Analysis of cartilage matrix fixed charge density and three-dimensional morphology via contrast-enhanced microcomputed tomography

Abstract

Small animal models of osteoarthritis are often used for evaluating the efficacy of pharmacologic treatments and cartilage repair strategies, but noninvasive techniques capable of monitoring matrix-level changes are limited by the joint size and the low radiopacity of soft tissues. Here we present a technique for the noninvasive imaging of cartilage at micrometer-level resolution based on detecting the equilibrium partitioning of an ionic contrast agent via microcomputed tomography. The approach exploits electrochemical interactions between the molecular charges present in the cartilage matrix and an ionic contrast agent, resulting in a nonuniform equilibrium partitioning of the ionic contrast agent reflecting the proteoglycan distribution. In an in vitro model of cartilage degeneration we observed changes in x-ray attenuation magnitude and distribution consistent with biochemical and histological analyses of sulfated glycosaminoglycans, and x-ray attenuation was found to be a strong predictor of sulfated glycosaminoglycan density. Equilibration with the contrast agent also permits direct in situ visualization and quantification of cartilage surface morphology. Equilibrium partitioning of an ionic contrast agent via microcomputed tomography thus provides a powerful approach to quantitatively assess 3D cartilage composition and morphology for studies of cartilage degradation and repair.

Fig. 1.

EPIC-μCT in an in vitro cartilage degradation model. (A) Representative 3D EPIC-μCT images of control and IL-1-stimulated explants at 2-day intervals demonstrate progressive increases in attenuation in IL-1-stimulated samples. (B) Representative safranin-O-stained sections indicate a progressive loss of sGAG content (red staining) in IL-1-stimulated samples, consistent with the EPIC-μCT images. (C) Average x-ray attenuation for control and IL-1-stimulated explants over the 10 days of culture. (D) sGAG release to the media over the 10 days of culture. (E) Inverse relationship between average x-ray attenuation and the measured sGAG density for combined control and IL-1-stimulated explants. SZ, superficial zone. ∗, P < 0.05 vs. control explants. (Scale bars: 1 mm.)

Fig. 2.

In situ imaging of a rabbit femur via EPIC-μCT. (A) Rabbit distal femur. (B) μCT scan taken after Hexabrix desorption reveals only the bone geometry. (C) μCT scan taken after Hexabrix equilibration demonstrates the ability to visualize the articular surface. Remnants of soft tissues (black arrows) and scalpel cuts to the cartilage (white arrows) can be seen both in the photograph and in the EPIC-μCT image. (D) A thickness map generated after segmentation of soft tissue and bone demonstrates the ability to quantitatively analyze soft tissue morphology. (Scale bars: 1 mm.)

Fig. 3.

Characterization of contrast agent for EPIC-μCT. (A) Linear decrease of x-ray attenuation with dilution of anionic CT contrast agents. At a given osmolality, attenuation of Hexabrix is higher than that of sodium diatrizoate because of the higher iodine content of the dimeric ioxaglate ion, resulting in a greater sensitivity to changes in matrix FCD. (B) Time course of Hexabrix equilibration in full-thickness immature bovine cartilage explants (P < 0.0001 for model). (C) X-ray attenuation histogram of a cross-sectional image slice from a rabbit distal femur equilibrated with 40%/60% Hexabrix/0.15 M PBS. Peaks corresponding to bone and cartilage could be easily distinguished, allowing segmentation of the cartilage layer for thickness analysis.