Quantitative determination of morphological and territorial structures of articular cartilage from both perpendicular and parallel sections by polarized light microscopy

Abstract

In order to investigate the three-dimensional 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 microm thick and from a different cartilage depth. Each section was imaged using polarized light microscopy with a 5x objective (2.0 microm pixel size), generating two quantitative images (angle and retardation). Selected sections were also imaged using a 40x objective (0.25 microm pixel size). At an increased depth from the articular surface, the angle and retardation results in the perpendicular sections showed the well-known 90 degrees change in fibril orientation between the surface and the deep cartilage. In contrast, the retardation results of the parallel sections decreased from the articular surface and remained approximately 0 through most of the radial zones, while the angle results of the parallel sections only changed about 30 degrees. 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 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 three-dimensional territorial and interterritorial fibrils in articular cartilage, the disturbance of which could signify the onset of degenerative cartilage diseases such as osteoarthritis.

Figure 1

(a) A schematic representation of the perpendicular (full thickness) and parallel sections from a block of articular cartilage. The short lines illustrate the orientation of collagen fibrils in three histological zones. The horizontal lines on the right face of the block illustrate the parallel sections. (b) The retardation and angle images of a perpendicular section using a 5x objective. Two arrows point to the tidemark line in the cartilage. (c) The retardation and angle images of four parallel sections using a 5x objective at cartilage depths of 18μm (SZ), 96μm (TZ), 270μm (upper RZ) and 468μm (lower RZ). The yellow boxes in the images show the regions where the cartilage was imaged using a 40x objective. All histograms refer to the angle images. The left histogram in (b) is from the first 182μm of the cartilage depth, where the double peaks represent the orientations of the superficial fibrils (centered at 0°) and the radial fibrils (centered at 90°), respectively; the right histogram in (b) is from the remaining deep cartilage, representing only the radial zone. All images are plotted using the same upper and lower limits as shown alongside the scales in (c). (A.S. is articular surface)

Figure 2

(a) Average angle profiles when a 5x objective was used. Data points are shown with their respective standard deviations and fitted with a hyperbolic tangent and a polynomial for the perpendicular and parallel sections, respectively. (b) Retardation profiles for the perpendicular and parallel sections processed identically as in (a). Both profiles are fitted using a polynomial to show depth-dependent birefringence.

Figure 3

The angle and retardation images of two chondrocyte clusters using a 40x objective. The vector maps show graphically the average orientation of collagen fibrils forming “cocoons” around the cell clusters (meshed areas).

Figure 4

The angle and retardation images using a 40x objective. (a) The angle (left) and retardation (right) images of a full-thickness perpendicular section. (b) The angle (left) and retardation (right) images of four parallel sections outlined in Fig 1c.

Figure 5

The depth-dependent angle (left) and retardation (right) images of the cell clusters using a 40x objective, for the perpendicular sections (a) and parallel sections (b) shown in Fig 4 (the yellow boxes). The yellow rings represent the approximate boundaries of the chondrocyte clusters.

Figure 6

(a) The ratio of the long axis (a) and the short axis (b) of the cell clusters at different depths in the perpendicular and parallel sections. The error bars of the parallel sections are the standard deviation from five clusters taken at each depth. (b) The orientation of the cell clusters at different depths in both perpendicular and parallel sections. Five different clusters of cells were analyzed at each tissue depth. Since the cells appeared circular and therefore have no consistent orientation after a depth of 300 μm in the parallel sections, there were no data points shown.

Figure 7

The schematics of the cell clusters in SZ (a) and RZ (c) and their 2D sections (b, d, e). a and b refer to the long and short axes of the chondrocyte clusters plotted in Fig. 6.