Full thickness blocks of canine humeral cartilage were microtomed into both perpendicular sections and a series of 100 parallel sections, each 6 μm thick. Fourier Transform Infrared Imaging (FTIRI) was used to image each tissue section eleven times under different infrared polarizations (from 0° to 180° polarization states in 20° increments and with an additional 90° polarization), at a spatial resolution of 6.25 μm and a wavenumber step of 8 cm−1. With increasing depth from the articular surface, amide anisotropies increased in the perpendicular sections and decreased in the parallel sections. Both types of tissue sectioning identified a 90° difference between amide I and amide II in the superficial zone of cartilage. The fibrillar distribution in the parallel sections from the superficial zone was shown to not be random. Sugar had the greatest anisotropy in the upper part of the radial zone in the perpendicular sections. The depth-dependent anisotropic data were fitted with a theoretical equation that contained three signature parameters, which illustrate the arcade structure of collagens with the aid of a fibril model. Infrared imaging of both perpendicular and parallel sections provides the possibility of determining the three-dimensional macromolecular structures in articular cartilage. Being sensitive to the orientation of the macromolecular structure in healthy articular cartilage aids the prospect of detecting the early onset of the tissue degradation that may lead to pathological conditions such as osteoarthritis.
In order to investigate the 3D structure of the collagen fibrils in articular cartilage, full thickness canine humeral cartilage was microtomed into perpendicular sections that included both the articular surface and the subchondral bone and approximately 100 successive parallel sections that were each 6 μm thick and from a different cartilage depth. Each section was imaged using polarized light microscopy (PLM) with a 5x objective (2.0μm pixel size), generating two quantitative images (angle and retardation). Selected sections were also imaged using a 40x objective (0.25μm pixel size). At an increased depth from the articular surface, the angle and retardation results in the perpendicular sections showed the well-known 90° change in fibril orientation between the surface and deep cartilage. In contrast, the retardation results of the parallel sections decreased from the articular surface and remained approximately zero through most of the radial zone, while the angle results of the parallel sections only changed about 30°. The territorial matrix morphology surrounding 61 chondrocyte clusters was quantified by its length, aspect ratio, and orientation. The cellular clusters in the surface cartilage were ellipsoidal in both the parallel and perpendicular sections. In the radial zone, the cellular clusters were oriented in vertical columns in the perpendicular sections and as circular groupings in the parallel sections. This orthogonal imaging technique could provide a better understanding of the 3D territorial and interterritorial fibrils in articular cartilage, the disturbance of which could signify the onset of degenerative cartilage diseases such as osteoarthritis.
ObjectiveA quantitative contrast-enhanced micro–computed tomography (qCECT) method was developed to investigate the depth dependency and heterogeneity of the glycosaminoglycan (GAG) concentration of ex vivo cartilage equilibrated with an anionic radiographic contrast agent, Hexabrix.DesignFull-thickness fresh native (n = 19 in 3 subgroups) and trypsin-degraded (n = 6) articular cartilage blocks were imaged using micro–computed tomography (μCT) at high resolution (13.4 μm3) before and after equilibration with various Hexabrix bathing concentrations. The GAG concentration was calculated depth-dependently based on Gibbs-Donnan equilibrium theory. Analysis of variance with Tukey’s post hoc was used to test for statistical significance (P < 0.05) for effect of Hexabrix bathing concentration, and for differences in bulk and zonal GAG concentrations individually and compared between native and trypsin-degraded cartilage.ResultsThe bulk GAG concentration was calculated to be 74.44 ± 6.09 and 11.99 ± 4.24 mg/mL for native and degraded cartilage, respectively. A statistical difference was demonstrated for bulk and zonal GAG between native and degraded cartilage (P < 0.032). A statistical difference was not demonstrated for bulk GAG when comparing Hexabrix bathing concentrations (P > 0.3214) for neither native nor degraded cartilage. Depth-dependent GAG analysis of native cartilage revealed a statistical difference only in the radial zone between 30% and 50% Hexabrix bathing concentrations.ConclusionsThis nondestructive qCECT methodology calculated the depth-dependent GAG concentration for both native and trypsin-degraded cartilage at high spatial resolution. qCECT allows for more detailed understanding of the topography and depth dependency, which could help diagnose health, degradation, and repair of native and contrived cartilage.
Background:The predictable outcome of the anterior cruciate ligament transection (ACLT) canine model, and the similarity to naturally occurring osteoarthritis (OA) in humans, provide a translatable method for studying OA. Still, evidence of direct meniscus-induced cartilaginous damage has not been identified, and gross-anatomical blinded scoring of early-stage OA has not been performed.Objective:A gross anatomical observation and statistical analysis of OA progression to determine meniscus induced cartilaginous damage, to measure the macroscopic progression of OA, and to address matters involving arthroscopic and surgical procedures of the knee.Method:Unblinded assessment and blinded scoring of meniscal, tibial, femoral, and patellar damage were performed for control and at four time points following unilateral ACLT: 3-week (N=4), 8-week (N=4), 12-week (N=5), and 25-week (N=4). Mixed-model statistics illustrates damage (score) progression; Wilcoxon rank-sum tests compared time-point scores; and Wilcoxon signed-rank tests compared ACLT and contralateral scores, and meniscus and tibia scores.Result:Damage was manifest first on the posterior aspect of the medial meniscus and subsequently on the tibia and femur, implying meniscal damage can precede, coincide with, and aggravate cartilage damage. Damage extent varied chronologically and was dependent upon the joint component. Meniscal damage was evident at 3 weeks and progressed through 25-weeks. Meniscal loose bodies corresponded to tibial cartilage damage location and extent through 12 weeks, followed by cartilage repair activity after complete meniscal degeneration.Conclusion:This study provides additional information for understanding OA progression, identifying OA biomarkers, and arthroscopic and meniscectomy procedures.
Background: Medical imaging has become an invaluable tool to diagnose damage to cartilage. Depletion of glycosaminoglycans (GAG) has been shown to be one of the early signs of cartilage degradation. In order to investigate the topographical changes in GAG concentration caused by the anterior cruciate ligament transection (ACLT) surgery in a canine model, microscopic magnetic resonance imaging (µMRI) and microscopic computed tomography (µCT) were used to measure the GAG concentration with correlation from a biochemical assay, inductively coupled plasma optical emission spectroscopy (ICP-OES), to understand where the topographical and depth-dependent changes in the GAG concentration occur.Methods: This study used eight knee joints from four canines, which were examined 3 weeks after ACLT surgery. From right (n=3) and left (n=1) medial tibias of the ACLT and the contralateral side, two ex vivo specimens from each of four locations (interior, central, exterior and posterior) were imaged before and after equilibration in contrast agents. The cartilage blocks imaged using µMRI were approximately 3 mm × 5 mm and were imaged before and after eight hours submersion in a gadolinium (Gd) contrast agent with an in-plane pixel resolution of 17.6 µm 2 and an image slice thickness of 1 mm. The cartilage blocks imaged using µCT were approximately 2 mm × 1 mm and were imaged before and after 24 hours submersed in ioxaglate with an isotropic voxel resolution of 13.4 µm 3 . ICP-OES was used to quantify the bulk GAG at each topographical location.Results: The pre-contrast µMRI and µCT results did not demonstrate significant differences in GAG between the ACLT and contralateral cartilage at all topographical locations. The post-contrast µMRI and µCT results demonstrated topographically similar significant differences in GAG concentrations between the ACLT and contralateral tibia. Using µMRI, the GAG concentrations (mg/mL) were measured for the ACLT and contralateral respectively, the exterior (54.0±3.
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