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.

Tailored Synthesis of PEGylated Bismuth Nanoparticles for X-ray Computed Tomography and Photothermal Therapy: One-Pot, Targeted Pyrolysis, and Self-Promotion.

Complex experimental design is a common problem in the preparation of theranostic nanoparticles, resulting in poor reaction control, expensive production cost, and low experiment success rate. The present study aims to develop PEGylated bismuth (PEG-Bi) nanoparticles with a precisely controlled one-pot approach, which contains only methoxy[(poly(ethylene glycol)]trimethoxy-silane (PEG-silane) and bismuth oxide (Bi2O3). A targeted pyrolysis of PEG-silane was achieved to realize its roles as both the reduction and PEGylation agents. The unwanted methoxy groups of PEG-silane were selectively pyrolyzed to form reductive agents, while the useful PEG-chain was fully preserved to enhance the biocompatibility of Bi nanoparticles. Moreover, Bi2O3 not only acted as the raw material of the Bi source but also presented a self-promotion in the production of Bi nanoparticles via catalyzing the pyrolysis of PEG-silane. The reaction mechanism was systematically validated with different methods such as nuclear magnetic resonance spectroscopy. The PEG-Bi nanoparticles showed better compatibility and photothermal conversion than those prepared by the complex multiple step approaches in literature studies. In addition, the PEG-Bi nanoparticles possessed prominent performance in X-ray computed tomography imaging and photothermal cancer therapy in vivo. The present study highlights the art of precise reaction control in the synthesis of PEGylated nanoparticles for biomedical applications.

Membrane-Dependent Binding and Entry Mechanism of Dopamine into Its Receptor.

Synaptic neurotransmission has recently been proposed to function via either a membrane-independent or a membrane-dependent mechanism, depending on the neurotransmitter type. In the membrane-dependent mechanism, amphipathic neurotransmitters first partition to the lipid headgroup region and then diffuse along the membrane plane to their membrane-buried receptors. However, to date, this mechanism has not been demonstrated for any neurotransmitter-receptor complex. Here, we combined isothermal calorimetry measurements with a diverse set of molecular dynamics simulation methods to investigate the partitioning of an amphipathic neurotransmitter (dopamine) and the mechanism of its entry into the ligand-binding site. Our results show that the binding of dopamine to its receptor is consistent with the membrane-dependent binding and entry mechanism. Both experimental and simulation results showed that dopamine favors binding to lipid membranes especially in the headgroup region. Moreover, our simulations revealed a ligand-entry pathway from the membrane to the binding site. This pathway passes through a lateral gate between transmembrane alpha-helices 5 and 6 on the membrane-facing side of the protein. All in all, our results demonstrate that dopamine binds to its receptor by a membrane-dependent mechanism, and this is complemented by the more traditional binding mechanism directly through the aqueous phase. The results suggest that the membrane-dependent mechanism is common in other synaptic receptors, too.

Synchrotron MicroCT Reveals the Potential of the Dual Contrast Technique for Quantitative Assessment of Human Articular Cartilage Composition.

Dual contrast micro computed tomography (CT) shows potential for detecting articular cartilage degeneration. However, the performance of conventional CT systems is limited by beam hardening, low image resolution (full-body CT), and long acquisition times (conventional microCT). Therefore, to reveal the full potential of the dual contrast technique for imaging cartilage composition we employ the technique using synchrotron microCT. We hypothesize that the above-mentioned limitations are overcome with synchrotron microCT utilizing monochromatic X-ray beam and fast image acquisition. Human osteochondral samples (n = 41, four cadavers) were immersed in a contrast agent solution containing two agents (cationic CA4+ and non-ionic gadoteridol) and imaged with synchrotron microCT at an early diffusion time point (2 h) and at diffusion equilibrium (72 h) using two monochromatic X-ray energies (32 and 34 keV). The dual contrast technique enabled simultaneous determination of CA4+ (i.e., proteoglycan content) and gadoteridol (i.e., water content) partitions within cartilage. Cartilage proteoglycan content and biomechanical properties correlated significantly (0.327 < r < 0.736, p < 0.05) with CA4+ partition in superficial and middle zones at both diffusion time points. Normalization of the CA4+ partition with gadoteridol partition within the cartilage significantly (p < 0.05) improved the detection sensitivity for human osteoarthritic cartilage proteoglycan content, biomechanical properties, and overall condition (Mankin, Osteoarthritis Research Society International, and International Cartilage Repair Society grading systems). The dual energy technique combined with the dual contrast agent enables assessment of human articular cartilage proteoglycan content and biomechanical properties based on CA4+ partition determined using synchrotron microCT. Additionally, the dual contrast technique is not limited by the beam hardening artifact of conventional CT systems. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 38:563-573, 2020.

Triple Contrast CT Method Enables Simultaneous Evaluation of Articular Cartilage Composition and Segmentation.

Early degenerative changes of articular cartilage are detected using contrast-enhanced computed tomography (CT) with a cationic contrast agent (CA). However, cationic CA diffusion into degenerated cartilage decreases with proteoglycan depletion and increases with elevated water content, thus hampering tissue evaluation at early diffusion time points. Furthermore, the contrast at synovial fluid-cartilage interface diminishes as a function of diffusion time hindering accurate cartilage segmentation. For the first time, we employ quantitative dual-energy CT (QDECT) imaging utilizing a mixture of three CAs (cationic CA4+ and non-ionic gadoteridol which are sensitive to proteoglycan and water contents, respectively, and bismuth nanoparticles which highlight the cartilage surface) to simultaneously segment the articulating surfaces and determine of the cartilage condition. Intact healthy, proteoglycan-depleted, and mechanically injured bovine cartilage samples (n = 27) were halved and imaged with synchrotron microCT 2-h post immersion in triple CA or in dual CA (CA4+ and gadoteridol). CA4+ and gadoteridol partitions were determined using QDECT, and pairwise evaluation of these partitions was conducted for samples immersed in dual and triple CAs. In conclusion, the triple CA method is sensitive to proteoglycan depletion while maintaining sufficient contrast at the articular surface to enable detection of cartilage lesions caused by mechanical impact.

Simultaneous Quantitation of Cationic and Non-ionic Contrast Agents in Articular Cartilage Using Synchrotron MicroCT Imaging.

Early diagnosis of acute cartilage injuries enables monitoring of disease progression and improved treatment option planning to prevent post-traumatic osteoarthritis. In contrast-enhanced computed tomography (CECT), the changes in cationic agent diffusion within the tissue reflect cartilage degeneration. The diffusion in degenerated cartilage depends on proteoglycan (PG) content and water content, but each having an opposite effect on diffusion, thus compromising the diagnostic sensitivity. To overcome this limitation, we propose the simultaneous imaging of cationic (sensitive to PG and water contents) and non-ionic (sensitive to water content) agents. In this study, quantitative dual-energy CT (QDECT) imaging of two agents is reported for the first time at clinically feasible imaging time points. Furthermore, this is the first time synchrotron microCT with monochromatic X-rays is employed in cartilage CECT. Imaging was conducted at 1 and 2 h post contrast agent immersion. Intact, PG-depleted, and mechanically injured + PG-depleted cartilage samples (n = 33) were imaged in a mixture of cationic (iodine-based CA4+) and non-ionic (gadolinium-based gadoteridol) agents. Concurrent evaluation of CA4+ and gadoteridol partitions in cartilage is accomplished using QDECT. Subsequent normalization of the CA4+ partition with that of the gadoteridol affords CA4+ attenuations that significantly correlate with PG content - a key marker of OA.

Quantitative Dual Contrast CT Technique for Evaluation of Articular Cartilage Properties.

Impact injuries of cartilage may initiate post-traumatic degeneration, making early detection of injury imperative for timely surgical or pharmaceutical interventions. Cationic (positively-charged) CT contrast agents detect loss of cartilage proteoglycans (PGs) more sensitively than anionic (negatively-charged) or non-ionic (non-charged, i.e., electrically neutral) agents. However, degeneration related loss of PGs and increase in water content have opposite effects on the diffusion of the cationic agent, lowering its sensitivity. In contrast to cationic agents, diffusion of non-ionic agents is governed only by steric hindrance and water content of cartilage. We hypothesize that sensitivity of an iodine(I)-based cationic agent may be enhanced by simultaneous use of a non-ionic gadolinium(Gd)-based agent. We introduce a quantitative dual energy CT technique (QDECT) for simultaneous quantification of two contrast agents in cartilage. We employ this technique to improve the sensitivity of cationic CA4+ (q =+4) by normalizing its partition in cartilage with that of non-ionic gadoteridol. The technique was evaluated with measurements of contrast agent mixtures of known composition and human osteochondral samples (n = 57) after immersion (72 h) in mixture of CA4+ and gadoteridol. Samples were arthroscopically graded and biomechanically tested prior to QDECT (50/100 kV). QDECT determined contrast agent mixture compositions correlated with the true compositions (R2= 0.99, average error = 2.27%). Normalizing CA4+ partition in cartilage with that of gadoteridol improved correlation with equilibrium modulus (from ρ = 0.701 to 0.795). To conclude, QDECT enables simultaneous quantification of I and Gd contrast agents improving diagnosis of cartilage integrity and biomechanical status.

Dual Contrast CT Method Enables Diagnostics of Cartilage Injuries and Degeneration Using a Single CT Image.

Cartilage injuries may be detected using contrast-enhanced computed tomography (CECT) by observing variations in distribution of anionic contrast agent within cartilage. Currently, clinical CECT enables detection of injuries and related post-traumatic degeneration based on two subsequent CT scans. The first scan allows segmentation of articular surfaces and lesions while the latter scan allows evaluation of tissue properties. Segmentation of articular surfaces from the latter scan is difficult since the contrast agent diffusion diminishes the image contrast at surfaces. We hypothesize that this can be overcome by mixing anionic contrast agent (ioxaglate) with bismuth oxide nanoparticles (BINPs) too large to diffuse into cartilage, inducing a high contrast at the surfaces. Here, a dual contrast method employing this mixture is evaluated by determining the depth-wise X-ray attenuation profiles in intact, enzymatically degraded, and mechanically injured osteochondral samples (n = 3 × 10) using a microCT immediately and at 45 min after immersion in contrast agent. BiNPs were unable to diffuse into cartilage, producing high contrast at articular surfaces. Ioxaglate enabled the detection of enzymatic and mechanical degeneration. In conclusion, the dual contrast method allowed detection of injuries and degeneration simultaneously with accurate cartilage segmentation using a single scan conducted at 45 min after contrast agent administration.