A Tissue-Penetrating Double Network Restores the Mechanical Properties of Degenerated Articular Cartilage.

Incorporation of an interpenetrating polymer network into an existing single polymer network enables augmentation of the original substrate's mechanical properties, and translation of this concept from purely synthetic materials to natural-synthetic hybrid systems provides the opportunity to reinforce mechanical properties of bulk biological substrates. In many disease states, the mechanical properties of bodily tissues deteriorate rendering them prone to further material failure. Herein, a tissue-supplementing technique is described in which an interpenetrating biomimetic hydrogel is polymerized in situ throughout cartilage tissue. The treatment restores the inferior compressive properties of osteoarthritic cartilage to that of healthy cartilage, preferentially localizing to weaker regions of tissue. Furthermore, the treatment technique preserves cartilage under harsh articulation conditions, showing promise as a materials-based treatment for early-stage osteoarthritis.

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.

Quantitative Evaluation of Equine Articular Cartilage Using Cationic Contrast-Enhanced Computed Tomography.

OBJECTIVE: To investigate the diffusion trajectory of a cationic contrast medium (CA4+) into equine articular cartilage, and to assess normal and degenerative equine articular cartilage using cationic contrast-enhanced computed tomography (CECT). DESIGN: In the first experiment (Exp1), equine osteochondral specimens were serially imaged with cationic CECT to establish the diffusion time constant and time to reach equilibrium in healthy articular cartilage. In a separate experiment (Exp2), articular cartilage defects were created on the femoral trochlea (defect joint) in a juvenile horse, while the opposite joint was a sham-operated control. After 7 weeks, osteochondral biopsies were collected throughout the articular surfaces of both joints. Biopsies were analyzed for cationic CECT attenuation, glycosaminoglycan (GAG) content, mechanical stiffness (Eeq), and histology. Imaging, biochemical and mechanical data were compared between defect and control joints. RESULTS: Exp1: The mean diffusion time constant was longer for medial condyle cartilage (3.05 ± 0.1 hours) than lateral condyle cartilage (1.54 ± 0.3 hours, P = 0.04). Exp2: Cationic CECT attenuation was lower in the defect joint than the control joint (P = 0.005) and also varied by anatomic location (P = 0.045). Mean cationic CECT attenuation from the lateral trochlear ridge was lower in the defect joint than in the control joint (2223 ± 329 HU and 2667 ± 540 HU, respectively; P = 0.02). Cationic CECT attenuation was strongly correlated with both GAG (ρ = 0.79, P < 0.0001) and Eeq (ρ = 0.61, P < 0.0001). CONCLUSIONS: The equilibration time of CA4+ into equine articular cartilage is affected by tissue volume. Quantitative cationic CECT imaging reflects the biochemical, biomechanical and histological state of normal and degenerative equine articular cartilage.

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.

Protocol development for synchrotron contrast-enhanced CT of human hip cartilage.

Understanding hip osteoarthritis requires new investigational tools for quantitative studies of biophysical and biomechanical properties as well as for determination of structure. Three new protocols to study pathological changes in cartilage and to measure cartilage thickness in intact human hips are described using synchrotron contrast enhanced computed tomography (sCECT) with the iodinated contrast agent CA4+. Ten human cadaver hips were prepared and injected with CA4+ using three different methods, all of which included rotation and distraction of the joint. CA4+ diffusion into cartilage was monitored using sCECT. The thickness of acetabular and femoral cartilage was also measured. Diffusion times ranged from 2 h to 75 h, depending on the injection protocol and the cartilage region. Direct single injection of the contrast through the labrum resulted in the fastest diffusion times. The iodine attenuation coefficient, which reflects the contrast agent distribution in the cartilage, ranged from 0.0142/cm to 0.1457/cm. Three injections at the head/neck conjunction area yielded the highest iodine attenuation coefficients in cartilage. The femoral cartilage in the Superior-Medial compartment was significantly thicker than in the other 3 femoral compartments, and femoral cartilage in the Superior-Anterior compartment was significantly thinner than the other 3 femoral compartments. The acetabular cartilage in the Superior compartment was significantly thicker than that in the Superior-Posterior compartment. sCECT with CA4+ allows assessment of hip cartilage thickness with 0.1 mm isotropic voxel size, sufficient for evaluating cartilage pathology and biomechanics.

Contrast-Enhanced Computed Tomography Scoring System for Distinguishing Early Osteoarthritis Disease States: A Feasibility Study.

Early detection of osteoarthritis (OA) remains a diagnostic challenge owing to insensitive diagnostic techniques currently available. Herein a new semiquantitative scoring system, based upon contrast-enhanced computed tomographic (CECT) imaging, is described for further refinement of early OA disease staging. Trochlear ridge cartilage defects were surgically created in the femoropatellar joint of an adult horse (ACUC approved protocols). Seven weeks post-surgery, CECT imaging was performed on a clinical scanner after intra-articular injection of a cationic iodinated contrast agent, CA4+, into both injured and control femoropatellar joint compartments. The femoral cartilage surface was densely biopsied, and specimens were assessed for visual (Outerbridge score), functional (equilibrium compressive modulus), and biochemical (glycosaminoglycan content) measures of cartilage quality. Cartilage CECT attenuation was compared with cartilage quality measures using receiver operating characteristic curve analysis to establish attenuation thresholds for distinguishing among cartilage quality levels. CECT imaging identifies macroscopically damaged cartilage regions and in morphologically identical tissue provides moderately sensitive and specific semiquantitative segregation of cartilage quality based upon CECT attenuation, reflecting both glycosaminoglycan content and compressive stiffness of cartilage area under the curve (AUC = 0.83 [95% confidence interval [CI]: 0.72-0.93] for distinguishing poor quality and AUC = 0.76 [95% CI: 0.65-0.90] for distinguishing healthy quality cartilage). A semiquantitative 6-point scoring system-the Osteoarthritis Attenuation and Morphological Assessment (OAMA) score-is proposed as a tool for assessing cartilage quality from CECT images. The OAMA scoring system expands the current disease staging capability of early OA by inclusion of morphological, biochemical, and biomechanical assessments. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2138-2148, 2019.

Contrast-enhanced computed tomography (CECT) attenuation is associated with stiffness of intact knee cartilage.

Contrast-enhanced computed tomography (CECT) using charged contrast-agents enables quantification of cartilage glycosaminoglycan content. Since glycosaminoglycan content is a key determinant of cartilage compressive stiffness, CECT measurements have the potential to non-invasively assess cartilage stiffness. The objective of this study was to determine whether CECT attenuation, using a cationic contrast-agent (CA4+), correlates with the stiffness of intact cartilage. Six fresh femoral and six fresh tibial compartments with intact cartilage were obtained from patients undergoing total knee replacement surgery. The instantaneous stiffness was determined for 25-50 points on the surface of each compartment using an established indentation technique. The samples were then immersed in CA4+ solution for 48 h, scanned in a micro-CT scanner, and the average CECT attenuation at each indentation site was found for the superficial cartilage. A significant (p < 0.01) and positive correlation was observed between stiffness and CECT attenuation for sites from each individual cartilage surface, with correlation coefficients ranging from r = 0.37-0.57 and r = 0.48-0.69 (p < 0.01) for the tibia and femur, respectively. When data for each type of cartilage surface were pooled together, the correlation coefficients were r = 0.73 for femoral condyle data points and r = 0.49 for tibial plateau data points. CECT provided a map of cartilage stiffness across each surface, which allows regions of low stiffness to be identified. These findings support continued evaluation and development of quantitative imaging techniques to assess the functional properties of cartilage. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2641-2647, 2018.

Contrast-enhanced CT imaging as a non-destructive tool for ex vivo examination of the biochemical content and structure of the human meniscus.

The biochemical and histopathological techniques used to investigate meniscal content and structure are destructive and time-consuming. Therefore, this study evaluated whether contrast-enhanced computed tomography (CECT) attenuation and contrast agent flux using the iodinated contrast agents CA4+ and ioxaglate correlate with the glycosaminoglycan (GAG) content/distribution and water content in human menisci. The optimal ioxaglate and CA4+ contrast agent concentrations for mapping meniscal GAG distribution were qualitatively determined by comparison of CECT color maps with Safranin-O stained histological sections. The associations between CECT attenuation and GAG content, CECT attenuation and water content, and flux and water content at various time points were determined using both contrast agents. Depth-wise analyses were also performed through each of the native surfaces to examine differences in contrast agent diffusion kinetics and equilibrium partitioning. The optimal concentrations for GAG depiction for ioxaglate and CA4+ were ≥80 and 12 mgI/ml, respectively. Using these concentrations, weak to moderate associations were found between ioxaglate attenuation and GAG content at all diffusion time points (1-48 h), while strong and significant associations were observed between CA4+ attenuation and GAG content as early as 7 h (R2 ≥ 0.67), being strongest at the equilibrium time point (48 h, R2 = 0.81). CECT attenuation for both agents did not significantly correlate with water content, but CA4+ flux correlated with water content (R2 = 0.56-0.64). CECT is a promising, non-destructive imaging technique for ex vivo assessment of meniscal GAG concentration and water content compared to traditional biochemical and histopathological methods. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1018-1028, 2017.

Synthesis and Preclinical Characterization of a Cationic Iodinated Imaging Contrast Agent (CA4+) and Its Use for Quantitative Computed Tomography of Ex Vivo Human Hip Cartilage.

Contrast agents that go beyond qualitative visualization and enable quantitative assessments of functional tissue performance represent the next generation of clinically useful imaging tools. An optimized and efficient large-scale synthesis of a cationic iodinated contrast agent (CA4+) is described for imaging articular cartilage. Contrast-enhanced CT (CECT) using CA4+ reveals significantly greater agent uptake of CA4+ in articular cartilage compared to that of similar anionic or nonionic agents, and CA4+ uptake follows Donnan equilibrium theory. The CA4+ CECT attenuation obtained from imaging ex vivo human hip cartilage correlates with the glycosaminoglycan content, equilibrium modulus, and coefficient of friction, which are key indicators of cartilage functional performance and osteoarthritis stage. Finally, preliminary toxicity studies in a rat model show no adverse events, and a pharmacokinetics study documents a peak plasma concentration 30 min after dosing, with the agent no longer present in vivo at 96 h via excretion in the urine.

Contrast-Enhanced Computed Tomography Enables Quantitative Evaluation of Tissue Properties at Intrajoint Regions in Cadaveric Knee Cartilage.

Objective The aim of this study was to investigate whether the concentration of the anionic contrast agent ioxaglate, as quantitated by contrast-enhanced computed tomography (CECT) using a clinical cone-beam CT (CBCT) instrument, reflects biochemical, histological, and biomechanical characteristics of articular cartilage imaged in an ex vivo, intact human knee joint. Design An osteoarthritic human cadaveric knee joint (91 years old) was injected with ioxaglate (36 mg I/mL) and imaged using CBCT over 61 hours of ioxaglate diffusion into cartilage. Following imaging, the joint surfaces were excised, rinsed to remove contrast agent, and compressive stiffness (equilibrium and instantaneous compressive moduli) was measured via indentation testing ( n = 17 sites). Each site was sectioned for histology and assessed for glycosaminoglycan content using digital densitometry of Safranin-O stained sections, Fourier transform infrared spectroscopy for collagen content, and morphology using both the Mankin and OARSI semiquantitative scoring systems. Water content was determined using mass change after lyophilization. Results CECT attenuation at all imaging time points, including those <1 hour of ioxaglate exposure, correlated significantly ( P < 0.05) with cartilage water and glycosaminoglycan contents, Mankin score, and both equilibrium and instantaneous compressive moduli. Early time points (<30 minutes) also correlated ( P < 0.05) with collagen content and OARSI score. Differences in cartilage quality between intrajoint regions were distinguishable at diffusion equilibrium and after brief ioxaglate exposure. Conclusions CECT with ioxaglate affords biochemical and biomechanical measurements of cartilage health and performance even after short, clinically relevant exposure times, and may be useful in the clinic as a means for detecting early signs of cartilage pathology.

The Expansion of Heterotopic Bone in Fibrodysplasia Ossificans Progressiva Is Activin A-Dependent.

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder that is characterized by episodic yet cumulative heterotopic ossification (HO) in skeletal muscles, tendons, and ligaments over a patient's lifetime. FOP is caused by missense mutations in the type I bone morphogenetic protein (BMP) receptor ACVR1. We have determined that the formation of heterotopic bone in FOP requires activation of mutant ACVR1 by Activin A, in part by showing that prophylactic inhibition of Activin A blocks HO in a mouse model of FOP. Here we piece together a natural history of developing HO lesions in mouse FOP, and determine where in the continuum of HO Activin A is required, using imaging (T2-MRI, µCT, 18 F-NaF PET/CT, histology) coupled with pharmacologic inhibition of Activin A at different times during the progression of HO. First, we show that expansion of HO lesions comes about through growth and fusion of independent HO events. These events tend to arise within a neighborhood of existing lesions, indicating that already formed HO likely triggers the formation of new events. The process of heterotopic bone expansion appears to be dependent on Activin A because inhibition of this ligand suppresses the growth of nascent HO lesions and stops the emergence of new HO events. Therefore, our results reveal that Activin A is required at least up to the point when nascent HO lesions mineralize and further demonstrate the therapeutic utility of Activin A inhibition in FOP. These results provide evidence for a model where HO is triggered by inflammation but becomes "self-propagating" by a process that requires Activin A. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

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 R(2) = 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.

Contrast enhanced CT attenuation correlates with the GAG content of bovine meniscus.

We determined whether contrast-enhanced computed tomography (CECT) attenuation obtained using a µCT scanner correlated with the glycosaminoglycan (GAG) content and distribution in ex vivo bovine menisci. Bovine samples were immersed in different concentrations of the contrast agents CA4+ and Ioxaglate, and the µCT images were compared to Safranin-O staining. CA4+ and Ioxaglate diffusion-in kinetics and the correlation between their CECT attenuations and GAG content were investigated. CA4+ and Ioxaglate both reached steady state in the meniscal regions within 95 h, with tau values of 20.6 ± 3.98 and 25.9 ± 3.71 h (mean ± SD), respectively. Both agents diffused preferentially through the proximal and secondarily through the distal surface. The CA4+ CECT attenuation was strongly and positively correlated with the GAG content of the meniscus regions (R(2) = 0.89, p < 0.001) at low concentrations (12 mgI/ml), while the Ioxaglate CECT attenuation was moderately and negatively correlated with the GAG content (R(2) = 0.51, p = 0.03) at 60 mgI/ml. CECT can image ex vivo menisci, and the CA4+, compared to Ioxaglate, enhanced attenuation strongly correlates with the GAG content and distribution in bovine meniscus.

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 (R(2) = 0.86, p < 0.0001 compared with R(2) = 0.62, p = 0.004). The natural inhomogeneous distribution of GAGs in the ECM could clearly be identified in undegraded samples.