Visible and Near-Infrared Spectroscopy Enables Differentiation of Normal and Early Osteoarthritic Human Knee Joint Articular Cartilage

Osteoarthritis degenerates cartilage and impairs joint function. Early intervention opportunities are missed as current diagnostic methods are insensitive to early tissue degeneration. We investigated the capability of visible light-near-infrared spectroscopy (Vis-NIRS) to differentiate normal human cartilage from early osteoarthritic one. Vis-NIRS spectra, biomechanical properties and the state of osteoarthritis (OARSI grade) were quantified from osteochondral samples harvested from different anatomical sites of human cadaver knees. Two support vector machines (SVM) classifiers were developed based on the Vis-NIRS spectra and OARSI scores. The first classifier was designed to distinguish normal (OARSI: 0–1) from general osteoarthritic cartilage (OARSI: 2–5) to check the general suitability of the approach yielding an average accuracy of 75% (AUC = 0.77). Then, the second classifier was designed to distinguish normal from early osteoarthritic cartilage (OARSI: 2–3) yielding an average accuracy of 71% (AUC = 0.73). Important wavelength regions for differentiating normal from early osteoarthritic cartilage were related to collagen organization (wavelength region: 400–600 nm), collagen content (1000–1300 nm) and proteoglycan content (1600–1850 nm). The findings suggest that Vis-NIRS allows objective differentiation of normal and early osteoarthritic tissue, e.g., during arthroscopic repair surgeries.Peer reviewe

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

Ultrasound speed varies in articular cartilage under indentation loading

In ultrasound elastography, tissue strains are determined by localizing changes in ultrasound echoes during mechanical loading. The technique has been proposed for arthroscopic quantification of the mechanical properties of cartilage. The accuracy of ultrasound elastography depends on the invariability of sound speed in loaded tissue. In unconfined geometry, mechanical compression has been shown to induce variation in sound speed, leading to errors in the determined mechanical properties. This phenomenon has not been confirmed in indentation geometry, the only loading geometry applicable in situ or in vivo. In the present study, ultrasound speed during indentation of articular cartilage was characterized and the effect of variable sound speed on the strain measurements was investigated. Osteochondral samples (n = 7, diameter = 25.4 mm), prepared from visually intact bovine patellae (n = 7), were indented with a plane-ended ultrasound transducer (diameter = 5.6 mm, peak frequency: 8.1 MHz). A sequence of three compression tests (strain-rate = 10%/s, 2700-s relaxation) was applied using the mean strains of 2.2%, 4.5%, and 6.4%. Then, ultrasound speed during the ramp and stress-relaxation phases was determined using the time-offlight technique. To investigate the role of cartilage structure and composition for sound speed in loaded articular cartilage, a sample-specific fibril-reinforced poroviscoelastic (FRPVE) finite element model was constructed and fitted to experimental mechanical data. Ultrasound speed in articular cartilage decreased significantly during dynamic indentation (p < 0.05). The magnitude of the decrease in speed during indentation was related to the applied strain. However, the relative error in acoustically determined tissue strain was inversely related to the magnitude of true strain. The modeling results suggested that the compression-related variation in sound speed is controlled by changes in the collagen architecture during dynamic indentation. To conclude, variation in sound speed during dynamic indentation of articular cartilage may lead to significant errors in the values of measured mechanical parameters. Because the relative errors are inversely proportional to applied strain, higher strains should be used to minimize the errors in, e.g., in vivo measurements

Bath concentration of anionic contrast agents does not affect their diffusion and distribution in articular cartilage in vitro

Objective: Differences in contrast agent diffusion reflect changes in composition and structure of articular cartilage. However, in clinical application the contrast agent concentration in the joint capsule varies, which may affect the reliability of contrast enhanced cartilage tomography (CECT). In the present study, effects of concentration of x-ray contrast agents on their diffusion and equilibrium distribution in cartilage were investigated. Design: Full-thickness cartilage discs (d = 4.0 mm, n = 120) were detached from bovine patellae (n = 24). The diffusion of various concentrations of ioxaglate (5, 10, 21, 50 mM) and iodide (30, 60, 126, 300 mM) was allowed only through the articular surface. Samples were imaged with a clinical peripheral quantitative computed tomography scanner before immersion in contrast agent, and after 1, 5, 9, 16, 25, and 29 hours in the bath. Results: Diffusion and partition coefficients were similar between different contrast agent concentrations. The diffusion coefficient of iodide (473 ± 133 μm2/s) was greater (P ≤ 0.001) than that of ioxaglate (92 ± 46 μm2/s). In full-thickness cartilage, the partition coefficient (at 29 h) of iodide (71 ± 5%) was greater (P ≤ 0.02 with most concentrations) than that of ioxaglate (62 ± 6%). Conclusions: Significant differences in partition and diffusion coefficient of two similarly charged (-1) contrast agents were detected, which shows the effect of steric interactions. However, the increase in solute concentration did not increase its partition coefficient. In clinical application, it is important that contrast agent concentration does not affect the interpretation of CECT imaging

Effects of Medium and Temperature on Cellular Responses in the Superficial Zone of Hypo-Osmotically Challenged Articular Cartilage

Osmotic loading of articular cartilage has been used to study cell-tissue interactions and mechanisms in chondrocyte volume regulation &lt;em&gt;in situ&lt;/em&gt;. Since cell volume changes are likely to affect cell’s mechanotransduction, it is important to understand how environmental factors, such as composition of the immersion medium and temperature affect cell volume changes &lt;em&gt;in situ&lt;/em&gt; in osmotically challenged articular cartilage. In this study, chondrocytes were imaged &lt;em&gt;in situ&lt;/em&gt; with a confocal laser scanning microscope (CLSM) through cartilage surface before and 3 min and 120 min after a hypo-osmotic challenge. Samples were measured either in phosphate buffered saline (PBS, without glucose and Ca&lt;sup&gt;2+&lt;/sup&gt;) or in Dulbecco’s modified Eagle’s medium (DMEM, with glucose and Ca&lt;sup&gt;2+&lt;/sup&gt;), and at 21 °C or at 37 °C. In all groups, cell volumes increased shortly after the hypotonic challenge and then recovered back to the original volumes. At both observation time points, cell volume changes as a result of the osmotic challenge were similar in PBS and DMEM in both temperatures. Our results indicate that the initial chondrocyte swelling and volume recovery as a result of the hypo-osmotic challenge of cartilage are not dependent on commonly used immersion media or temperature

Ultrasound backscattering is anisotropic in bovine articular cartilage

Collagen, proteoglycans and chondrocytes can contribute to ultrasound scattering in articular cartilage. However, anisotropy of ultrasound scattering in cartilage is not fully characterized.We investigate this using a clinical intravascular ultrasound device with ultrasound frequencies of 9 and 40 MHz. Osteochondral samples were obtained from intact bovine patellas, and cartilage was imaged in two perpendicular directions: through articular and lateral surfaces. At both frequencies, ultrasound backscattering was higher (p < 0.05) when measured through the lateral surface of cartilage. In addition, the composition and structure of articular cartilage were investigated with multiple reference methods involving light microscopy, digital densitometry, polarized light microscopy and Fourier infrared imaging. Reference methods indicated that acoustic anisotropy of ultrasound scattering arises mainly from non-uniform distribution of chondrocytes and anisotropic orientation of collagen fibers. To conclude, ultrasound backscattering in articular cartilage was found to be anisotropic and dependent on the frequency in use

Effects of freeze-thaw cycle with and without proteolysis inhibitors and cryopreservant on the biochemical and biomechanical properties of articular cartilage

Objective: We investigated the effects of freeze-thawing on the properties of articular cartilage. Design: The reproducibility of repeated biomechanical assay of the same osteochondral sample was first verified with 11 patellar plugs from 3 animals. Then, 4 osteochondral samples from 15 bovine patellae were divided into 4 groups. The reference samples were immersed in phosphate-buffered saline (PBS) containing proteolysis inhibitors and biomechanically tested before storage for further analyses. Samples of group 1 were biomechanically tested before and after freeze-thawing in PBS in the absence and those of group 2 in the presence of inhibitors. Samples of the group 3 were biomechanically tested in PBS-containing inhibitors, but frozen in 30% dimethyl sulfoxide/PBS and subsequently tested in PBS supplemented with the inhibitors. Glycosaminoglycan contents of the samples and immersion solutions were analyzed, and proteoglycan structures examined with SDS-agarose gel electrophoresis. Results: Freeze-thawing decreased slightly dynamic moduli in all 3 groups. The glycosaminoglycan contents and proteoglycan structures of the cartilage were similar in all experimental groups. Occasionally, the diffused proteoglycans were partly degraded in group 1. Digital densitometry revealed similar staining intensities for the glycosaminoglycans in all groups. Use of cryopreservant had no marked effect on the glycosaminoglycan loss during freeze-thawing. Conclusion: The freeze-thawed cartilage samples appear suitable for the biochemical and biomechanical studies

Ultrasound evaluation of mechanical injury of bovine knee articular cartilage under arthroscopic control

A local cartilage injury can trigger development of posttraumatic osteoarthritis (OA). Surgical methods have been developed for repairing cartilage injuries. Objective and sensitive methods are needed for planning an optimal surgery as well as for monitoring the surgical outcome. In this laboratory study, the feasibility of an arthroscopic ultrasound technique for diagnosing cartilage injuries was investigated. In bovine knees (n = 7) articular cartilage in the central patella and femoral sulcus was mechanically degraded with a steel brush modified for use under arthroscopic control. Subsequently, mechanically degraded and intact adjacent tissue was imaged with a high frequency (40 MHz) intravascular ultrasound device operated under arthroscopic guidance. After opening the knee joint, mechanical indentation measurements were also conducted with an arthroscopic device at each predefined anatomical site. Finally, cylindrical osteochondral samples were extracted from the measurement sites and prepared for histological analysis. Quantitative parameters, i.e., reflection coefficient (R), integrated reflection coefficient (IRC), apparent integrated backscattering (AIB), and ultrasound roughness index (URI) were calculated from the ultrasound signals. The reproducibilities (sCV %) of the measurements of ultrasound parameters were variable (3.7% to 26.1%). Reflection and roughness parameters were significantly different between mechanically degraded and adjacent intact tissue(p < 0.05). Surface fibrillation of mechanically degraded tissue could be visualized in ultrasound images. Furthermore, R and IRC correlated significantly with the indentation stiffness. The present results are encouraging; however, further technical development of the arthroscopic ultrasound technique is needed for evaluation of the integrity of human articular cartilage in vivo

Cluster analysis of infrared spectra can differentiate intact and repaired articular cartilage

SummaryObjectiveSuccessful repair of articular cartilage (AC) defects would be a major advantage due to the low ability of AC to heal spontaneously. Sensitive methods to determine changes in AC composition and structure are required to monitor the success of repair. This study evaluates the ability of unsupervised cluster analysis applied to Fourier transform infrared (FTIR) microspectroscopy to discriminate between healthy and repaired AC.MethodsOsteochondral lesions (3 mm in depth) were surgically created in patellar grooves of rabbit femurs and were either left to heal spontaneously (n = 6) or surgically repaired with autologous chondrocytes in type II collagen gel (n = 6). After 6 months, tissues were harvested, FTIR microspectroscopy was conducted and Fuzzy c-means (FCM) cluster analysis applied to spectra of pairs of intact and repaired AC samples from each rabbit. Two spectral regions [amide I and carbohydrate (CHO)] were analyzed and the results from the two types of repair were compared.ResultsTwo separate regions of repair were detected with FCM. The estimated proteoglycan content (from CHO region) in the repaired AC was significantly lower than that in intact AC. The spontaneously repaired AC was better distinguished from the intact AC than the collagen II gel repaired AC. The most distinct clustering was observed for spontaneously repaired samples using CHO region.ConclusionsThis study revealed that unsupervised cluster analysis applied to FTIR microspectroscopy can detect subtle differences in infrared spectra between normal and repaired AC. The method may help in evaluation and optimization of future AC repair strategies