Site- and Zone-Dependent Changes in Proteoglycan Content and Biomechanical Properties of Bluntly and Sharply Grooved Equine Articular Cartilage

In this study, we mapped and quantified changes of proteoglycan (PG) content and biomechanical properties in articular cartilage in which either blunt or sharp grooves had been made, both close to the groove and more remote of it, and at the opposing joint surface (kissing site) in equine carpal joints. In nine adult Shetland ponies, standardized blunt and sharp grooves were surgically made in the radiocarpal and middle carpal joints of a randomly chosen front limb. The contralateral control limb was sham-operated. At 39 weeks after surgery, ponies were euthanized. In 10 regions of interest (ROIs) (six remote from the grooves and four directly around the grooves), PG content as a function of tissue-depth and distance-to-groove was estimated using digital densitometry. Biomechanical properties of the cartilage were evaluated in the six ROIs remote from the grooves. Compared to control joints, whole tissue depth PG loss was found in sites adjacent to sharp and, to a larger extent, blunt grooves. Also, superficial PG loss of the surgically untouched kissing cartilage layers was observed. Significant PG loss was observed up to 300 µm (sharp) and at 500 µm (blunt) from the groove into the surrounding tissue. Equilibrium modulus was lower in grooved cartilage than in controls. Grooves, in particular blunt grooves, gave rise to severe PG loss close to the grooved sites and to mild degeneration more remote from the grooves in both sharply and bluntly grooved cartilage and at the kissing sites, resulting in loss of mechanical strength over the 9-month period

Dual-contrast micro-CT enables cartilage lesion detection and tissue condition evaluation ex vivo

Background: Post-traumatic osteoarthritis is a frequent joint disease in the horse. Currently, equine medicine lacks effective methods to diagnose the severity of chondral defects after an injury. Objectives: To investigate the capability of dual-contrast-enhanced computed tomography (dual-CECT) for detection of chondral lesions and evaluation of the severity of articular cartilage degeneration in the equine carpus ex vivo. Study design: Pre-clinical experimental study. Methods: In nine Shetland ponies, blunt and sharp grooves were randomly created (in vivo) in the cartilage of radiocarpal and middle carpal joints. The contralateral joint served as control. The ponies were subjected to an 8-week exercise protocol and euthanised 39 weeks after surgery. CECT scanning (ex vivo) of the joints was performed using a micro-CT scanner 1 hour after an intra-articular injection of a dual-contrast agent. The dual-contrast agent consisted of ioxaglate (negatively charged, q = −1) and bismuth nanoparticles (BiNPs, q = 0, diameter ≈ 0.2 µm). CECT results were compared to histological cartilage proteoglycan content maps acquired using digital densitometry. Results: BiNPs enabled prolonged visual detection of both groove types as they are too large to diffuse into the cartilage. Furthermore, proportional ioxaglate diffusion inside the tissue allowed differentiation between the lesion and ungrooved articular cartilage (3 mm from the lesion and contralateral joint). The mean ioxaglate partition in the lesion was 19 percentage points higher (P < 0.001) when compared with the contralateral joint. The digital densitometry and the dual-contrast CECT findings showed good subjective visual agreement. Main limitations: Ex vivo study protocol and a low number of investigated joints. Conclusions: The dual-CECT methodology, used in this study for the first time to image whole equine joints, is capable of effective lesion detection and simultaneous evaluation of the condition of the articular cartilage

Structural, compositional, and functional effects of blunt and sharp cartilage damage on the joint: a 9-month equine groove model study

This study aimed to quantify the long-term progression of blunt and sharp cartilage defects and their effect on joint homeostasis and function of the equine carpus. In nine adult Shetland ponies, the cartilage in the radiocarpal and middle carpal joint of one front limb was grooved (blunt or sharp randomized). The ponies were subjected to an 8-week exercise protocol and sacrificed at 39 weeks. Structural and compositional alterations in joint tissues were evaluated in vivo using serial radiographs, synovial biopsies, and synovial fluid samples. Joint function was monitored by quantitative gait analysis. Macroscopic, microscopic, and biomechanical evaluation of the cartilage, and assessment of subchondral bone parameters were performed ex vivo. Grooved cartilage showed higher OARSI microscopy scores than the contra-lateral sham-operated controls (p <0.0001). Blunt-grooved cartilage scored higher than sharp-grooved cartilage (p = 0.007) and fixed charge density around these grooves was lower (p = 0.006). Equilibrium and instantaneous moduli trended lower in grooved cartilage than their controls (significant for radiocarpal joints). Changes in other tissues included a 3 to 7-fold change in IL-6 expression in synovium from grooved joints at week 23 (p = 0.042) and an increased CPII/C2C ratio in synovial fluid from blunt-grooved joints at week 35 (p = 0.010). Gait analysis outcome revealed mild, gradually increasing lameness. In conclusion, blunt and, to a lesser extent, sharp grooves in combination with a period of moderate exercise, lead to mild degeneration in equine carpal cartilage over a 9-month period, but the effect on overall joint health remains limited. This article is protected by copyright. All rights reserved

Quantitative dual contrast photon-counting computed tomography for assessment of articular cartilage health

Photon-counting detector computed tomography (PCD-CT) is a modern spectral imaging technique utilizing photon-counting detectors (PCDs). PCDs detect individual photons and classify them into fixed energy bins, thus enabling energy selective imaging, contrary to energy integrating detectors that detects and sums the total energy from all photons during acquisition. The structure and composition of the articular cartilage cannot be detected with native CT imaging but can be assessed using contrast-enhancement. Spectral imaging allows simultaneous decomposition of multiple contrast agents, which can be used to target and highlight discrete cartilage properties. Here we report, for the first time, the use of PCD-CT to quantify a cationic iodinated CA4+(targeting proteoglycans) and a non-ionic gadolinium-based gadoteridol (reflecting water content) contrast agents inside human osteochondral tissue (n=53). We performed PCD-CT scanning at diffusion equilibrium and compared the results against reference data of biomechanical and optical density measurements, and Mankin scoring. PCD-CT enables simultaneous quantification of the two contrast agent concentrations inside cartilage and the results correlate with the structural and functional reference parameters. With improved soft tissue contrast and assessment of proteoglycan and water contents, PCD-CT with the dual contrast agent method is of potential use for the detection and monitoring of osteoarthritis.Peer reviewe

Effects of human articular cartilage constituents on simultaneous diffusion of cationic and nonionic contrast agents

Contrast‐enhanced computed tomography is an emerging diagnostic technique for osteoarthritis. However, the effects of increased water content, as well as decreased collagen and proteoglycan concentrations due to cartilage degeneration, on the diffusion of cationic and nonionic agents, are not fully understood. We hypothesize that for a cationic agent, these variations increase the diffusion rate while decreasing partition, whereas, for a nonionic agent, these changes increase both the rate of diffusion and partition. Thus, we examine the diffusion of cationic and nonionic contrast agents within degraded tissue in time‐ and depth‐dependent manners. Osteochondral plugs (N = 15,\ua0d = 8 mm) were extracted from human cadaver knee joints, immersed in a mixture of cationic CA4+ and nonionic gadoteridol contrast agents, and imaged at multiple time‐points, using the dual‐contrast method. Water content, and collagen and proteoglycan concentrations were determined using lyophilization, infrared spectroscopy, and digital densitometry, respectively. Superficial to mid (0%‐60% depth) cartilage CA4+ partitions correlated with water content (R

Anterior cruciate ligament transection of rabbits alters composition, structure and biomechanics of articular cartilage and chondrocyte deformation 2 weeks post-surgery in a site-specific manner

Abstract Anterior cruciate ligament (ACL) injury often leads to post-traumatic osteoarthritis (OA) and articular cartilage degradation, changing biomechanics of the tissue and chondrocytes, and altering the fixed charged density (FCD) and collagen network. However, changes in these properties are not known at a very early time point after ACL rupture, but recognizing early changes might be crucial for successful intervention. We investigated the effects of ACL transection (ACLT) in rabbits on the site-specific biomechanical properties of articular cartilage and chondrocytes, FCD content and collagen network organization, two weeks post-surgery. Unilateral ACLT was performed in eight rabbits, and femoral condyles, tibial plateaus, femoral grooves and patellae were harvested from experimental and contralateral knee joints. An intact control group was used as a reference. We analyzed chondrocyte morphology under pre- and static loading, cartilage biomechanical properties, FCD content and collagen fibril orientation. ACLT caused FCD loss in the lateral and medial femoral condyle, lateral tibial plateau, femoral groove and patellar cartilage (p &lt; 0.05). Minor changes in the collagen orientation occurred in the femoral groove and lateral and medial femoral condyle cartilage (p &lt; 0.05). Cartilage stiffness was reduced in the lateral and medial femoral condyles, and chondrocyte biomechanics was altered in the lateral femoral condyle and patellar cartilage (p &lt; 0.05). We observed loss of FCD from articular cartilage two weeks after ACLT at several joint locations. These changes may have led to decreased cartilage stiffness and altered cell deformation behavior, especially in the femoral condyles

Functional and structural properties of human patellar articular cartilage in osteoarthritis

Abstract Changes in the fibril-reinforced poroelastic (FRPE) mechanical material parameters of human patellar cartilage at different stages of osteoarthritis (OA) are not known. Further, the patellofemoral joint loading is thought to include more sliding and shear compared to other knee joint locations, thus, the relations between structural and functional changes may differ in OA. Thus, our aim was to determine the patellar cartilage FRPE properties followed by associating them with the structure and composition. Osteochondral plugs (n = 14) were harvested from the patellae of six cadavers. Then, the FRPE material properties were determined, and those properties were associated with proteoglycan content, collagen fibril orientation angle, optical retardation (fibril parallelism), and the state of OA of the samples. The initial fibril network modulus and permeability strain-dependency factor were 72% and 63% smaller in advanced OA samples when compared to early OA samples. Further, we observed a negative association between the initial fibril network modulus and optical retardation (r = -0.537, p &lt; 0.05). We also observed positive associations between 1) the initial permeability and optical retardation (r = 0.547, p &lt; 0.05), and 2) the initial fibril network modulus and optical density (r = 0.670, p &lt; 0.01).These results suggest that the reduced pretension of the collagen fibrils, as shown by the reduced initial fibril network modulus, is linked with the loss of proteoglycans and cartilage swelling in human patellofemoral OA. The characterization of these changes is important to improve the representativeness of knee joint models in tissue and cell scale

Effects of human articular cartilage constituents on simultaneous diffusion of cationic and nonionic contrast agents

Contrast-enhanced computed tomography is an emerging diagnostic technique for osteoarthritis. However, the effects of increased water content, as well as decreased collagen and proteoglycan concentrations due to cartilage degeneration, on the diffusion of cationic and nonionic agents, are not fully understood. We hypothesize that for a cationic agent, these variations increase the diffusion rate while decreasing partition, whereas, for a nonionic agent, these changes increase both the rate of diffusion and partition. Thus, we examine the diffusion of cationic and nonionic contrast agents within degraded tissue in time- and depth-dependent manners. Osteochondral plugs (N = 15, d = 8 mm) were extracted from human cadaver knee joints, immersed in a mixture of cationic CA4+ and nonionic gadoteridol contrast agents, and imaged at multiple time-points, using the dual-contrast method. Water content, and collagen and proteoglycan concentrations were determined using lyophilization, infrared spectroscopy, and digital densitometry, respectively. Superficial to mid (0%-60% depth) cartilage CA4+ partitions correlated with water content (R &lt; −0.521, P &lt;.05), whereas in deeper (40%-100%) cartilage, CA4+ correlated only with proteoglycans (R &gt; 0.671, P &lt;.01). Gadoteridol partition correlated inversely with collagen concentration (0%-100%, R &lt; −0.514, P &lt;.05). Cartilage degeneration substantially increased the time for CA4+ compared with healthy tissue (248 ± 171 vs 175 ± 95 minute) to reach the bone-cartilage interface, whereas for gadoteridol the time (111 ± 63 vs 179 ± 163 minute) decreased. The work clarifies the diffusion mechanisms of two different contrast agents and presents depth and time-dependent effects resulting from articular cartilage constituents. The results will inform the development of new contrast agents and optimal timing between agent administration and joint imaging.Biomaterials & Tissue Biomechanic