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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Osteoarthritis Cartilage. Author manuscript; available in PMC Jan 29, 2012.
Published in final edited form as:
PMCID: PMC3268049
NIHMSID: NIHMS156597

Nondestructive Assessment of sGAG Content and Distribution in Normal and Degraded Rat Articular Cartilage via EPIC-μCT

Liqin Xie, Ph.D.,+ Angela S.P. Lin, M.S.,+ Robert E. Guldberg, Ph.D.,+ and Marc E. Levenston, Ph.D.*,1

Summary

Objective

The objective of this study was to evaluate the feasibility of quantifying the Equilibrium Partitioning of an Ionic Contrast agent via μCT (EPIC-μCT) to nondestructively assess sulfated glycosaminoglycan (sGAG) content and distribution in rat articular cartilage ex vivo, and in doing so to establish a paradigm for extension of this technique to other small animal models.

Design

After determination of an appropriate incubation time for the anionic contrast agent, EPIC-μCT was used to examine age-related differences in cartilage sGAG content between 4-, 8-, and 16-week old (n=5 each) male Wistar rats and to evaluate sGAG depletion in the right femora of each age group after 60 minutes of digestion with chondroitinase ABC. The EPIC-μCT measurements were validated by histological safranin-O staining, and reproducibility was evaluated by triplicate scans of six femora.

Results

Cartilage attenuation gradually increased with cumulative digestion time and reached a plateau at approximately 60 minutes with a 16.0% temporal increase (p<0.01). Average femoral articular cartilage attenuation increased by 14.2% from 4 to 8 weeks of age (p<0.01) and further increased by 2.5% from 8 to 16 weeks (p<0.05). After 60 minutes of digestion, femoral articular cartilage attenuations increased by 15-17% in each age group (p<0.01). Correspondingly, sGAG optical density decreased with age and digestion, and showed a linear correlation (r=−0.88, slope=−1.26, p<0.01, n=30) with EPIC-μCT cartilage attenuation. High reproducibility was indicated by a low coefficient of variation (1.5%) in cartilage attenuation.

Conclusions

EPIC-μCT imaging provides high spatial resolution and sensitivity to assess sGAG content and three-dimensional distribution in rat femoral articular cartilage.

Keywords: EPIC-μCT, microcomputed tomography, Cartilage development, Cartilage degradation, Cartilage imaging

Introduction

Proteoglycans (PGs), primarily aggrecan, comprise about 40% of articular cartilage by dry weight [1] and carry a net negative charge due to a high density of sulfated glycosaminoglycans (sGAGs). PGs maintain hydration of the extracellular matrix and contribute to mechanical properties in part through electrochemical interactions [2, 3]. The PG content changes substantially during cartilage development [4], degeneration and repair [5, 6]. PG loss is one of the earliest symptoms of osteoarthritis and usually occurs before gross morphological changes [7]. Studies involving animal tissues indicate that even moderate decreases in PG concentration can impair the load-bearing capacity of cartilage [8-11], and softened articular cartilage is susceptible to damage during normal activity that contributes to cartilage erosion [11]. The earliest degenerative changes may be reversible [12-14], but current therapies are unable to regenerate cartilage once collagen damage or gross tissue erosion begins. Detection of early PG depletion (and potentially repletion) would thus be valuable for the development and evaluation of novel regenerative therapies.

Experimental small animal models are necessary and complementary to clinical studies. A variety of small animal models have been used for understanding pathogenesis and evaluating the efficacy of disease modifying drugs, including surgical disruption models, intraarticular injection of various agents, and spontaneous and genetically modified models of osteoarthritis [15, 16]. However, typical histological and biochemical evaluation techniques are destructive, time consuming, and incapable of describing the three-dimensional (3-D) spatial distribution of tissue constituents. Current MRI techniques, including delayed gadolinium-enhanced MRI of cartilage (dGEMRIC), can produce 3-D, nondestructive measurements of PG concentration in cartilage in vitro and in clinical applications [17-19]. However, even high-powered research MRI systems may not be adequate to detect local cartilage changes in small animal joints. Therefore, there is a clear need for imaging techniques that are capable of providing three-dimensional, high resolution and nondestructive measurements of cartilage composition in small animal joints (either in vivo or ex vivo).

We previously introduced a novel imaging technique measuring the Equilibrium Partitioning of an Ionic Contrast agent via Microcomputed Tomography (EPIC-μCT), based on the principle that a negatively charged, radioopaque contrast agent will be preferentially excluded from regions of higher negative fixed charge density associated with higher PG concentration [20]. EPIC-μCT detected PG concentration and distribution in cultured bovine cartilage explants, providing high resolution, 3-D images of degeneration patterns in vitro. The studies presented here aimed to establish appropriate parameters for ex vivo imaging of the much thinner cartilage of the rat distal femur, to evaluate the ability to track previously described differences in sGAG concentration with age and in vitro chondroitinase ABC digestion, and to validate the accuracy of EPIC-μCT through qualitative and quantitative comparison to histological analyses. In doing so, these studies demonstrate an approach to determination of appropriate parameters and validation that will facilitate application of EPIC- μCT to other joints or animal models. These studies extend the application of EPIC-μCT, establishing a nondestructive technique to quantitatively evaluate the articular cartilage sGAG content and 3-D distribution for future hypothesis-driven studies of joint degradation and repair in small animal models.

Method

Specimen Preparation

The Georgia Institute of Technology Institutional Animal Care and Use Committee approved experimental procedures. Male Wistar rats 4-, 8- and 16-weeks old (Charles River Laboratories, Sparks, NV) were sacrificed via CO2 inhalation. The femora were harvested, dissected free of surrounding tissues, and stored in phosphate buffered saline (PBS) with proteinase inhibitors (PI, Cocktail I, CalBiochem, San Diego, CA) at 4°C. For studies involving enzymatic digestion, the distal femur was immersed in 1ml (0.1 U/ml) chondroitinase ABC (Sigma, St. Louis, MO) at 37°C [21].

μCT Scanning and Analysis

Each femur was consistently secured within a scanning tube such that the diaphyseal axis was aligned with the vertical axis of the μCT scanning tube. To reduce dehydration, the tube contained a small amount of PBS at the bottom (not contacting the sample) and was sealed with Parafilm. Samples were scanned using a μCT 40 (Scanco Medical, Bassersdorf, Switzerland) at 45 kVp, 177 μA and 200-ms integration time. Characterization studies used a 12 mm scanning tube and a 12 μm voxel size, while subsequent studies used a 16 mm scanning tube and a 16 μm voxel size to accommodate the largest samples. For EPIC-μCT, samples were immersed in a solution of 40% Hexabrix 320 contrast agent (Mallinckrodt, Hazelwood, MO) and 60% PBS at 37°C [22] and gently patted dry before scanning.

A central region was defined for each condyle by manually identifying the scan slice first intersecting cartilage and then selecting a fixed number (see below) of sequential slices from the cartilage surface. This region included cartilage, subchondral bone, and some trabecular bone. Using Scanco Medical software, a histogram of the X-ray attenuation values was produced, revealing two peaks corresponding to contrast-enhanced articular cartilage and calcified bone. Lower and upper thresholds were manually assigned to isolate the voxels within the cartilage peak, which also contained a few (<3%) bone marrow voxels. The central cartilage attenuation for an individual condyle was defined as the average attenuation for the thresholded cartilage voxels of the central region, while the central cartilage attenuation for an entire femur was similarly determined for pooled central cartilage voxels from both condyles.

To determine the average attenuation for the entire articular cartilage layer, cartilage was segmented as previously described [22]. Femoral cross-sections were transformed into sagittal sections via 3-D rotation of the grayscale image file, and contour lines were manually drawn on every 3-10 slices to eliminate marrow space. After semi-automatic contouring was applied to the remaining slices, appropriate thresholds were identified from an attenuation histogram to segment cartilage from bone. The 3-D morphology of the entire articular cartilage layer was then produced and the average attenuation was determined.

For evaluation of depth-dependence, a sagittal slice through the center of a condyle was isolated and the cartilage thickness at the highest point on the surface was measured. A 400μm wide region was subdivided into 10 equal increments through the thickness, and the average attenuation of each increment was determined.

Histology and Analysis

Following scanning, femora were fixed in 10% neutral buffered formalin overnight and decalcified in 2.5% formic acid (pH 4.2) for 10 days. Dehydrated samples were embedded in glycol methacrylate according to the manual of the JB-4 embedding kit (Polysciences, Warrington, PA). For comparison with EPIC-μCT images, sagittal sections were cut at 8μm thickness and one section through the center of each condyle was examined. Sections were stained for sGAGs with a 0.5% safranin-O solution and a 0.2% aqueous solution of fast green as a counterstain. Digital images of each section were captured at 0.5μm resolution under controlled ambient light. The images were exported to Matlab (MathWorks, Natick, MA), red, green and blue image components were isolated, and a red content parameter (Rc,) representing the sGAG optical density (sGAG-OD) was determined by nonlinear weighting of the fractional intensity of the red component [23]. The cartilage was manually outlined and the femur's sGAG-OD was determined by averaging the average Rc values from the two condyles. Depth-dependent sGAG-OD was examined by dividing a 400μm wide central of cartilage into 10 equal increments and determining the average Rc for each increment.

Characterization Studies

As the time required for equilibration varies depending on the contrast agent, tissue composition, and tissue thickness, the appropriate equilibration time for rat femoral cartilage was first determined. Femora (n=3) from 8-week old rats were pre-scanned before exposure to the contrast agent. Each sample was then immersed in contrast solution for 5 min and rescanned. This process was repeated for cumulative immersion times of 0, 5, 10, 15, 30, and 60 min. As handling and scanning required approximately 30 minutes, it should be noted that intermittent exposure may have produced somewhat different kinetics than continuous exposure. Central cartilage attenuation was determined for a region extending 372 μm (31 slices) from the cartilage surface.

To determine an appropriate digestion time, femora (n=4) from 8-week old rats were immersed in contrast solution for 30 min (based on results of the time-course study) and scanned.

Samples were incubated in PBS for 30 min to desorb the contrast agent, digested with chondroitinase ABC for 10 min, then re-immersed in contrast solution and rescanned. This process was repeated for cumulative digestion times of 10, 20, 30, 40, 60, 80, and 120 min to simulate progressive degradation. Cartilage attenuation for each femur was determined as described above.

To evaluate the reproducibility of EPIC-μCT attenuation measurements, femora from 4-week old (n=4) and 8-week old (n=2) rats were immersed in contrast solution for 30 min and scanned. After incubation for 1 hour in PBS (sufficient to desorb the contrast agent), the femora were re-immersed in contrast solution and re-scanned. After overnight storage in PBS at 4°C, a third immersion and scan was performed. The attenuation averaged over the entire cartilage layer was determined for each scan, and the coefficient of variation (100 times the standard deviation divided by the mean) was calculated for each sample. The root mean square coefficient of variation (RMS-CV) and root mean square standard deviation (RMS-SD) [24] were calculated and averaged for the 6 samples.

sGAG Variation with Age and Digestion

Both femora were harvested from 4-week, 8-week, and 16-week old (n=5 each) rats, incubated in contrast solution for 30 min and scanned. The average attenuation of the entire cartilage layer and the central cartilage attenuations for both condyles were determined for each femur. Reflecting the decrease in cartilage thickness with age, the central regions extended from the cartilage surface to depths of 496μm (31 slices) in 4-week old rats, 336μm (21 slices) in 8-week old rats and 256μm (16 slices) in 16-week old rats. After scanning, right femora were incubated in PBS for 30 min to desorb the contrast agent, digested with chondroitinase ABC for 60 min, re-incubated with the contrast agent for 30 min and rescanned. Following scanning, undigested left and digested right femora were fixed, embedded and stained, and analyzed to determine the sGAG-OD.

Statistical Analysis

Cartilage attenuations in the characterization studies were evaluated via one-way repeated measures ANOVA. General linear models (factors in parentheses) were used to evaluate effects of age on cartilage attenuation (age, animal, limb) or sGAG-OD in undigested femurs (age), effects of digestion on cartilage attenuation (age, animal, digestion) or sGAG-OD (age, animal, digestion), with age as a between-groups factor and animal nested within age. Regional cartilage attenuation was evaluated with a two factor mixed general linear model (age, condyle) with condyle as a repeated measure. Tukey's test was used for post-hoc analyses. The relationship between cartilage attenuation and sGAG-OD was examined via linear regression analysis. Statistical significance was set at p <0.05 (SPSS 11, SPSS Inc., Chicago, IL or Minitab 15, Minitab Inc., State College, PA). All data are presented as mean±SD.

Results

Characterization Studies

The average attenuation of femoral articular cartilage without incubation (65±2, expressed in threshold units) was close to that of PBS (58), and increased over 60 minutes to an attenuation of 193±7. Cartilage attenuation achieved 69% of the final increase by 5 min (Fig. 1), 85% by 10 min (p<0.01 vs. 5 min), 92% by 15 min (p<0.01 vs. 5-min), and 99% by 30 min (p<0.05 vs. 10 min). There were no significant differences in attenuation among incubation times of 15, 30, and 60 min. Consequently, incubation for 30 min was used in subsequent studies. Using this protocol, the root mean square coefficient of variation for cartilage attenuation determined by triplicate scanning of six samples was 1.5%, and the root mean square standard deviation was 2.1%.

Figure 1
Optimization of incubation time for EPIC-μCT in the rat distal femur. (A) Representative EPIC-μCT images of femoral articular cartilage after incubation with 40% Hexabrix at 0, 5, 10, 15, 30 and 60 min cumulative exposure times. Central ...

Color images representing the range of X-ray attenuation values were generated using Scanco Medical image processing software. As expected, the baseline μCT scan failed to distinguish cartilage from other soft tissues, as all of the soft tissues exhibited similar attenuations (65, blue, Fig. 2 left). However, EPIC-μCT clearly identified cartilage from bone and adjacent non-cartilaginous soft tissues such as ligaments. Due to the higher negative fixed charge density, articular cartilage had lower contrast agent content and thus lower X-ray attenuation (140, green and yellow, Fig. 2 middle) compared to other soft tissues (240, dim red, Fig. 2 middle). Safranin-O staining of the same femur showed cartilage morphology consistent with the EPIC-μCT images, with cartilage appearing dark red while other soft tissues appeared green (Fig. 2 right).

Figure 2
Sagittal sections of baseline μCT scan (Left); EPIC-μCT Scan (Middle); and histological staining with safranin-O (Right) of the same femur from a 4-week old rat. The baseline μCT scan without contrast agent failed to distinguish ...

Color-coded images were generated to visualize variations in attenuation during digestion (Fig. 3A). As expected for decreasing sGAG content, the average central cartilage attenuation increased from 187±10 before digestion to a plateau (217±7) at approximately 60 min of digestion (Fig. 3B). The 16.0% increase in average cartilage attenuation (p<0.05 vs. original measurement) was maintained from 60-120 min of digestion. Consequently, digestion for 60 min was used in subsequent studies.

Figure 3
Time course of central cartilage attenuation during chondroitinase ABC digestion. (A) Representative EPIC-μCT images of femoral articular cartilage after digestion with chondroitinase ABC for 0, 10, 30 and 60 min cumulative exposure times. Average ...

sGAG Variation with Age and Digestion

Representative sagittal EPIC-μCT images (Fig. 4A-C) demonstrate that cartilage attenuation increased with age, while representative safranin-O stained images (Fig. 4G-I) illustrate that sGAG-OD decreased with age. Consistent with the visual assessment, attenuation of the distal femoral cartilage layer increased by 14.2% from 4- to 8-weeks of age (157.6±4.8 vs. 138.1±1.8, n=10, p<0.0001, Fig. 5A), and further increased by 2.5% from 8- to 16-weeks of age (161.5±2.3 vs. 157.6±4.8, n=10, p=0.002). The means of relative differences [25] (absolute difference between left and right femora divided by the mean of both femora) for central cartilage attenuation for a given age group ranged from 0.9% to 2.0%, without significant differences between right and left limbs (Fig. 5A). sGAG-OD in the left femoral articular cartilage decreased by 13.9% from 4- to 8-weeks of age (65.4±3.2 vs. 76.0±2.9, n=5, p<0.001, Fig. 5B), but did not significantly decrease from 8- to 16-weeks of age (60.2±4.0 vs. 65.4±3.2, n=5, p=0.08).

Figure 4
EPIC-μCT images and corresponding histological sections show similar patterns of changes in sGAG content with age and digestion. (A-F): Representative sagittal sections of undigested (A-C) and digested (D-F) distal femora from EPIC-μCT ...
Figure 5
Quantitative analysis of changes in cartilage attenuation and sGAG optical density with age and digestion. (A) Average EPIC-μCT attenuation of the cartilage layer for undigested left femora, undigested right femora, and digested right femora for ...

EPIC-μCT revealed progressive differences with maturation between medial and lateral condyle attenuations. The central cartilage attenuation of medial and lateral condyles did not significantly differ in 4-week old rats (141.5±3.6 vs 140.1±4.3, p=0.41, n=10). However, the attenuations in medial condyles were 8.5% (195.7±13.6 vs 180.5±11.6, p<0.01, n=10) and 9.9% (211.4±12.6 vs 192.3±12.3, p<0.01, n=10) higher in 8-week and 16-week old rats, respectively, indicating a lower sGAG content in the medial condyles at these ages.

Representative sagittal EPIC-μCT images (Fig. 4D-F) and safranin-O stained images (Fig. 4J-L) demonstrate that chondroitinase ABC digestion increased cartilage attenuation and decreased sGAG-OD across ages. After 60 min of digestion, the cartilage attenuation in the right femora increased by 17% to 160.8±10.4 (n=5, p<0.01, Fig. 5A) compared to normal cartilage in left or right femora in the 4-week age group, by 16% to 182.8±6.6 (n=5, p<0.01) in the 8-week age group, and by 15% to 185.2±4.2 (n=5, p<0.01) in the 16-week age group. Compared to normal cartilage in the left femora, sGAG-OD of digested articular cartilage in the right femora was lower by 18.9% at 61.6±3.4 (n=5, p<0.01, Fig. 5B) in the 4-week age group, by 30.6% at 45.4±3.4 (n=5, p<0.01) in the 8-week age group, and by 29.6% at 42.4±3.0 (n=5, p<0.01) in the 16-week age group. There was a strong, linear relationship (r=−0.88, slope=−1.26, p<0.01, n=30, Fig. 5C) between cartilage attenuation and sGAG-OD for cartilage pooled across age and digestion states.

In 4-week old rats, the attenuation (Fig. 6A) decreased from the superficial zone (177±7) to the deep zone (101±6) and reached the lowest value in the bottom of middle zone (97±6), corresponded to the highest sGAG level. As expected, the contrast agent achieved higher concentrations in digested cartilage, with increased X-ray attenuation in the superficial (211±9), middle (171±7) and deep zones (163±8). Histological sections revealed consistent sGAG distributions in normal and digested cartilage (Fig. 6B). In normal cartilage, sGAG-OD increased from the superficial zone (41±5) to the deep zone (83±4). After digestion, the cartilage exhibited decreases in sGAG-OD progressing from the superficial zone (26±5) to the deep zone (73±5). The poorly stained regions (superficial zone) were consistent with the locations of increased attenuation in the EPIC-μCT images (Fig. 4).

Figure 6
sGAG distribution through the thickness of normal and digested cartilage from the medial condyles of femora from 4-week old rats. (A) Average EPIC-μCT attenuation vs. depth determined from a 2D image slice through the center of the medial condyle ...

Discussion

Nondestructive techniques capable of assessing cartilage composition and distribution in rodents would greatly benefit the development of effective osteoarthritis treatments and cartilage repair strategies. These studies demonstrated that EPIC-μCT provides high spatial resolution and sensitivity to assess changes in sGAG concentration within rat femoral articular cartilage, expanding the application of μCT from mineralized hard tissues to soft tissues. EPIC-μCT was able to clearly distinguish articular cartilage from bone and adjacent non-cartilaginous soft tissues (although the distinction from other soft tissues may diminish with degeneration), and allowed visualization of the spatial distribution of sGAGs in the thin articular cartilage of the rat. EPIC-μCT had high precision in repeated measurements and demonstrated high sensitivity for detecting differences in sGAG content of rat articular cartilage with maturation and in vitro enzymatic digestion. Importantly, the inverse relationship between EPIC-μCT attenuation and sGAG content was validated by histological analysis, demonstrating a strong linear relationship between sGAG-OD and cartilage attenuation.

Current quantitative analyses for sGAG concentration include histological staining and biochemical assays using cationic dyes, dGEMRIC [26], sodium MRI[27], and the EPIC-μCT technique employed in this study, all of which measure the negative electrical charge of the extracellular matrix as an indication of sGAG concentration. EPIC-μCT, with inherently higher spatial resolution than dGEMRIC, was shown in this study to provide the 3-D spatial distribution of X-ray attenuation and therefore sGAG concentration throughout the articular cartilage layer in rats. The mechanical properties of articular cartilage are site-dependent [28], and EPIC-μCT images may be helpful in exploring structure-function relationships between local mechanical properties and sGAG concentration [10, 29, 30]. Additionally, measurement of sGAG concentration may be a useful surrogate for tissue mechanical properties in small animals where it is difficult to directly assess material properties.

Depth-dependent patterns of cartilage attenuation could arise from incomplete diffusion of the contrast agent. In that case the cartilage attenuation would be lowest in the deep zone, but the lowest attenuation was found towards the bottom of middle zone (Fig. 6) where the sGAG content is highest [31, 32]. Observations of higher cartilage attenuation in digested cartilage than in normal cartilage further indicate that the matrix charge enabled the contrast agent to distribute in accordance with sGAG concentration [33]. A previous MRI study demonstrated a nonuniform distribution of an ionic contrast agent (gadolinium) in cartilage but a uniform distribution of a similarly sized neutral contrast agent (gadoteridol) that was independent of regional sGAG concentration [33, 34]. These findings suggest that the non-uniform distributions were governed by the electrical charge of contrast agents and not by the effect of molecular filtering within the cartilage matrix [35].

The staining intensity of safranin-O, a cationic dye that binds to sGAG, has been used to semi-quantitatively evaluate sGAG concentration with high accuracy and reproducibility [23, 36]. In this study, the close correspondence between EPIC-μCT images and safranin-O staining and the strong, negative relationship between EPIC-μCT attenuation and sGAG-OD suggest that EPIC-μCT could be a sensitive and specific measure of local sGAG content, with potential for in situ estimation of relative sGAG concentrations in rat articular cartilage.

Precision is a critical factor for animal studies, influencing both the number of experimental animals needed and the required duration of the study. Measurements with low precision may fail to detect the slow evolution of arthritis progression [24, 37]. The small coefficients of variation and standard deviations demonstrated a high level of precision for the EPIC-μCT technique. Based on our results, EPIC-μCT is expected to have 80% power and 95% confidence to detect a 1.3% cartilage attenuation difference with a sample size of 10, while the changes of cartilage attenuation in this study were over 2.5% during cartilage development and over 15% during digestion. The high precision was further indirectly validated by the small mean relative difference (<2%) in cartilage attenuation between left and right femora.

Cartilage attenuation from EPIC-μCT was used to semi-quantitatively evaluate cartilage maturation and degradation in this study, and the results demonstrated that EPIC-μCT has high sensitivity to assess the change of sGAG content associated with age and digestion. EPIC-μCT images showed that cartilage attenuation increased with the age of rats, indicating that sGAG concentration gradually decreased during cartilage maturation. This decrease was validated by safranin-O staining and is consistent with previous biochemical studies [4]. In the ex vivo degradation study, both EPIC-μCT imaging and histology methods indicated that sGAG concentration significantly decreased for digested cartilage. Based on a baseline characterization of normal cartilage attenuation across an age range, EPIC-μCT may have the capability to nondestructively determine the degree of degradation within the cartilage in rat models of arthritis, facilitating the evaluation of disease progression and the efficacy of cartilage defect therapies. For example, based on results presented here, a cartilage region in an 8-week old rat could be tentatively classified as degraded if the local attenuation exceeded the upper 95% confidence limit of 167.2.

In addition to the evaluation of the average attenuation (sGAG concentration) of the entire femoral articular cartilage layer, EPIC-μCT demonstrated the capability to assess sGAG content in focal regions. The results indicating lower sGAG contents in the medial condyles are consistent with previous studies [38-40], possibly reflecting lower stiffness in medial condyle [41]. It is also feasible to assess and compare other focal regions, such as condyles vs. groove and weight bearing vs. non-weight bearing regions [42]. Considering the high resolution (up to submicrometer) [43], EPIC-μCT also has potential to analyze the sGAG concentration in mouse cartilage, which has an approximate articular cartilage thickness of 100 μm or less [44, 45].

Chondroitinase ABC specifically degrades chondroitin sulfate and dermatan sulfate chains and provides a reproducible experimental model of decreased sGAG content [21, 46]. In the enzymatic digestion study, sGAG depletion was primarily observed in the superficial zone because the enzyme was large enough (150kD) to restrict early access to the deep zone [47]. sGAG depletion preceding apparent loss of tissue was consistent with early osteoarthritic symptoms [10, 32]. These data suggest that EPIC-μCT has the capability to nondestructively assess local sGAG depletion that may occur in early stages of focal defect formation in experimental osteoarthritis.

Although sGAG concentration was indirectly validated by histological analysis, questions still remain regarding the direct correspondence of cartilage attenuation to absolute sGAG concentration. The limited thickness and volume of rat articular cartilage makes it difficult or impossible to extract enough samples to calibrate sGAG concentration as a function of cartilage attenuation. According to electrochemical equilibrium (Donnan) theory, cartilage attenuation for a given level of tissue hydration is determined by the sGAG concentration with a given incubation condition. This theoretical speculation was validated by a previous biochemical assay in calf cartilage explants which demonstrated a negative linear relationship between sGAG concentration and cartilage attenuation [20]. However, the calibration curve may vary with factors such as collagen content and orientation, fluid volume fraction, and status of degradation. Once these factors are understood, development of an appropriate calibration curve will allow EPIC-μCT to provide a 3-D distribution map of absolute sGAG concentration in the rat. It is important to note that the quantitative assessment of sGAG content relies on an equilibrium contrast agent distribution that currently can only be imposed ex vivo. The potential for in vivo application is supported by a recent report that contrast enhanced in vivo μCT arthrography was able to detect cartilage degeneration in rat patellae in an experimental osteoarthritis model [48]. While issues such as contrast agent clearance from the joint space make in vivo quantification of sGAG content challenging, combined application of complementary in vivo and ex vivo implementations may be provide the best results.

In summary, the present study established a semi-quantitative, high-resolution, 3-D imaging technique capable of nondestructively assessing sGAG concentration and distribution (as well as cartilage morphology [22]) for end-stage analysis in small animals. EPIC-μCT will dramatically benefit studies of cartilage maturation and aging, degradation and regeneration [18], tissue engineering constructs, pathogenesis of osteoarthritis, and the efficacy of pharmacologic therapies in small animal models.

Acknowledgements

This project was funded by NIH grant R21AR053716. The authors thank Dr. Ashley W. Palmer for expert technical advice. The study sponsor had no involvement in the work described.

Footnotes

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Conflict of Interest Statement

The authors have no conflicts of interest to disclose.

References

1. Torchia DA, Hasson MA, Hascall VC. Investigation of molecular motion of proteoglycans in cartilage by 13C magnetic resonance. J Biol Chem. 1977;252:3617–3625. [PubMed]
2. Laurent D, Wasvary J, O'Byrne E, Rudin M. In vivo qualitative assessments of articular cartilage in the rabbit knee with high-resolution MRI at 3 T. Magn Reson Med. 2003;50:541–549. [PubMed]
3. Grodzinsky AJ. Electromechanical and physicochemical properties of connective tissue. Crit Rev Biomed Eng. 1983;9:133–199. [PubMed]
4. Watrin A, Ruaud JP, Olivier PT, Guingamp NC, Gonord PD, Netter PA, et al. T2 mapping of rat patellar cartilage. Radiology. 2001;219:395–402. [PubMed]
5. Watrin-Pinzano A, Ruaud JP, Olivier P, Grossin L, Gonord P, Blum A, et al. Effect of proteoglycan depletion on T2 mapping in rat patellar cartilage. Radiology. 2005;234:162–170. [PubMed]
6. Williams A, Oppenheimer RA, Gray ML, Burstein D. Differential recovery of glycosaminoglycan after IL-1-induced degradation of bovine articular cartilage depends on degree of degradation. Arthritis Res Ther. 2003;5:R97–105. [PMC free article] [PubMed]
7. Buckwalter JA, Mankin HJ. Articular cartilage: degeneration and osteoarthritis, repair, regeneration, and transplantation. Instr Course Lect. 1998;47:487–504. [PubMed]
8. Armstrong CG, Mow VC. Variations in the intrinsic mechanical properties of human articular cartilage with age, degeneration, and water content. J Bone Joint Surg Am. 1982;64:88–94. [PubMed]
9. Williamson AK, Chen AC, Sah RL. Compressive properties and function-composition relationships of developing bovine articular cartilage. J Orthop Res. 2001;19:1113–1121. [PubMed]
10. Rieppo J, Toyras J, Nieminen MT, Kovanen V, Hyttinen MM, Korhonen RK, et al. Structure-function relationships in enzymatically modified articular cartilage. Cells Tissues Organs. 2003;175:121–132. [PubMed]
11. LeRoux MA, Arokoski J, Vail TP, Guilak F, Hyttinen MM, Kiviranta I, et al. Simultaneous changes in the mechanical properties, quantitative collagen organization, and proteoglycan concentration of articular cartilage following canine meniscectomy. J Orthop Res. 2000;18:383–392. [PubMed]
12. Ossendorf C, Kaps C, Kreuz PC, Burmester GR, Sittinger M, Erggelet C. Treatment of posttraumatic and focal osteoarthritic cartilage defects of the knee with autologous polymer-based three-dimensional chondrocyte grafts: 2-year clinical results. Arthritis Res Ther. 2007;9:R41. [PMC free article] [PubMed]
13. Park YS, Lim SW, Lee IH, Lee TJ, Kim JS, Han JS. Intra-articular injection of a nutritive mixture solution protects articular cartilage from osteoarthritic progression induced by anterior cruciate ligament transection in mature rabbits: a randomized controlled trial. Arthritis Res Ther. 2007;9:R8. [PMC free article] [PubMed]
14. Richter W. Cell-based cartilage repair: illusion or solution for osteoarthritis. Curr Opin Rheumatol. 2007;19:451–456. [PubMed]
15. Bendele AM. Animal models of osteoarthritis. J Musculoskelet Neuronal Interact. 2001;1:363–376. [PubMed]
16. Wooley PH. The usefulness and the limitations of animal models in identifying targets for therapy in arthritis. Best Pract Res Clin Rheumatol. 2004;18:47–58. [PubMed]
17. Burstein D, Velyvis J, Scott KT, Stock KW, Kim YJ, Jaramillo D, et al. Protocol issues for delayed Gd(DTPA)(2-)-enhanced MRI (dGEMRIC) for clinical evaluation of articular cartilage. Magn Reson Med. 2001;45:36–41. [PubMed]
18. Kurkijarvi JE, Mattila L, Ojala RO, Vasara AI, Jurvelin JS, Kiviranta I, et al. Evaluation of cartilage repair in the distal femur after autologous chondrocyte transplantation using T2 relaxation time and dGEMRIC. Osteoarthritis Cartilage. 2007;15:372–378. [PubMed]
19. Trattnig S, Marlovits S, Gebetsroither S, Szomolanyi P, Welsch GH, Salomonowitz E, et al. Three-dimensional delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) for in vivo evaluation of reparative cartilage after matrix-associated autologous chondrocyte transplantation at 3.0T: Preliminary results. J Magn Reson Imaging. 2007;26:974–982. [PubMed]
20. Palmer AW, Guldberg RE, Levenston ME. Analysis of cartilage matrix fixed charge density and three-dimensional morphology via contrast-enhanced microcomputed tomography. Proc Natl Acad Sci U S A. 2006;103:19255–19260. [PMC free article] [PubMed]
21. Nieminen MT, Toyras J, Rieppo J, Hakumaki JM, Silvennoinen J, Helminen HJ, et al. Quantitative MR microscopy of enzymatically degraded articular cartilage. Magn Reson Med. 2000;43:676–681. [PubMed]
22. Xie L, Lin AS, Levenston ME, Guldberg RE. Quantitative assessment of articular cartilage morphology via EPIC-microCT. Osteoarthritis Cartilage. 2009;17:313–320. [PMC free article] [PubMed]
23. Martin I, Obradovic B, Freed LE, Vunjak-Novakovic G. Method for quantitative analysis of glycosaminoglycan distribution in cultured natural and engineered cartilage. Ann Biomed Eng. 1999;27:656–662. [PubMed]
24. Eckstein F, Cicuttini F, Raynauld JP, Waterton JC, Peterfy C. Magnetic resonance imaging (MRI) of articular cartilage in knee osteoarthritis (OA): morphological assessment. Osteoarthritis Cartilage. 2006;14(Suppl A):A46–75. [PubMed]
25. Dam EB, Folkesson J, Pettersen PC, Christiansen C. Automatic morphometric cartilage quantification in the medial tibial plateau from MRI for osteoarthritis grading. Osteoarthritis Cartilage. 2007;15:808–818. [PubMed]
26. Gray ML, Burstein D, Kim YJ, Maroudas A. 2007 Elizabeth Winston Lanier Award Winner. Magnetic resonance imaging of cartilage glycosaminoglycan: basic principles, imaging technique, and clinical applications. J Orthop Res. 2008;26:281–291. [PubMed]
27. Wheaton AJ, Borthakur A, Dodge GR, Kneeland JB, Schumacher HR, Reddy R. Sodium magnetic resonance imaging of proteoglycan depletion in an in vivo model of osteoarthritis. Acad Radiol. 2004;11:21–28. [PubMed]
28. Laasanen MS, Saarakkala S, Toyras J, Hirvonen J, Rieppo J, Korhonen RK, et al. Ultrasound indentation of bovine knee articular cartilage in situ. J Biomech. 2003;36:1259–1267. [PubMed]
29. Lammentausta E, Kiviranta P, Nissi MJ, Laasanen MS, Kiviranta I, Nieminen MT, et al. T2 relaxation time and delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) of human patellar cartilage at 1.5 T and 9.4 T: Relationships with tissue mechanical properties. J Orthop Res. 2006;24:366–374. [PubMed]
30. Samosky JT, Burstein D, Eric Grimson W, Howe R, Martin S, Gray ML. Spatially-localized correlation of dGEMRIC-measured GAG distribution and mechanical stiffness in the human tibial plateau. J Orthop Res. 2005;23:93–101. [PubMed]
31. Venn M, Maroudas A. Chemical composition and swelling of normal and osteoarthrotic femoral head cartilage. I. Chemical composition. Ann Rheum Dis. 1977;36:121–129. [PMC free article] [PubMed]
32. Waldschmidt JG, Rilling RJ, Kajdacsy-Balla AA, Boynton MD, Erickson SJ. In vitro and in vivo MR imaging of hyaline cartilage: zonal anatomy, imaging pitfalls, and pathologic conditions. Radiographics. 1997;17:1387–1402. [PubMed]
33. Bashir A, Gray ML, Boutin RD, Burstein D. Glycosaminoglycan in articular cartilage: in vivo assessment with delayed Gd(DTPA)(2-)-enhanced MR imaging. Radiology. 1997;205:551–558. [PubMed]
34. Gillis A, Gray M, Burstein D. Relaxivity and diffusion of gadolinium agents in cartilage. Magn Reson Med. 2002;48:1068–1071. [PubMed]
35. Nimer E, Schneiderman R, Maroudas A. Diffusion and partition of solutes in cartilage under static load. Biophys Chem. 2003;106:125–146. [PubMed]
36. Kiraly K, Lapvetelainen T, Arokoski J, Torronen K, Modis L, Kiviranta I, et al. Application of selected cationic dyes for the semiquantitative estimation of glycosaminoglycans in histological sections of articular cartilage by microspectrophotometry. Histochem J. 1996;28:577–590. [PubMed]
37. Gandy SJ, Dieppe PA, Keen MC, Maciewicz RA, Watt I, Waterton JC. No loss of cartilage volume over three years in patients with knee osteoarthritis as assessed by magnetic resonance imaging. Osteoarthritis Cartilage. 2002;10:929–937. [PubMed]
38. Tiderius CJ, Olsson LE, de Verdier H, Leander P, Ekberg O, Dahlberg L. Gd-DTPA2)-enhanced MRI of femoral knee cartilage: a dose-response study in healthy volunteers. Magn Reson Med. 2001;46:1067–1071. [PubMed]
39. Gold GE, Burstein D, Dardzinski B, Lang P, Boada F, Mosher T. MRI of articular cartilage in OA: novel pulse sequences and compositional/functional markers. Osteoarthritis Cartilage. 2006;14(Suppl A):A76–86. [PubMed]
40. Tiderius CJ, Svensson J, Leander P, Ola T, Dahlberg L. dGEMRIC (delayed gadolinium-enhanced MRI of cartilage) indicates adaptive capacity of human knee cartilage. Magn Reson Med. 2004;51:286–290. [PubMed]
41. Lyyra T, Kiviranta I, Vaatainen U, Helminen HJ, Jurvelin JS. In vivo characterization of indentation stiffness of articular cartilage in the normal human knee. J Biomed Mater Res. 1999;48:482–487. [PubMed]
42. Koo S, Gold GE, Andriacchi TP. Considerations in measuring cartilage thickness using MRI: factors influencing reproducibility and accuracy. Osteoarthritis Cartilage. 2005;13:782–789. [PubMed]
43. Schneider P, Stauber M, Voide R, Stampanoni M, Donahue LR, Muller R. Ultrastructural properties in cortical bone vary greatly in two inbred strains of mice as assessed by synchrotron light based micro- and nano-CT. J Bone Miner Res. 2007;22:1557–1570. [PubMed]
44. Botter SM, van Osch GJ, Waarsing JH, van der Linden JC, Verhaar JA, Pols HA, et al. Cartilage damage pattern in relation to subchondral plate thickness in a collagenase-induced model of osteoarthritis. Osteoarthritis Cartilage. 2007 [PubMed]
45. Lapvetelainen T, Hyttinen MM, Saamanen AM, Langsjo T, Sahlman J, Felszeghy S, et al. Lifelong voluntary joint loading increases osteoarthritis in mice housing a deletion mutation in type II procollagen gene, and slightly also in non-transgenic mice. Ann Rheum Dis. 2002;61:810–817. [PMC free article] [PubMed]
46. Toyras J, Rieppo J, Nieminen MT, Helminen HJ, Jurvelin JS. Characterization of enzymatically induced degradation of articular cartilage using high frequency ultrasound. Phys Med Biol. 1999;44:2723–2733. [PubMed]
47. Torzilli PA, Arduino JM, Gregory JD, Bansal M. Effect of proteoglycan removal on solute mobility in articular cartilage. J Biomech. 1997;30:895–902. [PubMed]
48. Piscaer TM, Waarsing JH, Kops N, Pavljasevic P, Verhaar JA, van Osch GJ, et al. In vivo imaging of cartilage degeneration using μCT-arthrography. Osteoarthritis Cartilage. 2008;16:1011–1017. [PubMed]
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