Raman needle arthroscopy for in vivo molecular assessment of cartilage.

The development of treatments for osteoarthritis (OA) is burdened by the lack of standardized biomarkers of cartilage health that can be applied in clinical trials. We present a novel arthroscopic Raman probe that can "optically biopsy" cartilage and quantify key extracellular matrix (ECM) biomarkers for determining cartilage composition, structure, and material properties in health and disease. Technological and analytical innovations to optimize Raman analysis include (1) multivariate decomposition of cartilage Raman spectra into ECM-constituent-specific biomarkers (glycosaminoglycan [GAG], collagen [COL], water [H2 O] scores), and (2) multiplexed polarized Raman spectroscopy to quantify superficial zone (SZ) COL anisotropy via a partial least squares-discriminant analysis-derived Raman collagen alignment factor (RCAF). Raman measurements were performed on a series of ex vivo cartilage models: (1) chemically GAG-depleted bovine cartilage explants (n = 40), (2) mechanically abraded bovine cartilage explants (n = 30), (3) aging human cartilage explants (n = 14), and (4) anatomical-site-varied ovine osteochondral explants (n = 6). Derived Raman GAG score biomarkers predicted 95%, 66%, and 96% of the variation in GAG content of GAG-depleted bovine explants, human explants, and ovine explants, respectively (p < 0.001). RCAF values were significantly different for explants with abrasion-induced SZ COL loss (p < 0.001). The multivariate linear regression of Raman-derived ECM biomarkers (GAG and H2 O scores) predicted 94% of the variation in elastic modulus of ovine explants (p < 0.001). Finally, we demonstrated the first in vivo Raman arthroscopy assessment of an ovine femoral condyle through intraarticular entry into the synovial capsule. This study advances Raman arthroscopy toward a transformative low-cost, minimally invasive diagnostic platform for objective monitoring of treatment outcomes from emerging OA therapies.

Tantalum Oxide Nanoparticles for the Quantitative Contrast-Enhanced Computed Tomography of Ex Vivo Human Cartilage: Assessment of Biochemical Composition and Biomechanics.

Nanoparticle-based contrast agents, when used in concert with imaging modalities such as computed tomography (CT), enhance the visualization of tissues and boundary interfaces. However, the ability to determine the physiological state of the tissue via the quantitative assessment of biochemical or biomechanical properties remains elusive. We report the synthesis and characterization of tantalum oxide (Ta2O5) nanoparticle (NP) contrast agents for rapid, nondestructive, and quantitative contrast-enhanced computed tomography (CECT) to assess both the glycosaminoglycan (GAG) content and the biomechanical integrity of human metacarpal phalangeal joint (MCPJ) articular cartilage. Ta2O5 NPs 3-6 nm in diameter and coated with either nonionic poly(ethylene) glycol (PEG) or cationic trimethylammonium ligands readily diffuse into both healthy and osteoarthritic MCPJ cartilage. The CECT attenuation for the cationic and neutral NPs correlates with the glycosaminoglycan (GAG) content (R2 = 0.8975, p < 0.05 and 0.7054, respectively) and the equilibrium modulus (R2 = 0.8285, p < 0.05 and 0.9312, p < 0.05, respectively). The results highlight the importance of the surface charge and size in the design of NP agents for targeting and imaging articular cartilage. Further, nanoparticle CECT offers the visualization of both soft tissue and underlying bone unlike plain radiography, which is the standard for imaging bone in musculoskeletal diseases, and the ability to provide a real-time quantitative assessment of both hard and soft tissues to provide a comprehensive image of the disease stage, as demonstrated herein.

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.

A Markov chain model of particle deposition in the lung.

Particle deposition in the lung during inhalation is of interest to a wide range of biomedical sciences due to the noninvasive therapeutic route to deliver drugs to the lung and other organs via the blood stream. Before reaching the alveoli, particles must transverse the bifurcating network of airways. Computational fluid mechanical studies are often used to estimate high-fidelity flow patterns through the large conducting airways, but there is a need for reduced-dimensional modeling that enables rapid parameter optimization while accommodating the complete airway network. Here, we introduce a Markov chain model with each state corresponding to an airway segment in which a particle may be located. The local flows and transition probabilities of the Markov chain, verified against computational fluid dynamics simulations, indicate that the independent effects of three fundamental forces (gravity, fluid drag, diffusion) provide a sufficient approximation of overall particle behavior. The model enables fast computation of how different inhalation strategies, called flow policies, determine total particle escape rates and local particle deposition. In a 3-dimensional airway tree model, the optimal flow policy minimizing the risk of deposition at each generation, compared to other inlet flow waveforms, predicted significantly higher probability of escape defined as the fraction of particles exiting the tree. The model also predicts a small influence of body orientation with respect to a gravitational field on total escape probability, but a significant effect of airway narrowing on regional deposition. In summary, this model provides insight into inhalation strategies for targeted drug delivery.

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.

Assessment of healthy trapeziometacarpal cartilage properties using indentation testing and contrast-enhanced computed tomography.

BACKGROUND: The trapeziometacarpal joint is a common site for osteoarthritis development in the hand. When osteoarthritis is present, it results in significant functional disabilities due to the broad range of activities performed by this joint. However, our understanding of osteoarthritis initiation and progression at this joint is limited because of the current lack of knowledge regarding the properties and structure of the corresponding cartilage layers. The objective of this study is to assess the morphological and mechanical properties of trapeziometacarpal cartilage via the combination of indentation testing and contrast-enhanced computed tomography. Such research may lead to the development of medical imaging-based approaches to measure cartilage properties in vivo. METHODS: Intact first metacarpals and trapezia were extracted from 16 fresh-frozen human cadaver hands. For each specimen, load-displacement behavior was measured at 9 testing sites using a standardized indentation testing device to calculate the normal force and Young's modulus of the cartilage sub-regions. The specimens were then immersed in CA4+ contrast agent solution for 48 h and subsequently scanned with a resolution of 41 µm in a HR-pQCT scanner to measure cartilage thickness and attenuation. Finally, correlations between compressive Young's modulus and contrast-enhanced computed tomography attenuation of the cartilage were assessed. FINDINGS: No significant difference was found in cartilage thickness between the trapezium and first metacarpal, but the comparison between articular regions showed thinner cartilage around the volar aspect of both the first metacarpal and the trapezium. The first metacarpal cartilage was stiffer than the trapezial cartilage. A significant positive correlation was observed between Young's modulus and mean contrast-enhanced CT attenuations in superficial and full-depth cartilage in both the first metacarpal and the trapezium cartilage. INTERPRETATION: The quantitative measurements of trapeziometacarpal thickness and stiffness as well as a correlation between Young's modulus and contrast-enhanced computed tomography attenuation provides a method for the non-destructive in vivo assessment of cartilage properties, a greater understanding of thumb cartilage behavior, and a dataset for the development of more accurate computer models.

Contrast-enhanced CT using a cationic contrast agent enables non-destructive assessment of the biochemical and biomechanical properties of mouse tibial plateau cartilage.

Mouse models of osteoarthritis (OA) are commonly used to study the disease's pathogenesis and efficacy of potential treatments. However, measuring the biochemical and mechanical properties of articular cartilage in these models currently requires destructive and time-consuming histology and mechanical testing. Therefore, we examined the feasibility of using contrast-enhanced CT (CECT) to rapidly and non-destructively image and assess the glycosaminoglycan (GAG) content. Using three ex vivo C57BL/6 mouse tibial plateaus, we determined the time required for the cationic contrast agent CA4+ to equilibrate in the cartilage. The whole-joint coefficient of friction (µ) of 10 mouse knees (some digested with Chondroitenase ABC to introduce variation in GAG) was evaluated using a modified Stanton pendulum. For both the medial and lateral tibial plateau cartilage of these knees, linear regression was used to compare the equilibrium CECT attenuations to µ, as well as each side's indentation equilibrium modulus (E) and Safranin-O determined GAG content. CA4+ equilibrated in the cartilage in 30.9 ± 0.95 min (mean ± SD, tau value of 6.17 ± 0.19 min). The mean medial and lateral CECT attenuation was correlated with µ (R(2) = 0.69, p < 0.05), and the individual medial and lateral CECT attenuations correlated with their respective GAG contents (R(2) ≥ 0.63, p < 0.05) and E (R(2) ≥ 0.63, p < 0.05). In conclusion, CECT using CA4+ is a simple, non-destructive technique for three-dimensional imaging of ex vivo mouse cartilage, and significant correlations between CECT attenuation and GAG, E, and µ are observed. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1130-1138, 2016.

Assessment of contrast-enhanced computed tomography for imaging of cartilage during fracture healing.

Assessment of the early stages of fracture healing via X-rays and computed tomography is limited by the low radio-opacity of cartilage. We validated a method of contrast-enhanced computed tomography (CECT) for non-destructive identification of cartilage within a healing fracture callus. Closed, stabilized fractures in femora of C57BL/6 mice were harvested on post-operative day 9.5 and imaged ex vivo with micro-computed tomography (µCT) before and after incubation in a cationic contrast agent that preferentially accumulates in cartilage due to the high concentration of sulfated glycosaminoglycans in the tissue. Co-registration of the pre- and post-incubation images, followed by image subtraction, enabled two- and three-dimensional delineation of mineralized tissue, soft callus, and cartilage. The areas of cartilage and callus identified with CECT were compared to those identified with the gold-standard method of histomorphometry. No difference was found between the areas of cartilage measured by the two methods (p = 0.999). Callus area measured by CECT was smaller than, but strongly predictive of (R(2) = 0.80, p < 0.001), the corresponding histomorphometric measurements. CECT also enabled qualitative identification of mineralized cartilage. These findings indicate that the CECT method provides accurate, quantitative, and non-destructive visualization of the shape and composition of the fracture callus, even during the early stages of repair when little mineralized tissue is present. The non-destructive nature of this method would allow subsequent analyses, such as mechanical testing, to be performed on the callus, thus enabling higher-throughput, comprehensive investigations of bone healing.