 20 Four site‐ and donor‐matched pairs of CEP samples were incubated in the ribose solution (one sample from each pair for 0 days [control] and one for 9 days) and then scanned at baseline with a micro‐CT scanner (Scanco, μCT50).…”

A Cationic Contrast Agent in X-ray Imaging of Articular Cartilage: Pre-Clinical Evaluation of Diffusion and Attenuation Properties

“…A novel cationic iodinated contrast agent, CA4+, was specifically designed to electrostatically interact with glycosaminoglycans (GAGs) bound in proteoglycans [ 16 ]. In particular, the Coulomb attraction between negative fixed charges of GAGs and positively-charged CA4+ molecules showed superior contrast enhancement compared to commercially available contrast agents, (even at lower concentrations [ 17 , 18 , 19 ]), in animal [ 20 , 21 , 22 , 23 ] and human [ 24 , 25 , 26 , 27 , 28 , 29 ] samples, on several types of tissue containing GAGs [ 13 , 30 , 31 ] and cartilage tissue-engineered constructs [ 32 ]. The use of CA4+ was investigated in combination with synchrotron radiation as well, exploiting the high brilliance and monochromaticity of X-ray beam [ 33 , 34 , 35 , 36 ].…”