Characteristics of topographical heterogeneity of articular cartilage over the joint surface of a humeral head

Osteoarthritis and Cartilage (2002) 10, 370–380 © 2002 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved. doi:10.1053/joca.2002.0523, available online at http://www.idealibrary.com on

1063–4584/02/$35.00/0

Characteristics of topographical heterogeneity of articular cartilage over the joint surface of a humeral head Y. Xia*, J. B. Moody*, H. Alhadlaq*, N. Burton-Wurster† and G. Lust† *Department of Physics and Center for Biomedical Research, Oakland University, Rochester, Michigan 48309, U.S.A. †James A. Baker Institute for Animal Health, Cornell University, Ithaca, New York 14853, U.S.A. Summary Objective: To investigate the topographical variations among the morphological and physical properties of articular cartilage over a canine humeral head. Design: Nine side-by-side specimens from a canine humeral head were used in the combined polarized light microscopy (PLM) and microscopic magnetic resonance imaging (
MRI). Sixteen images were acquired from each specimen in
MRI. Subsequently, ten to fourteen histological slices were prepared at the location of the
MRI slice from each specimen. Four optical images were acquired from each histological slice. Using these images, the images of the T2 relaxation, the optical retardation, and the collagen-fiber orientation were constructed in two dimensions. Results: Along the medial/lateral direction of the humeral head, we have observed a number of topographical variations. These include the total thickness of the cartilage tissue, the thickness of the individual histological zones, the depth of the minimum optical retardation in the tissue, and the depth of the maximum T2 relaxation in the tissue. We found that the depth of the minimum retardation from PLM occurs at the geometric middle point of the transitional zone in histology, and that the depth of the maximum T2 relaxation from
MRI is closely correlated with the minimum retardation depth in PLM. In addition, although the thickness of the total tissue increases approximately by the same amount on both the lateral and medial sides of the joint, a slight asymmetry exists between the measurements from the medial and lateral halves of the humerus. Conclusions: Cartilage from different sites over a single joint could have different properties, possibly the consequence of the variation in load-bearing status. Because of these topographical variations, one must keep in mind that a different sampling site within a relatively small area of the same joint may significantly influence the results of the study. © 2002 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved. Key words: Cartilage, MRI, Polarized light microscopy, Topographical variation.

the collagen fibers in the tissue15, and the mechanical properties of the tissue9. It is also reasonable to consider variations in the load-bearing status over a joint surface: some sites bear more loads than others. The variations in molecular structure and content may simply reflect the differences in the mechanical demands on the cartilage sites over the joint. In addition to variations within a single joint, cartilage from different types of joints in the same animal may vary because of the different load-bearing patterns of the different joints. Likewise, cartilage from the same type of joint in different animal species might also vary as a consequence of variations in their natural way of motion. For example, a human shoulder joint functions very differently to that of a dog. Hence, the site-to-site variations within a single joint appear to optimally meet the mechanical demands of a given joint in a given animal species. Quantitative knowledge of the topographical variations of cartilage over a joint surface provides a basis to understand the mechanisms underlining the tissue’s functions and activities at different loading sites, and to evaluate the degradation and failure of the tissue in the event of disease and injury. Magnetic Resonance Imaging (MRI) has been used in recent decades to diagnose the cartilage diseases16 and to study the mechanisms in the tissue degradation13,17–21. In

Introduction The major biological and biomechanical functions of hyaline articular cartilage in a synovial (diarthrodial) joint are to act as a resilient, load-bearing medium that absorbs shocks and distributes stress, and to provide a smooth and curved two-dimensional (2D) surface for joint motion1,2. Although it is convenient to consider articular cartilage as having a one-dimensional (1D) variation that consists of three histological zones across the depth of the tissue (superficial, transitional, radial), cartilage tissue taken from different sites in a single joint surface should not be considered identical3–5. A number of topographical variations have been known to exist in cartilage from different sites within a single joint. These variations include the thickness of the histological zones and the total tissue6–10, the content of major molecular components8,11–14, the organization of Received 15 June 2001; accepted 15 October 2001. Source of Support: Research Excellence Fund in Biotechnology from Oakland University (YX). An instrument endorsement from R. B. and J. N. Bennett (YX). R01 grant (AR 45172) from NIH (YX). *Address correspondence to: Yang Xia, Ph.D., Department of Physics, Oakland University, Rochester, Michigan 48309, U.S.A. Tel: (248) 370-3420; Fax: (248) 370-3408; E-mail: [email protected]

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Osteoarthritis and Cartilage Vol. 10, No. 5 MRI studies of articular cartilage, it is known that normal cartilage can appear laminated when placed at certain orientations with respect to the external magnetic field (B0), a phenomenon known as the laminar appearance of articular cartilage in MRI5,22,23. When the normal axis of the articular surface of the tissue (the radial direction) is approximately 55° with respect to B0, the cartilage does not appear laminated but appears homogeneous and brighter than at other angles. Because the angle of 54.74° is known as the magic angle in Nuclear Magnetic Resonance (NMR), this disappearance of the cartilage laminae associated with the 55° angle is known as the magic angle effect in MRI of cartilage. The origin of the magic angle effect in MRI of cartilage is the T2 relaxation anisotropy in the tissue5. T2 relaxation corresponds to the decay in phase coherence between the individual nuclear spins in a sample (protons in our case). For samples containing molecules that are less mobile, the dipolar spin Hamiltonian is no longer averaged to zero and could make a significant contribution to the spin relaxation, resulting in a shorter T2. The equation for the dipolar spin Hamiltonian contains a geometrical factor, (3cos2−1), where  is the angle between the position vector joining the two spins and the external magnetic field. Fullerton et al.24 showed that T2 of bovine tendons has two components and that the long T2 component varies approximately with the curve defined by (3cos2−1). The reason for this variation is that water molecules near collagen fibrils are no longer as mobile as they are in non-viscous solutions. Instead, these water molecules are loosely bound to the fibrils. Consequently, their motion is restricted and may become anisotropic. Several microscopic MRI (
MRI) studies showed that articular cartilage possesses a distinct T2 anisotropy that is linked closely to the local collagen ultrastructure, and to the water–proteoglycan interaction that ‘amplifies’ the prevailing directional orientation of the collagen fibrils by influencing the mobility of water20,25. This T2 anisotropy translates into the orientational dependence and laminated appearance of the MR image intensity of the cartilage. In a recent study of canine cartilage using
MRI and polarized light microscopy (PLM)26, we have shown that the three
MRI zones based on the T2 characteristics are statistically equivalent to the histological zones based on the collagen fiber orientation. Just as non-calcified cartilage can be conceptually subdivided based on the orientation of the collagen fibers into three distinct structural zones in histology and optical microscopy, we show that cartilage can also be subdivided based on the regional characteristics of the T2 relaxation in MRI into three structural zones. These findings suggest that it is now possible to study the tissue structures and molecular activities in individual loading sites of the tissue using
MRI and the conventional PLM, and to study the quantitative correlation between the results from
MRI and PLM26. The goal of this study is to apply our quantitative correlation techniques to characterize the topographical variations in the morphological and physical properties of articular cartilage over a joint surface accurately and quantitatively, at the highest possible MRI resolution, and based on the same piece of tissue.

Material and methods CARTILAGE SAMPLES

Nine cartilage–bone plugs were harvested from the right humerus in the shoulder joint of an 8-month-old dog

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Lesser tubercle

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L42 L32 L22 L12 C02 R12 R22 R32 R42

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Caudal Fig. 1. The schematic joint surface of a right humeral head with the locations of the cartilage–bone plugs in the imaging experiments. The arrow points to the slice locations for both
MRI and histology studies.

(female, a cross of 3/4 Greyhound and 1/4 Labrador retriever, about 100 pounds). The animal was healthy and sacrificed for an unrelated study. The joint was frozen at −80°C immediately after necropsy and thawed at 4°C before the opening. Each plug has a dimension about 2 mm by 2 mm by 10 mm. These nine plugs were adjacent to each other and across nearly the entire width of the humerus (Fig. 1). The left edge of the L42 plug and the right edge of the R42 plug were about 1–2 mm from the margins of the joint surface. A marker was placed on each plug to indicate the original orientation of the plug on the joint surface. The plugs, consisting of the full thickness of the articular cartilage attached to the underlying bone, were bathed in physiological saline and sealed in precision NMR tubes. The samples in the glass tubes were placed at room temperature for about 4 h before the
MRI experiments. The Institutional Review Boards have approved the protocols handling the animal subjects. MICROSCOPIC MRI (
MRI) PROTOCOL

The
MRI experiments were performed on a Bruker AMX 300 NMR spectrometer equipped with a 7-Tesla/89-mm vertical-bore superconducting magnet and micro-imaging accessory (Bruker Instrument, Billerica, MA). Quantitative T2-imaging experiments were repeated for each sample at four angular orientations with respect to the B0 field (0°, 35°, 55°, 90°). The angular orientation  is defined as the angle between the normal to the articular surface (i.e., the radial direction) and the direction of the B0 field. The T2 relaxation was calculated by a single exponential fitting of the data on a pixel-by-pixel basis at each orientation. The echo time (TE) of the imaging segment (TEi) was 8.86 ms; the echo time of the T2-contrast segment (TEc) were 0, 16, 30, and 60 ms; and the repetition time (TR) of the experiments was 2 s. The in-plane pixel resolution, which was across the depth of the cartilage tissue, was 13.7
m, and the slice thickness was 1 mm. The imaging planes in
MRI and later in PLM are parallel with the central transverse

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direction across the lateral-medial width of the joint. Other experimental details have been described earlier20,23.

POLARIZED LIGHT MICROSCOPY (PLM) PROTOCOL

After the
MRI experiments, each cartilage–bone plug was cut approximately at the same location of the imaging slice so that the same plane that had been just examined by
MRI could be examined by histology. A marker was created on each half of the plug to identify the end of the imaging plane. The specimens were prepared using the standard histological methods26. Ten to fourteen histological sections (6-
m thickness) were made from each cartilage–bone plug. Four optical images were acquired for each histological section. Our PLM system has a thermoelectrically cooled Sensys 12-bit CCD camera (Photometrics, Tucson, AZ) mounted on a Leica DM RXP Polarized Light Microscope (Leica Microsystems Wetzlar GmbH, Wetzlar, Germany). The output of the CCD camera was digitized and sent to a personal computer (Power Macintosh G3/266, Apple Computers, Cupertino, CA) for analysis. Our PLM protocol uses the method of Oldenbourg and Mei27 with circular polarized light and a liquid crystal compensator (Cambridge Research and Instrumentation Inc., Cambridge, MA). This protocol allows the calculation of images representing the optical retardance and the angular orientation of birefrigent elements in the tissue. At 5× magnification, a retardance sensitivity of (0.32± 0.17) nm was found in our system (the rms noise level±the standard deviation of the noise level). The pixel resolution in our PLM measurements was 2.72
m by 2.72
m. Other experimental details have been described earlier26,28.

DATA ANALYSIS AND PRESENTATION

The results of this study are presented both as 2D images and as one-dimensional (1D) cross-sectional profiles through the depth of the cartilage tissue. The crosssectional profiles enable us to examine the results quantitatively and to compare the profiles from different imaging experiments. To enhance the signal-to-noise ratio of the 1D profiles, parallel neighboring columns of data from a rectangular region-of-interest (ROI) in the 2D images were averaged to obtain the 1D cross-sectional profiles presented in this report. The width and the location of the ROI were approximately the same in both
MRI and PLM. All 1D profiles in a comparative plot shown in this study were processed with identical data-processing procedures. No manual scaling or adjustment was made when plotting several cross-sectional profiles, which came from independent experiments, into one graph. (In the descriptions related to the data analysis and presentation in this manuscript, the word ‘column’ refers to a single-pixel-wide 1D data array; the word ‘profile’ refers to a 1D data array averaged from the columns of data within a rectangular ROI.) For
MRI experiments, 20 parallel neighboring columns of data from the 2D-
MR images were averaged to obtain the 1D cross-sectional profiles presented in this report. Because the averaging process occurs perpendicular to the tissue depth, the spatial resolution of the 1D profiles along the direction of the tissue depth is still 13.7
m. For PLM experiments, neighboring columns with the same width in the ROI as in the
MRI profiles were averaged to produce one 1D profile from each histological section. The

1D profiles from 10–14 histological sections from the same specimen were then averaged to produce the 1D PLM profiles shown here. The pixel resolution in the averaged PLM profiles along the tissue depth is still 2.72
m. The data for comparison were analysed using the Student t-test in KaleidaGraph.

Results The MR proton images of all nine samples for the three orientations of the tissue in the magnetic field at the shortest TE are shown in Fig. 2. These proton images clearly show the classic laminar appearance of the tissue in MRI5,20. Close examination reveals that a subtle feature of these MR proton images is the topographical variation of the tissue thickness along the medial/lateral direction. The cartilage tissue was thin at the central location and thicker at both lateral and medial peripheral sites. The PLM intensity images of all nine samples under linearly polarized light are shown in Fig. 3. Because of the approximate 90° difference in the collagen orientations in the superficial and radial zones, the maximum contrast among the superficial/ transitional/radial zones occurs when the articular surface of the tissue is about 45° to the transmission axis of the linear polarizer. These intensity images in PLM also showed a similar medial/lateral variation of the tissue thickness over the joint. The total thickness of the tissue was measured from the central region of each MR image and each PLM image, at approximately the same physical location in the image, and compared in Fig. 4. The correlation between the MRI thickness and the PLM thickness was excellent (t-probability=0.8439). It is interesting to note that the cartilage in this humeral head (of a 100-lb dog) exhibits a consistent laminar appearance across the entire width of the joint. This is quite different from one of our earlier studies where several distinct heterogeneities of the laminar pattern were observed in the humeral heads of the much smaller beagles (about 20 lb)10. 2D images of the fiber angle and the optical retardation in the cartilage specimens were calculated for all histological sections from each specimen. To compare quantitatively the detailed variations of the angle and the retardation across the depth of the tissue, cross-sectional profiles are plotted in Fig. 5 for both the retardation and the angle from all nine samples, taken from approximately the same location in the images of the specimens. The angle maps in our measurement represent the pixel-averaged orientations of the collagen fibers in the tissue. The angle profiles show that the collagen fibers have an approximately 90° difference between the surface and the deep zones, as expected. The retardation maps are influenced by several factors in the measurements, including the randomness of the collagen fibrils, the fibril diameter, the packing density of the fibrils, and the thickness of the histological section. For a single section, a smaller retardation value generally indicates that the collagen fibers are less ordered. The retardation profiles show that the optical retardation of articular cartilage is a strong function of the tissue depth. A striking feature of these profiles is the difference between the images from the lateral half and the medial half of the joint. For the medial half of the joint, there is a gradual shift of the center of the transitional zone towards the deep part of the tissue [Fig. 5(a),(b)]. However, although the thickness of the total tissue increases by approximately the same amount on both the sides of the

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Fig. 2. The MRI images of the nine cartilage–bone plugs from the humeral head placed side by side in their original order on the joint surface. Each specimen was imaged at four different angles in the magnetic field (B0). At each angle, each specimen was imaged at four different T2 weightings. The intensity images with the least T2 weighting and at three angles are shown here (labeled as Pro1). (The image at the fourth angle is needed for the tissue zone division. It looks very similar to the 90° image.) Two T2 images with the most and the least dipolar attenuation were also shown. Note that the angle  is defined as the angle between the normal to the articular surface of cartilage (i.e., the radial direction of the surface) and the direction of the B0. The angle at about 55° is the magic angle where the laminar appearance of the cartilage disappears and the tissue appears homogeneous. After the experiments, each image was rotated individually so that the articular surface is leveled.

joint, the similar shift was absent from the lateral half of the joint [Fig. 5(c),(d)]. Using the quantitative criteria that we have established in our previous study26, we calculated the histological zone thickness for each specimen based on the angle profiles [Fig. 5(b),(d)]. The thickness of each histological zone exhibits different degrees of topographical variation, as shown in Fig. 6. Moving from the medial peripheral to the lateral peripheral transversely on the humeral surface, the thickness of the superficial zone decreases slightly; the thickness of the radial zone increases; and the thickness of the transitional zone has a concave shape and is the thinnest near the central location. We also found that the minimums of the optical retardation profiles of the tissues exhibit excellent correlation (t-probability=0.9596) with the middles of the transitional zones (Fig. 7). This result shows

that the collagen fibers reach their most random state at the middle of the transitional zones. A quantitative T2 map was calculated for each specimen that was oriented with its normal to the articular surface parallel with the magnetic field. 1D T2 profiles when the specimens were oriented at about 0° with the B0 field were extracted from the 2D images so that the detailed features of the T2 profiles can be compared quantitatively (Fig. 8). The T2 profiles in our experiments reflect the state of the water molecules in the tissue. The T2 relaxation in articular cartilage is known to have a unique anisotropy that is linked closely to the local collagen ultra-structure, and to the water–proteoglycan interaction that ‘amplifies’ the prevailing directional orientation of the collagen fibers by influencing the mobility of water20,25,26. As we have described in our previous studies, these T2 profiles also appear to be a

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Fig. 3. The PLM intensity images of the nine cartilage–bone plugs from the humeral head placed side by side in their original order on the joint surface. Ten to fourteen unstained sections were made from each specimen at the location of the MRI slice. The images shown here were obtained using the linearly polarized light.

Total tissue thickness (µm)

ties of the cartilage tissue over a single canine humeral head. Although one right humerus was used, the findings in this report should be generally applicable to other humeri, for the following five reasons.

0

Articular surface

200 400 600 800 1000

Medial

Central

Lateral

L42 L32 L22 L12 C02 R12 R22 R32 R42 Specimen Fig. 4. The correlation of the total tissue thickness as measured by PLM and by MRI. The tissue is the thinnest at the central location and thicker at both the lateral and medial peripherals. PLM thickness (
m; ); MRI thickness (
m, ); t-probability=0.8439.

strong function of the tissue depth. Under a close examination, the locations of the maximum T2 also exhibit a gradual shift across the width of the humeral surface [Fig. 9(a)]. Although the locations of the maximum T2 and the minimum retardation did not occur at the same depths, they follow a similar trend. If the locations of the maximum T2 were multiplied by a factor of 1.5, the maximum T2 locations could be correlated well with the minimum retardation locations (t-probability=0.9417) in Fig. 9(b). Table I summarizes the topographical variations of the tissue properties observed in this report.

Discussion We have observed in this study several topographical variations among the morphological and physical proper-

(1) We have acquired nine side-by-side specimens from the humeral head. Each specimen was studied independently by multiple
MRI experiments (16 independent images) and multiple PLM experiments (10–14 independent histological slices, each has four optical images). These individual images and profiles for each specimen are highly consistent. The smoothness in our pieced-together images shown in Figs 2 and 3 has clearly demonstrated the quality and repeatability of our independent measurements. All characteristic profiles and the data in Table I are the averaged results from the independent experiments for each specimen. The multiple 2D images enable us to apply the statistical procedures to analyse the characteristics of the tissue. (2) Our
MRI technique has the highest spatial resolution (13.7
m) reported in literature10,20,23. We have applied our techniques to several previous studies and obtained highly consistent results. The features of the tissue seen in this study is consistent with our previous observations. (3) Our PLM technique has also been used to study the canine and bovine tissues extensively. We have demonstrated that the determination of three histological zones and the total tissue thickness for canine humeral heads are statistically equivalent in MRI and PLM26,28. (4) The shoulder joint used in this experiment came from a dog that belongs to a group of similar dogs for an unrelated genetic study for the hips. This group of dogs is believed to have normal shoulder joints, a fact confirmed by repeated inspection of the shoulder joints from many of its siblings in our laboratories at two universities.

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Fig. 5. The 1D optical retardation profiles (a) and (c) and the collagen angle profiles (b) and (d) from a rectangular region-of-interest in the middle of the 2D angle and retardation images from all nine specimens. The pixel resolution along the tissue depth is 2.72
m (only one in every ten pixels was plotted in the profiles). While there is a gradual shift of the center of the transitional zone towards the deep part of the tissue in the medial half of the joint (a),(b), a similar shift was absent from the lateral half of the joint (c),(d). Two notes about the angle profiles: (1) Outside the tissue, the angle calculation for each individual histological section returns an arbitrary value and the average among the 10–14 histological sections from each specimen has a large variation. (2) The angle calculation in our PLM technique has a range from 0° to 180°. This is equivalent to the fact that a collagen fibril has neither head nor tail. (a) and (b) L42, — —; L32, — —; L22, — —; L12, ×; CO2 — —. (c) and (d) R42, — —; R32, — —; R22, — —; R12 ×; CO2, — —.

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Fig. 6. The topographical variations of the individual histological zones: the squares are the superficial zone (SZ); the open circles are the transitional zone (TZ); and the solid circles are the radial zone (RZ).

Fig. 7. The comparison of the minimum retardation in the tissue (
m, ) and the geometric middle point (SZ+TZ/2; ) of the transitional zone.

(5) It has been reported in literature9 that there is no significant differences in the mechanical properties and thickness in cartilage between right and left

humerus for a given geometric location on the joint surface, and that there is no significant differences for a given location between dogs. For example, the

Y. Xia et al.: Characteristics of topographical variations in cartilage )

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Fig. 8. (a) The T2 relaxation profiles from the nine specimens: (a) medial and (b) lateral. The angles were between the normal to the articular surface (i.e., the radial direction) and the direction of the B0 field. (a) CO2. T2 at 1°, ; L12. T2 at −3.5°, +; L22. T2 at −0.5°, ; L32. T2 at 3.5°, ×; L42. T2 at −0.5°, . (b) CO2. T2 at 1°, ; R12. T2 at 0°, +; R22. T2 at 1.5°, ; R32. T2 at 2°, ×; R42. T2 at 4°, .

percentage variations in our total tissue thickness at different sites are highly consistent with three previous investigations of canine humeral heads7,9,12. THE CORRELATION BETWEEN THE MIN RETARDATION AND THE MAX T2 IN THE TISSUE

In articular cartilage, there are several distinct ‘pools’ of water molecules, such as water molecules associated with the collagen fibrils, water molecules hydrogen-bonded to proteoglycans (PG) via electrostatic attraction, and water molecules relatively free to move around in the tissue29. Although the motion of water molecules associated with collagen is known to be strongly anisotropic20,24,30, this pool is a relatively small fraction of the total (ca. 11% wet-weight)31. Most water molecules are associated with the PG or are ‘free’. When more than one pool of spins exists in a voxel, the MRI signal is the weighted ensemble average of all MRI-visible spins. Because of the finite echo time in MRI (at least several milliseconds), the signal from the collagen waters is not directly visible in MRI. However, because of the exchange processes between the tightlybound collagen waters and the rest of the ‘loosely-bound’ waters, the T2 relaxation in articular cartilage exhibits a

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Fig. 9. The correlation between the min retardation in PLM and the max T2 in
MRI in the specimens. Although the locations of the maximum T2 and the minimum retardation did not occur at the same depths (see text for discussion), they follow a similar trend in (b).

strong anisotropy, which has a directional characteristic that reflects the prevailing collagen fibril orientation20. The results in this study show that (1) the minimum optical retardation in each specimen is the geometric middle point of the transitional zone in histology; and (2) the minimum optical retardation in each specimen occurs at a deeper location (by about 30–70
m) than the location of the maximum T2 relaxation calculated from the 0° orientation with the magnetic field. In our experiments, the minimum retardation location indicates the least ordered location of the collagen fibers in the tissue. By comparison, the maximum T2 location at 0° indicates the location of the least-ordered water molecules in the tissue when the specimen is oriented with its articular surface perpendicular to the B0. Hence, our data imply that the location of the least-ordered collagen fibers occur deeper than the leastordered water molecules in the tissue. Note that Fig. 8 only shows the T2 profiles from the nine samples at ∼0°. For each sample, there are three additional T2 profiles at three other angles. Because of the reduction of the nuclear dipolar interaction, the T2 relaxations at these additional angles are less attenuated at the surface and deep zones than at ∼0°. However, the four peak locations of the T2 profiles are consistent for each sample. We hypothesize that this relaxation–retardation correlation is related to the gradients of the PG and water concentrations in the tissue, as follows. We know the minimum optical retardation occurs at the geometric center of the transitional zone in the tissue, which indicates the

808±27 764±31 95.4±2.6 289.9±4.8 378.2±24.4 164±14 252±3

L42 712±27 734±28 176.5±10.0 178.9±22.0 379.0±32.0 168±14 273±3

726±27 715±34 64.6±4.6 221.2±9.5 428.8±48.1 100±14 175±3

L22

Medial half (left) L32 671±27 673±22 87.8±1.5 86.1±9.6 499.6±33.2 110±14 133±3

L12 617±27 622±47 55.0±1.4 119.0±13.5 447.9±34.9 102±14 115±3

Central C02 644±27 661±28 56.8±2.0 83.1±7.4 521.4±22.6 73±14 101±3

R12

671±27 701±24 71.6±2.4 89.0±2.3 540.8±17.6 71±14 115±3

891±27 844±36 73.4±2.1 71.4±7.3 699.2±30.7 64±14 108±3

R32

Lateral half (right) R22

836±27 792±50 18.7±0.5 200.1±23.5 572.7±27.0 83±14 112±3

R42

Note on the errors. The error for the total MRI thickness equals the size of two MRI pixels, because the surface and deep boundary of the total tissue have to be defined separately. The error for the total PLM thickness is the standard deviation from the average of 10–14 histological profiles for each specimen. Because we calculate the individual PLM zones based on the hyperbolic tangent fit to the angle profile of each specimen in PLM, the error for the individual PLM zone is the pixel location where an additional 1° deviation in the angle profile. For the depths of the maximum T2 and the minimum retardance, the error equals the size of 1 pixel. The details of the data fitting and error analysis have been described earlier26.

Total MRI thickness (
m) Total PLM Thickness (
m) PLM SZ thickness (
m) PLM TZ thickness (
m) PLM RZ thickness (
m) Depth of maximum T2 at 0° (
m) Depth of the minimum retardance (
m)

Plug no.

Table I Variations of the tissue properties in a right humeral head

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location where the collagen fibers are least ordered. If the PG and water in the tissue have a constant concentration across the depth of the tissue, one could expect that the maximum T2 relaxation occurs at the same location of the minimum retardation. However, we also know that the PG concentration has a gradient across the depth of the tissue: being less concentrated near the surface (ca. 4% wet weight) and more concentrated towards the middle of the radial zone (ca. 7% wet weight)5. In addition, the water concentration also has a gradient across the depth of the tissue: being more concentrated near the surface (ca. 75% wet weight) and less concentrated towards the radial zone (ca. 65% wet weight)5. Because of these two concentration gradients, there will be more ‘free’ or ‘loosely-bound’ waters above the least-ordered collagen location toward the surface than below the least-ordered collagen location toward the deeper part of the tissue. This will shift the leastordered water location away from the least-ordered collagen location towards the surface. The fact that the locations of the least ordered waters were shifted by about the same percentage amount further indicates that these shifts were not simply random variations in our measurement. Because the depth of the minimum retardation is highly correlated with the geometric middle point of the transitional zone (Fig. 7), both the minimum retardation depth in PLM and the maximum T2 depth in
MRI actually track the geometric center of the transitional zone in the tissue, except one directly and one indirectly. Note that Nieminen and coworkers have recently reported a study of enzymatically degraded bovine patellar articular cartilage using
MRI32. Collagenase was used to digest the collagen network and chondroitinase ABC to remove proteoglycans. They found that T2 in superficial cartilage increased significantly only in samples treated with collagenase. Although their findings seem to suggest the existence of an alternative explanation to our analysis (above) regarding the correlation between the T2 maximum and the retardation minimum, one possible cause for this location could be the image resolution: 78
m pixel size in Nieminen et al. and 13.7
m in our study. Figure 9 shows that the differences between the T2 maxima and the retardation minima are in the range of 30–70
m for all nine specimens in the joints. These differences are smaller than the size of 1 pixel for the experiments by Nieminen et al., but are more than 2–5 pixels for our experiments. Because articular cartilage is essentially a thin 2D surface, any quantitative work to characterize the tissue across the depth demands high resolution in imaging.

THE CONCAVE SHAPE OF THE THICKNESS OF THE TRANSITIONAL ZONE OVER THE HUMERUS

In this study, we have observed by both
MRI and PLM that imagings that the thickness of the cartilage varies across the transverse width of the canine humerus: thinner near the center of the humeral head and thicker at the periphery on both sides of the humerus. Since the humerus has a convex surface, our result is opposite to a general understanding that convex joint surfaces should have a thicker central portion2. However, our result is highly consistent with three previous studies of canine humeral heads along the central transverse direction7,9,12. In addition to the variation in the total tissue thickness, we have also observed different types of variations in the individual thickness of three histological zones. A notable feature of the zone variation is the transitional zone, where the

thickness of the zone has a variation with a concave shape and is the thinnest near the central location. Since the thickness of the superficial zone is thin and its variation is nearly linear, this concave variation in the thickness of the transitional zone is largely responsible for the variations in the total thickness of the total tissue. Since articular cartilage is a load-bearing tissue, it is natural to ask the question: how does the variation in the tissue thickness from different sites within one joint surface relate to the local load-bearing status? From our observations of humeri, the total tissue thickness at the heavy load-bearing sites centrally tends to be thinner than that at the light load-bearing sites peripherally from the same joint surface. This is again opposite to the conclusion in the study of the human first metatarsal bone by Muehleman et al.: the greatest total thickness is on the region that is subjected to the greatest load during the normal gait cycle33. The discrepancy may be related to the difference in the congruency of the different joints. Simon et al. suggested that variations in thickness of articular cartilage may be related to the congruency of opposing surfaces in joints34. Thus, a thin cartilage is sufficient in a highly congruent joint because of the efficient distribution/transfer of the load over a large area, while a thick cartilage is needed in a less congruent joint because of the less efficient distribution/transfer of the load over a small area. The measurement of the Poisson’s ratio in canine humeral head showed that the Poisson’s ratio is about double in the central joint region than in the joint periphery9. Because Poisson’s ratio measures the apparent compressibility of the tissue35, the smaller Poisson’s ratio in the peripherals means the tissue at these sites, where the transitional zone is thicker, is more easily compressible.

ASYMMETRY OF THE TISSUE PROPERTIES BETWEEN THE MEDIAL AND LATERAL HALVES OF THE JOINT

Although the total tissue thickness, the zone thickness, the optical retardation, and the T2 relaxation were measured independently and from different imaging techniques, the topographical variations of these measurements all exhibit a slight asymmetry between the medial and lateral halves of the humeral head. We do not know the origin of this asymmetry, and can only speculate that they are related to the load-bearing status of the tissue at different sites. In a study of mechanical properties in canine humeral heads by Korvick and Athanasiou9, the shear modulus in canine humeral heads was found to be higher at the peripheral sites than at the central sites, and higher at the lateral peripheral sites than at the medial peripheral sites. Since the shear modulus is a measure of tissue stiffness under shear load35, it is possible that the asymmetries in the thickness of the histological zones constitutes a tissue structure to meet the biological and biomechanical demand on the joint. The biomechanical properties of the cartilage are also directly related to the molecular compositions in the tissue. It is reasonable to state that there exist some topographical variations in the molecular compositions in articular cartilage. Two recent reports13,15 studied the relationships among biochemical composition, collagen organization, and MRI appearance in porcine humerus along the central sagittal direction: the central location of the humeral head being labeled as the ‘unloaded’ region and the caudal end of the humeral heads as the ‘loaded’ region. Hence, their regional variations are along a direction approximately

Osteoarthritis and Cartilage Vol. 10, No. 5 perpendicular to the direction in this study. They reported that the caudal end has more than twice the GAG content than the central regions. In addition, although the two regions have about the same collagen content, the structure and distribution of collagen fibers appear to be different15. Further work is needed to correlate the biomechanical and biochemical properties of the tissue with the imaging capabilities of
MRI and PLM.

SUMMARY

In summary, our imaging techniques used in this report are unique in two aspects. First, in contrast to most clinical MRI studies of cartilage for which the imaging resolution is about several hundreds of microns or poorer, our transverse resolution in
MRI is 13.7
m across the depth of the cartilage tissue, the highest spatial resolution that has been achieved in the studies of articular cartilage. This microscopic resolution enables us to examine the tissue properties in individual histological zones in in vitro cartilage at microscopic resolution. Note that because articular cartilage is a thin surface, any quantitative work to characterize the tissue across the depth demands microscopic resolution in imaging. Second, in contrast to most histological studies of cartilage using light microscopy for which the results are graphical and descriptive, we have used the digital imaging technique in PLM to construct 2D images of the optical retardation and the angular orientation of the collagen fibers in the tissue. Our quantitative PLM techniques provide an accurate means to examine a number of the properties in the tissue. Using these unique imaging techniques, we have observed several topographical variations among the morphological and physical properties of the tissue across the transverse section of the canine humerus. These variations include the total thickness of the cartilage tissue, the width of the individual histological zones, the depth of the minimum optical retardation in the tissue, and the depth of the maximum T2 relaxation in the tissue. We found that the depth of the minimum retardation from PLM occurs at the geometric center of the transitional zone in histology, and that the depth of the maximum T2 relaxation from
MRI is closely correlated with the minimum retardation depth in PLM. In addition, although the thickness of the total tissue increases approximately by the same amount on both sides of the joint, a slight asymmetry exists between the measurements from the medial and lateral halves of the humeral head. Considering these topographical variations and asymmetry together with other known variations in literature, therefore, one must keep in mind that a different sampling site within a relatively small area of the same joint surface may significantly alter the outcomes of the study, regardless of whether the study is morphological or mechanical, physical or chemical, in vivo or in vitro, clinical or in laboratory, and spectroscopic or imaging. Quantitative knowledge of the topographical variations of cartilage over a joint surface can provide helpful insights to the evaluation of the degradation and failure of the tissue. For example, Stoop et al.36 recently reported two phases of damage and degenerative changes in rat cartilage; the early phase being located mainly on the peripheral and margins of the tissue and the later phase being located in the load-bearing part of the joints. Furthermore, the tissue thickness is known to remain stable with aging3 but can be used as a measurement of health status of articular joints37–40. Because we now show that
MRI and PLM

379 techniques can generate definitive characteristic profiles of the tissue, and because MRI is intrinsically non-invasive, a deeper understanding of these fundamental topographical variations will guide clinical research and practice.

Acknowledgments The authors thank Clifford Les (Bone and Joint Center, Henry Ford Hospital, Detroit, Michigan, U.S.A.) for his help in identifying several references cited in this report, and to Elizabeth Robbins for her assistance in polarized light microscopy.

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