Dual‐contrast computed tomography enables detection of equine posttraumatic osteoarthritis in vitro

To prevent the progression of posttraumatic osteoarthritis, assessment of cartilage composition is critical for effective treatment planning. Posttraumatic changes include proteoglycan (PG) loss and elevated water content. Quantitative dual‐energy computed tomography (QDECT) provides a means to diagnose these changes. Here, we determine the potential of QDECT to evaluate tissue quality surrounding cartilage lesions in an equine model, hypothesizing that QDECT allows detection of posttraumatic degeneration by providing quantitative information on PG and water contents based on the partitions of cationic and nonionic agents in a contrast mixture. Posttraumatic osteoarthritic samples were obtained from a cartilage repair study in which full‐thickness chondral defects were created surgically in both stifles of seven Shetland ponies. Control samples were collected from three nonoperated ponies. The experimental (n = 14) and control samples (n = 6) were immersed in the contrast agent mixture and the distributions of the agents were determined at various diffusion time points. As a reference, equilibrium moduli, dynamic moduli, and PG content were measured. Significant differences (p < 0.05) in partitions between the experimental and control samples were demonstrated with cationic contrast agent at 30 min, 60 min, and 20 h, and with non‐ionic agent at 60 and 120 min. Significant Spearman's rank correlations were obtained at 20 and 24 h (ρ = 0.482–0.693) between the partition of cationic contrast agent, cartilage biomechanical properties, and PG content. QDECT enables evaluation of posttraumatic changes surrounding a lesion and quantification of PG content, thus advancing the diagnostics of the extent and severity of cartilage injuries.

and PG content were measured. Significant differences (p < 0.05) in partitions be- highly from as short as 2-5 years but for some injuries, the timeframe can be even longer. 1 The earlier the cartilage degeneration can be detected in the preclinical phase, the better are chances for effective treatment. The focus of this study is to evaluate a new contrastenhanced computed tomography (CECT) technique for the detection of the initial changes in cartilage adjacent to chondral defects.
Magnetic resonance imaging (MRI) is an important imaging tool in cartilage damage diagnostics as it features excellent soft-tissue contrast and can assess proteoglycan (PG) content, collagen orientation, and water content. 3,4 Unfortunately, MRI has a relatively low spatial resolution in vivo (1-2 mm) and long image acquisition times with the length of a typical knee MRI protocol varying from 20 to 30 min. 4,5 As an alternative to MRI, CECT enables the detection of cartilage degeneration in acute injuries [6][7][8][9][10] and has recently been introduced in the clinic. The advantages of CECT over MRI include better spatial resolution (0.5-0.625 mm), shorter acquisition time (<1 min), lower costs, and better availability. The CECT of the knee typically involves two subsequent CT scans acquired immediately (arthrography) and 45 min (delayed arthrography) after the intra-articular injection of a contrast agent. 6,7,10 The first scan allows segmentation of the articulating surface and lesions, while the second scan reveals internal cartilage changes related to the initiation of PTOA (e.g., increased water content and decreased PG content). In CECT, contrast agents enhance the contrast at the synovial fluid-cartilage interface since the natural contrast at this interface is almost nonexistent in CT. Contrast agents also enable the detection of degenerative changes by examining their uptake and partitioning in the cartilage. [11][12][13][14][15][16] The early degenerative changes of cartilage include PG loss, disruption of the superficial collagen network, and increased water content. 17,18 These changes increase the uptake of anionic contrast agent (most commonly ioxaglate), enabling the evaluation of the internal cartilage changes and degeneration. 19 A recently introduced cationic contrast agent (CA4+) has a superior sensitivity for revealing tissue PG content at diffusion equilibrium compared with the currently used anionic contrast agents. 13,[20][21][22] Cationic contrast agents distribute in cartilage proportionally to the PG content due to the electrostatic attraction caused by the negative fixed charge density of the PG molecules. At the onset of diffusion, cationic contrast agent diffusion is also controlled by two other degeneration-related factors: increased water content and decreased steric hindrance (i.e., the physical diffusion barrier formed by the dense collagen network and the interspersed PGs in the matrix). Degeneration of the extracellular matrix has opposite effects on the diffusion of cationic agents: the loss of PGs decreases the diffusion while the increased water content and decreased steric hindrance increase the diffusion. This phenomenon diminishes the sensitivity of detecting cartilage injuries and osteoarthritic degeneration at clinically feasible imaging time points (30 min up to 2 h after the administration of the contrast agent 6 ).
We previously reported that this weakness in clinically feasible time points is minimized by employing a quantitative dualenergy CT (QDECT) technique, together with a contrast agent mixture consisting of cationic, iodinated contrast agent (CA4+) and nonionic, gadolinium-based contrast agent (gadoteridol). 23,24 In the mixture, the cationic contrast agent is sensitive to the changes in PG content and the non-ionic contrast agent is sensitive to the tissue water content and altered steric hindrance.
Thus, with non-ionic agent, the effect of water content and altered steric hindrance into CA4+ diffusion can be assessed.
QDECT is based on dual-energy CT that exploits the "absorption k-edges," that is, sharp element-specific changes in the photoelectric X-ray absorption spectrum. In this method, the energies and filtration of the X-ray beam are selected so that the resulting X-ray energy spectra fall on both sides of either gadolinium (50.2 keV) or iodine (33.2 keV) k-edge. The technique enables simultaneous quantification of the uptake of cationic and nonionic contrast agents in cartilage and, thus, improved diagnosis of cartilage degeneration and injuries. 23,24 Previous research on QDECT has focused on studying cartilage injuries in lesion sites of osteoarthritic human cartilage and bovine cartilage with artificial injuries and degeneration ex vivo. [23][24][25][26][27] In this study, we extend our examinations from the lesion site to the surrounding tissue to evaluate the capability of the technique to reveal changes related to PTOA. This is of in-

| MicroCT imaging
The QDECT measurements were conducted using a microCT scanner

| Digital densitometry
Preceding sample preparation for histological digital densitometry where α E is the attenuation coefficient in the medium at an energy The attenuation of native cartilage tissue was removed by subtracting the images obtained before the contrast agent immersion from the image obtained at each time point with the same energy.
The contrast agent partitions were obtained by dividing the contrast agent concentration inside the cartilage with the concentration in the immersion bath.
In addition, depth-wise contrast agent concentration profiles were calculated from the locations of biomechanical testing to investigate potential differences between the contrast agents' partitioning and DD

| RESULTS
Mean CA4+ partitions in full thickness cartilage were significantly higher in experimental samples than in control samples at 30 min

| DISCUSSION
In our previous studies, we showed that QDECT can simultaneously quantify CA4+ and gadoteridol partitions in cartilage, and differentiate healthy from injured cartilage tissue. 23,24,27,27,35