Murine articular cartilage morphology and compositional quantification with high resolution cationic contrast‐enhanced μCT

Abstract

Articular cartilage lines the load-bearing surfaces of long bones and undergoes compositional and structural degeneration during osteoarthritis progression. Contrast enhanced microcomputed tomography (μCT) is being applied to a variety of preclinical models, including the mouse, to map structural and compositional properties in 3-D. The thinness (~30–50 μm) and high cellularity of mouse articular cartilage presents a significant imaging challenge. Our group previously showed that mouse articular cartilage and proteoglycan (PG) content can be assessed by μCT with the ioxagalate-based contrast agent Hexabrix, but the voxel size used (6 μm) was deemed to be barely adequate. The objective of the present study is to assess the utility of a novel contrast agent, CA4+, to quantify mouse articular cartilage morphology and composition with high resolution μCT imaging (3 μm voxels) and to compare the sensitivity of CA4+ and Hexabrix to detect between-group differences. While both contrast agents are iodine-based, Hexabrix is anionic and CA4+ is cationic so they interact differently with negatively charged PGs. With CA4+, a strong correlation was found between non-calcified articular cartilage thickness measurements made with histology and μCT (R2 = 0.72, p < 0.001). Cartilage degeneration – as assessed by loss in volume, thickness and PG content – was observed in 34-week old mice when compared to both 7- and 12-week old mice. High measurement precision was observed with CA4+, with the coefficient of variation after repositioning and re-imaging samples equaling 2.8%, 4.5%, 7.4% and 5.9% for attenuation, thickness, volume, and PG content, respectively. Use of CA4+ allowed increased sensitivity for assessing PG content compared to Hexabrix, but had no advantage for measurement of cartilage thickness or volume. This improvement in imaging should prove useful in preclinical studies of cartilage degeneration and regeneration.

Multi-scale imaging techniques to investigate solute transport across articular cartilage

“…Collagen distribution in ex-vivo non-calcified cartilage was successfully determined using micro -CT (voxel size = 3.2×3.2×3.2 µm 3 (Nieminen et al, 2015) and 17.4×17.4×17.4 µm 3 (Karhula et al, 2017)) after equilibration with phosphotungstic acid and phosphomolybdic acid). Most recent ly, contrast-enhanced micro-CT with high spatial resolution (voxel size = 3.0×3.0×3.0 µm 3 ) has proven advantageous for assessing GAG content of murine articular cartilage but was not accurate enough to measure cartilage thickness (Mashiatulla et al, 2017). The studies on diffusion involving CT techniques have used a wide range of voxel sizes ranging between approximately 0.2×0.2×2.3 mm 3 and 3.0×3.0×3.0 µm 3 , indicating the feasibility of CT contrast agent imaging in laboratory, pre-clinical and clinical studies.…”

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

“…Finally, the resulting mean grayscale values were normalized inside groups before fitting the equation 1 to datapoints. Equation (1) was derived from the previous studies [6,12].…”

Propagation phase-contrast micro-computed tomography allows laboratory-based three-dimensional imaging of articular cartilage down to the cellular level

“…Contrast agents, typically composed of high-density heavy metals, bind to extracellular components, increasing density and thereby increase the resulting signal 8 . Articular cartilage has been stained for micro-CT to study morphological changes 9,10 , glycosaminoglycan content 11,12 and to infer the spatial distribution of collagen and assessment of degradation 13 . Pauwels et al, conducted an investigation of 28 potential contrast agents and found that the most promising contrast agents for soft-tissue staining were phosphomolybdic acid (H 3 PMo 12 O 40 , PMA), phosphotungstic acid (H 3 PW 12 O 40 , PTA) and mercury chloride (HgCl 2 ) 6 .…”