Prashant N. Bansal

Contrast Enhanced Computed Tomography can predict the glycosaminoglycan content and biomechanical properties of articular cartilage

OBJECTIVE An early hallmark of osteoarthritis (OA) is the progressive loss of glycosaminoglycans (GAGs), the extracellular matrix (ECM) component of articular cartilage that confers it with compressive stiffness. Our aim in this work is to establish the feasibility of using Contrast Enhanced Computed Tomography (CECT) with an anionic iodinated contrast agent - Cysto Conray II - as a minimally invasive tool to measure the changes in the GAG content as well as the compressive stiffness of articular cartilage. METHODS The GAG content of mated osteochondral plugs excised from bovine patello-femoral joints was progressively degraded using chondroitinase ABC. The mated plugs were then immersed in an anionic, tri-iodinated contrast agent, imaged using peripheral quantitative computed tomography (pQCT), subjected to an unconfined compressive stress relaxation test and the GAG content measured using 1,9-dimethylmethylene blue (DMMB) assay. Partial correlation analysis was performed to compare the variation in X-ray attenuation measured by pQCT to the variation in GAG content and in equilibrium compressive modulus. RESULTS The X-ray attenuation of cartilage exposed to an anionic, tri-iodinated, contrast agent measured by quantitative computed tomography (QCT) accounted for 83% of the variation in GAG content (r(2)=0.83, P<0.0001) and 93% of the variation in the equilibrium compressive modulus (r(2)=0.93, P<0.0001). CONCLUSION Using a mated osteochondral plug model to evaluate the biochemical composition and biomechanical properties of cartilage, this study demonstrates the interrelationships between X-ray attenuation, GAG content, and equilibrium compressive modulus, and that CECT can be used to monitor and quantify changes in the GAG content and biomechanical properties of articular cartilage.

Cationic contrast agents improve quantification of glycosaminoglycan (GAG) content by contrast enhanced CT imaging of cartilage

Minimally invasive and non‐destructive methods to quantify glycosaminoglycans (GAGs) in articular cartilage extracellular matrix are of significant interest for the biochemical analysis of cartilage and diagnosis and tracking of osteoarthritis in vivo. Here, we report the use of cationic iodinated contrast agents in comparison to conventional anionic contrast agents for the quantitative monitoring of GAG concentrations with peripheral quantitative computed tomography. Using an ex vivo bovine osteochondral plug model, the cationic contrast agents were evaluated for their ability to distribute into articular cartilage and generate a positive relationship with GAG content. The cationic agents resulted in much higher equilibrium X‐ray attenuations in cartilage extracellular matrix (ECM) than anionic agents. Experiments with samples subjected to enzymatic GAG degradation demonstrated that the cationic agents were up to five times more sensitive (p = 0.0001) to changes in GAG content and had a 24% higher correlation (p = 0.002) compared to the anionic agent (R2 = 0.86, p < 0.0001 compared with R2 = 0.62, p = 0.004). The natural inhomogeneous distribution of GAGs in the ECM could clearly be identified in undegraded samples.

Hydrogels for Osteochondral Repair Based on Photocrosslinkable Carbamate Dendrimers

First generation, photocrosslinkable dendrimers consisting of natural metabolites (i.e., succinic acid, glycerol, and beta-alanine) and nonimmunogenic poly(ethylene glycol) (PEG) were synthesized divergently in high yields using ester and carbamate forming reactions. Aqueous solutions of these dendrimers were photocrosslinked with an eosin-based photoinitiator to afford hydrogels. The hydrogels displayed a range of mechanical properties based on their structure, generation size, and concentration in solution. All of the hydrogels showed minimal swelling characteristics. The dendrimer solutions were then photocrosslinked in situ in an ex vivo rabbit osteochondral defect (3 mm diameter and 10 mm depth), and the resulting hydrogels were subjected to physiologically relevant dynamic loads. Magnetic resonance imaging (MRI) showed the hydrogels to be fixated in the defect site after the repetitive loading regimen. The ([G1]-PGLBA-MA) 2-PEG hydrogel was chosen for the 6 month pilot in vivo rabbit study because this hydrogel scaffold could be prepared at low polymer weight (10 wt %) and possessed the largest compressive modulus of the 10% formulations, a low swelling ratio, and contained carbamate linkages, which are more hydrolytically stable than the ester linkages. The hydrogel-treated osteochondral defects showed good attachment in the defect site and histological analysis showed the presence of collagen II and glycosaminoglycans (GAGs) in the treated defects. By contrast, the contralateral unfilled defects showed poor healing and negligible GAG or collagen II production. Good mechanical properties, low swelling, good attachment to the defect site, and positive in vivo results illustrate the potential of these dendrimer-based hydrogels as scaffolds for osteochondral defect repair.

Effect of Contrast Agent Charge on Visualization of Articular Cartilage Using Computed Tomography: Exploiting Electrostatic Interactions for Improved Sensitivity

The synthesis and evaluation of a new class of cationic iodinated contrast agents for the imaging of cartilage using computed tomography (CT) are described. In direct comparisons with anionic contrast agents, the cationic contrast agents afforded higher equilibrium concentrations in the articular cartilage of ex vivo rabbit femurs and thus greater imaging sensitivity. Variations in CT intensity across the sample reflected the inhomogeneous distribution of glycosaminoglycans in the tissue as confirmed by histological analysis. We anticipate that this work represents the first step in the development of sensitive, nondestructive CT-based methods to characterize the biochemical properties of cartilage using cationic contrast agents.

Contrast-enhanced CT with a High-Affinity Cationic Contrast Agent for Imaging ex Vivo Bovine, Intact ex Vivo Rabbit, and in Vivo Rabbit Cartilage

PURPOSE To quantify the affinity of a cationic computed tomography (CT) contrast agent (CA(4+)) and that of an anionic contrast agent (ioxaglate) to glycosaminoglycans (GAGs) in ex vivo cartilage tissue explants and to characterize the in vivo diffusion kinetics of CA(4+) and ioxaglate in a rabbit model. MATERIALS AND METHODS All in vivo procedures were approved by the institutional animal care and use committee. The affinities of ioxaglate and CA(4+) to GAGs in cartilage (six bovine osteochondral plugs) were quantified by means of a modified binding assay using micro-CT after plug equilibration in serial dilutions of each agent. The contrast agents were administered intraarticularly to the knee joints of five New Zealand white rabbits to determine the in vivo diffusion kinetics and cartilage tissue imaging capabilities. Kinetics of diffusion into the femoral groove cartilage and relative contrast agent uptake into bovine plugs were characterized by means of nonlinear mixed-effects models. Diffusion time constants (τ) were compared by using a Student t test. RESULTS The uptake of CA(4+) in cartilage was consistently over 100% of the reservoir concentration, whereas it was only 59% for ioxaglate. In vivo, the contrast material-enhanced cartilage reached a steady CT attenuation for both CA(4+) and ioxaglate, with τ values of 13.8 and 6.5 minutes, respectively (P = .04). The cartilage was easily distinguishable from the surrounding tissues for CA(4+) (12 mg of iodine per milliliter); comparatively, the anionic contrast agent provided less favorable imaging results, even when a higher concentration was used (80 mg of iodine per milliliter). CONCLUSION The affinity of the cationic contrast agent CA(4+) to GAGs enables high-quality imaging and segmentation of ex vivo bovine and rabbit cartilage, as well as in vivo rabbit cartilage. SUPPLEMENTAL MATERIAL http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.12112246/-/DC1.

A Large-Molecular-Weight Polyanion, Synthesized via Ring-Opening Metathesis Polymerization, as a Lubricant for Human Articular Cartilage

A large-molecular-weight polyanion is found to possess lubricating properties for cartilage. The polyanion, sodium poly(7-oxanorbornene-2-carboxylate), is synthesized by ring-opening metathesis polymerization of methyl 5-oxanorbornene-2-carboxylate. When dissolved in aqueous solution and applied to the surface of human cartilage it reduces the friction at the interface and acts as a lubricant. Its performance is similar to that of synovial fluid and superior to those of saline and Synvisc in an ex vivo human cartilage plug-on-plug model. The polymer is also not readily degraded by hyaluronidase or cytotoxic to human chondrocytes in vitro. As such, this polymer is a new type of viscosupplement, and the results provide insight into the design requirements for synthesizing highly efficacious synthetic biolubricants.

Cationic agent contrast-enhanced computed tomography imaging of cartilage correlates with the compressive modulus and coefficient of friction

OBJECTIVE The aim of this study is to evaluate whether contrast-enhanced computed tomography (CECT) attenuation, using a cationic contrast agent (CA4+), correlates with the equilibrium compressive modulus (E) and coefficient of friction (μ) of ex vivo bovine articular cartilage. METHODS Correlations between CECT attenuation and E (Group 1, n = 12) and μ (Group 2, n = 10) were determined using 7 mm diameter bovine osteochondral plugs from the stifle joints of six freshly slaughtered, skeletally mature cows. The equilibrium compressive modulus was measured using a four-step, unconfined, compressive stress-relaxation test, and the coefficients of friction were determined from a torsional friction test. Following mechanical testing, samples were immersed in CA4+, imaged using μCT, rinsed, and analyzed for glycosaminoglycan (GAG) content using the 1,9-dimethylmethylene blue (DMMB) assay. RESULTS The CECT attenuation was positively correlated with the GAG content of bovine cartilage (R(2) = 0.87, P < 0.0001 for Group 1 and R(2) = 0.74, P = 0.001 for Group 2). Strong and significant positive correlations were observed between E and GAG content (R(2) = 0.90, P < 0.0001) as well as CECT attenuation and E (R(2) = 0.90, P < 0.0001). The CECT attenuation was negatively correlated with the three coefficients of friction: CECT vs μ(static) (R(2) = 0.71, P = 0.002), CECT vs μ(static_equilibrium) (R(2) = 0.79, P < 0.001), and CECT vs μ(kinetic) (R(2) = 0.69, P = 0.003). CONCLUSIONS CECT with CA4+ is a useful tool for determining the mechanical properties of ex vivo cartilage tissue as the attenuation significantly correlates with the compressive modulus and coefficient of friction.

Effect of mechanical convection on the partitioning of an anionic iodinated contrast agent in intact patellar cartilage

To determine if mechanical convection accelerates partitioning of an anionic contrast agent into cartilage while maintaining its ability to reflect the glycosaminoglycan (GAG) content in contrast‐enhanced computed tomography (CECT) of cartilage. Bovine patellae (N = 4) were immersed in iothalamate and serially imaged over 24 h of passive diffusion at 34°C. Following saline washing for 14 h, each patella was serially imaged over 2.5 h of mechanical convection by cyclic compressive loading (120N, 1 Hz) while immersed in iothalamate at 34°C. After similar saline washing, each patella was sectioned into 15 blocks (n = 60) and contrast concentration per time point as well as GAG content were determined for each cartilage block. Mechanical convection produced 70.6%, 34.4%, and 16.4% higher contrast concentration at 30, 60, and 90 min, respectively, compared to passive diffusion (p < 0.001) and boosted initial contrast flux 330%. The correlation between contrast concentration and GAG content was significant at all time points and correlation coefficients improved with time, reaching R2 = 0.60 after 180 min of passive diffusion and 22.5 min of mechanical convection. Mechanical convection significantly accelerated partitioning of a contrast agent into healthy cartilage while maintaining strong correlations with GAG content, providing an evidence‐based rationale for adopting walking regimens in CECT imaging protocols.

A synthetic polymeric biolubricant imparts chondroprotection in a rat meniscal tear model

Intra-articular injection of hyaluronic acid (HA) is used to treat osteoarthritis (OA) as a viscosupplement, yet it only provides short-term benefit because HA is cleaved by hyaluronidase and cleared out of the joint after several days. Therefore, we developed a new polymer biolubricant based on poly-oxanorbornane carboxylate to enhance joint lubrication for a prolonged time. Rheological and biotribological studies of the biolubricant reveal viscoelastic properties and coefficient of friction equivalent and superior to that of healthy synovial fluid, respectively. Furthermore, in an ex vivo bovine cartilage plug model, the biolubricant exhibits superior long-term reduction of friction and wear prevention compared to saline and healthy synovial fluid. ISO 10993 biocompatibility tests demonstrate that the biolubricant polymer is non-toxic. In an in vivo rat medial meniscal tear OA model, where the performance of the leading HA viscosupplement (Synvisc-one®) is comparable to the saline control, treatment with the biolubricant affords significant chondroprotection compared to the saline control.