Radiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Published online before print November 24, 2004, 10.1148/radiol.2341030394
This Article
Right arrow Abstract Freely available
Right arrow Figures Only
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2341030394v1
234/1/162    most recent
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Watrin-Pinzano, A.
Right arrow Articles by Loeuille, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Watrin-Pinzano, A.
Right arrow Articles by Loeuille, D.
(Radiology 2005;234:162-170.)
© RSNA, 2004

Experimental Studies

Effect of Proteoglycan Depletion on T2 Mapping in Rat Patellar Cartilage1

Astrid Watrin-Pinzano, PhD, Jean-Pierre Ruaud, PhD, Pierre Olivier, PhD, Laurent Grossin, PhD, Patrick Gonord, PhD, Alain Blum, MD, Patrick Netter, MD, PhD, Genevieve Guillot, PhD, Pierre Gillet, MD, PhD and Damien Loeuille, MD, PhD

1 From the Department of Pharmacology, Physiopathologie et Pharmacologie Articulaires (UMR 7561), Centre National de la Recherche Scientifique (CNRS)–Université Henri Poincaré Nancy I, Faculté de Médecine, BP 184, Avenue de la Forêt de Haye, F 54505 Vandoeuvre les Nancy, France (A.W.P., P.O., L.G., A.B., P.N., P. Gillet, D.L.); Unité de Recherches en Résonance Magnétique Médicale (UMR 8081), CNRS–Université Paris-Sud, Paris, France (J.P.R., P. Gonord, G.G.); Department of Radiology, Service d’Imagerie Guilloz, Hôpital Central, Centre Hospitalier Universitaire (CHU), Nancy, France (A.B.); and Department of Rheumatology, Hôpitaux de Brabois, CHU, Nancy, France (D.L.). Received March 4, 2003; revision requested May 2; final revision received June 23, 2004; accepted July 12. Supported in part by Projet Hospitalier de Recherche Clinique (1998), Contrat de Projet de Recherche Clinique (2000), Pole Européen de Santé, and Groupement de Recherches CNRS 2237. Address correspondence to P. Gillet (e-mail: pierre.gillet@medecine.uhp-nancy.fr).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
PURPOSE: To evaluate experimentally the sensitivity of T2 mapping with magnetic resonance (MR) imaging at 8.5 T in depicting variations in proteoglycan content and concurrent extracellular matrix of rat patellar cartilage.

MATERIALS AND METHODS: The study was performed in 36 immature (age, 5 weeks) and 36 mature (age, 10 weeks) Wistar rats. Maintenance and care of the rats were conducted in accordance with National Institutes of Health guidelines. Fifty-six rats underwent T2 mapping in 28 right patellae degraded with hyaluronidase for 1 and 6 hours and in 28 undegraded age-matched patellae that served as controls. After MR mapping, the rats were sacrificed, and the patellae were studied histologically to evaluate proteoglycan and collagen content and collagen network organization in cartilage. Biochemical analysis was performed in 88 patellae to quantify sulfated glycosaminoglycan and hydroxyproline content. Effects of age and/or degree of degradation were evaluated after rank transformation of continuous data by using rank analysis of variance (ANOVA). Associations between continuous variables were assessed with the Spearman rank correlation coefficient.

RESULTS: Results of histologic analysis showed proteoglycan loss after hyaluronidase degradation without alteration of collagen network. No significant variation in hydroxyproline sulfate content was observed with depletion of proteoglycan. Proteoglycan losses of 19% and 13%, found after 1-hour degradation in immature and mature groups, respectively, were associated with significantly increased global T2 values (ANOVA, P < .001). Six-hour degradation resulted in more severe proteoglycan losses of 45% and 53% in immature and mature groups, respectively, inducing significant increases in global T2 values in immature and mature groups (ANOVA, P < .001). Variations in T2 values between superficial and deep cartilage zones were not affected by proteoglycan depletion.

CONCLUSION: In rat patellar cartilage, T2 mapping permits detection of slight or severe proteoglycan depletion and concurrent changes of extracellular matrix when age-matched samples are compared.

© RSNA, 2004


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Proteoglycans, composed of proteins and negatively charged sugars, constitute, with type II collagen, the main extracellular matrix in cartilage, which is responsible both for the deformability of the cartilage and for its ability to retain water molecules. In pathologic conditions such as arthritis or osteoarthritis, proteoglycan loss is observed in association with an increase in water content, while the collagen network remains undisturbed. Proteoglycan loss is usually considered one of the earliest manifestations of osteoarthritis and occurs before any morphologic change can be detected with radiography (1). Conventional hydrogen 1 magnetic resonance (MR) imaging enables accurate morphologic assessment of chondral lesions and provides information about biochemical variations in the extracellular matrix, especially variations in proteoglycan content.

Magnetization transfer imaging, which can be performed with clinical MR imagers, depends mainly on type II collagen content, while proteoglycan content plays a minor role (2). T1 mapping with contrast agent injection, also available in clinical practice, is based on the electric interaction between contrast agents and negatively charged proteoglycans (3–6). T1 mapping is very accurate for determining proteoglycan content but not for evaluating the collagen network; in addition, it is time-consuming. T1{rho} mapping, also an accurate method for determining proteoglycan loss, demonstrates variations in T1{rho} values according to cartilage depth; although promising, this method is still used only in experimental studies (7,8).

Similar spatial variations also may be observed by using T2 mapping, which shows different patterns in normal cartilage (9,10) and in osteoarthritic cartilage (11). Moreover, we recently demonstrated the usefulness of T2 mapping at 8.5 T for evaluation of variations in the extracellular matrix content in rat patellar cartilage with chondral maturation and aging (12). In a recent study conducted in the pediatric knee with a clinical 1.5-T MR imaging system, precise patterns of spatial variation were shown with T2 mapping (13). In clinical studies, however, in which the imaging characteristics of tissue cannot routinely be compared with the results of biochemical assay for extracellular matrix component and of histologic analyses (with standard staining and immunohistochemistry methods), the respective roles of extracellular matrix contents, type II collagen, and proteoglycans, as well as ancillary interactions of water with extracellular matrix, in determining spatial variation in T2 values remains to be determined. Thus, the purpose of our study was to evaluate experimentally the sensitivity of T2 mapping with MR imaging at 8.5 T in depicting variations in proteoglycan content and concurrent extracellular matrix of rat patellar cartilage.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Animals
Two groups of Wistar rats (Iffa-Credo, St Aubin les Elbeuf, France) of different ages were used for this study: 36 rats aged 5 weeks (mean weight, 150 g), an age at which cartilage is not yet mature; and 36 rats aged 10 weeks (mean weight, 300 g), an age at which cartilage has matured. As in our previous study (12), these groups were evaluated for proteoglycan loss in two different age-related collagen networks. The maintenance and care of the experimental rats were conducted as recommended in National Institutes of Health guidelines. Rats were anesthetized and sacrificed with cervical dislocation, and their knees were dissected for MR imaging and histologic and biochemical analyses (A.W.P.). The right patella in each rat was degraded with testicular hyaluronidase, while the left patella served as a control. The patellae were then kept frozen (–20°C) either for 2 months, until MR imaging followed by histologic analysis (56 patellae), or for 2 weeks, until biochemical analysis (88 patellae).

Hyaluronidase Degradation
To evaluate the effect of the degree of proteoglycan depletion on T2 relaxation time, degradation was performed in 72 right patellae for either 1 hour (slight depletion, 36 patellae) or 6 hours (severe depletion, 36 patellae). These time points were based on the results of a preliminary experiment (14). Each right patella was incubated in 1 mL saline solution containing 0.1 mol/L anhydrous sodium acetate and 1000 U/mL ovine testicular hyaluronidase (2950 U/mg; Sigma Aldrich, St Quentin Fallavier, France) for either 1 hour or 6 hours at pH 5.1 and 37°C (15). After degradation of the right patellae, all of the patellae (including the 72 undegraded left patellae) were rinsed with the saline solution containing anhydrous sodium acetate and placed for 2 minutes in a solution composed of proteinase inhibitors: phenylmethanesulfonyl fluoride, for serine-dependent proteases; benzamidine hydrochloride, for trypsinlike activity; 6-aminohexanoic acid, for cathepsin D–like activity; N-ethylmaleimide, for thiol-dependent proteases; and ethylenediaminetetraacetic acid, for metalloproteases (Sigma Aldrich) (16). Last, the patellae were washed with sterile saline solution and frozen (–20°C). The 144 frozen patellae were randomly assigned either to biochemical analysis (n = 88) or to T2 mapping followed by histologic analysis (n = 56). Patellae designated for T2 mapping remained frozen for a longer period than those subjected to biochemical analysis (Figure 1). The number of patellae subjected to biochemical analysis was larger to permit independent extracellular matrix analysis of glycosaminoglycans and hydroxyproline in separate patellae. Grushko et al (17) and Rubenstein et al (18) previously showed that the chemical composition and physicochemical properties of cartilage that has been frozen and thawed are identical to those of fresh cartilage.



View larger version (61K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Flowchart shows experimental design. The unit of analysis is the patella, which has undergone one of two evaluation processes (biochemical analysis, or T2 mapping followed by histologic analysis). Comparisons were made with different sets of patellae, independent of each other. Only one comparison at a time was conducted in each subset for each outcome criterion, and no multiple comparisons were necessary. GAG = glycosaminoglycans, L = left, OH proline = hydroxyproline, R = right.

 
Quantitative T2 Mapping
Prior to MR imaging, 56 patellae (28 patellae degraded for either 1 or 6 hours, and 28 control patellae) (Fig 1) were thawed and then rehydrated in saline solution for 16 hours at 4°C. Preliminary results showed that 16 hours of rehydration were needed to correct freezing-related dehydration. All MR images of the patellae were obtained with a specially designed 8.5-T micro-MR imager (19) at a 360-MHz proton Larmor frequency. The micro-imager was a prototype that included a specially constructed MR imaging microscope, a computer (MicroVAX II; Digital Equipment, Maynard, Mass), and a cryomagnet (Oxford Instruments, Osney Mead, Oxford, England) with a vertical bore and a specific coil (20). Each patella was placed in a closed atmosphere in a sample tube. All patellae were oriented in the same direction with regard to the magnetic field (anatomic axis parallel to B0) to limit the potential anisotropic effect. Eight images, each acquired with a different echo time (TE) in the same section (12), were acquired in each patella by applying a two-dimensional section-selective single spin-echo pulse sequence in the transverse plane through the middle of the patella to obtain T2 maps. Imaging parameters were as follows: repetition time, 1.5 seconds; TE, 5.5, 7.5, 10.5, 12.5, 15, 20, 25, and 30 msec; number of signals acquired, four; total acquisition time, 105 minutes. T2 maps were processed with binary encoding of T2 values as 2-bit integers in microseconds (range, 0–32 767 µsec). The range of values derived from encoding was convenient with respect to the actual T2 values found in cartilage, as can be seen in Figure 2. Moreover, binary encoding provided for ease of data manipulation. In our previous study (12), the most convenient range of TE values was found to be 5.5–30.0 msec. The cartilage signal-to-noise ratio with the shortest TE (5.5 msec) was typically 20:1. The section thickness was 1 mm, the field of view was 4 x 4 mm, and the matrix was 128 x 128. The spatial resolution was 31 x 31 x 1000 µm. During MR image acquisitions, the temperature in the magnet bore was 25°–30°C.



View larger version (55K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. T2 maps obtained with an 8.5-T micro-MR imager in immature (5 weeks) and mature (10 weeks) patellar cartilage. Color-coded images show the spatial distribution of the T2 values in normal undegraded (Control) cartilage and in cartilage with slight (Hyalu 1H) or severe (Hyalu 6H) depletion of proteoglycans after hyaluronidase degradation. Cartilage appears as a thin blue-green structure (yellow arrow) surrounded by two structures in black corresponding to the air ({circ}) and subchondral bone (curved arrow). Trabecular bone is identified as a structure with high signal intensity (*).

 
T2 values were calculated from the eight spin-echo images by fitting a nonlinear least-squares curve to each pixel on the image. The signal intensity (SI) for the ith jth pixel as a function of TE can be expressed as follows: SIij(TE) = SOij · exp(–TE/T2ij), where SOij is the signal intensity for pixel ij at TE = 0, and T2ij is the T2 constant for pixel ij.

The T2 map is a color-coded representation of T2 relaxation times in the cartilage itself but also in adjacent structures such as subchondral and trabecular bone, with T2 values displayed in color, pixel by pixel, by using software (Transform; Spyglass, Savoy, Ill). Since the results of preliminary studies with the same micro-imager suggested a slight effect of hyaluronidase degradation on T2 values, T2 mapping was adapted to T2 values ranging from 0 to 32.76 msec for normal cartilage. The superficial zone of the cartilage was easily defined because of the marked contrast between the signal in air and that in cartilage. No clear landmark could be observed, however, between the calcified cartilage and the subchondral bone, because both tissue types are characterized by low T2 values. The value of 4 msec was considered the lowest threshold of T2, for the deepest layer of noncalcified cartilage. According to a method previously validated in rat patellar cartilage (12), superficial- and deep-cartilage T2 values were defined as T2 of the first superficial-cartilage pixel and the last deep-cartilage pixel, respectively. Zonal variations in T2 values were considered to indicate differences between superficial- and deep-cartilage T2 values. Global T2 values were calculated separately for degraded and control patellae as the means for all cartilage pixels (A.W.P.).

Cartilage thickness measurements were performed on T2 maps with a personal computer by using imaging software (Scion Image Beta 3b; Scion, Frederick, Md). The cartilage thickness was defined as the average value of 10 measurements performed at regular intervals on T2 maps, in a direction perpendicular to the cartilage surface, from the superficial to the calcified zone. All measurements were made by the same experienced operator (A.W.P.), in accordance with previously published procedures (12).

Histologic Study
Immediately after MR imaging, the control and degraded patellae were fixed in 12% neutral buffered formalin solution, decalcified for 7 days in ethylenediaminetetraacetic acid, dehydrated by using an automated tissue processing apparatus and serial applications of ethanol in gradually increasing increments, and embedded in paraffin. A series of 5-µm-thick slices were obtained in the longitudinal plane in the central region of each patella with a microtome. Histologic sections from each group of animals were stained with hematoxylin-eosin to assess cellularity, toluidine blue to assess proteoglycan content, and picrosirius red to evaluate type II collagen. The organization of the collagen fibers (collagen network) in the picrosirius red–stained slices was examined with polarized light microscopy.

For thickness measurements, slices stained with hematoxylin-eosin were photographed with a color digital video camera (WV-CL350; Panasonic, Osaka, Japan), saved in tagged image file format at a workstation (Indy; Silicon Graphics, Mountain View, Calif), and measured with the imaging software used for T2 map display and analysis. The cartilage thickness was defined as the average value of 10 measurements performed at regular intervals on the same image (A.W.P.), perpendicular to the cartilage surface, from the superficial to the calcified zone. We cannot be certain that MR and histologic measurements were performed in exactly the same locations in the patellae, but both MR and histologic measurements were made in the middle of each patella as determined with visual control. (To avoid any confusion between findings with different methods of cartilage analysis, we have used region to describe the location of sections at MR imaging and zone to describe the location of histologic slices.)

Biochemical Analysis
Eighty-eight patellae (22 degraded either 1 hour or 6 hours, and 22 controls from each of the two age groups) were used for biochemical analysis; 40 patellae were used for assessment of proteoglycan content, and 48 patellae were used for assessment of collagen content. Then the patellae were decalcified for 18 hours in 5% formic acid before the cartilage was stripped from the underlying bone, dried for 1 day, and weighed.

Quantification of sulfated glycosaminoglycans.—The sulfated glycosaminoglycan content, which represents a measurement of proteoglycan content, was quantified with a colorimetric method after hydrolysis of articular cartilage (21). Each dried sample was digested at 60°C for 2 hours in 210 µL of 20 mmol/L sodium phosphate buffer containing 1 mmol/L ethylenediaminetetraacetic acid, 2 mmol/L 1,4-dithiotreitol, and 60 µg papain (Sigma Aldrich). The reaction was terminated with 220 mmol/L iodoacetic acid. The volume then was increased to 1 mL by adding 50 mmol/L tris(hydroxymethyl)aminomethane hydrochloride (pH 8.0). The assay was calibrated by using reagent blanks and standards containing 5–100 µg/mL chondroitin sulfate in the same solvent as the one used for preparation of the samples. The metachromatic reaction of dimethyl-methylene blue was measured at 525 nm with a spectrophotometer (Dynatech, Guyancourt, France) connected to a computer (486 SX; Packard Bell, Paris, France) for data analysis.

Quantification of hydroxyproline.—The hydroxyproline content, which reflects the tissue collagen content, was quantified with a colorimetric method after hydrolysis of articular cartilage (22). Each dried cartilage sample was hydrolyzed in 6N HCl at 130°C for 3 hours in a small sealed borosilicate glass test tube and then neutralized with 6N NaOH solution (pH 5.0–6.0) before 200 µL of hydrolysate was withdrawn from each tube. Hydroxyproline oxidation was initiated by adding 500 µL of chloramine T to the remaining contents of each tube; the contents then were mixed and incubated for 20 minutes at room temperature. Finally, 500 µL of aldehyde–perchloric acid reagent was added, and the mixture was incubated for 20 minutes at 70°C. The assay was calibrated by using reagent blanks and standards that contained 0.5–8.0 µg of hydroxyproline in the same solvent as that used for the samples. The absorbance was determined with a spectrometer (UV-1601; Shimadzu, Duisburg, Germany) set at 550 nm. The spectrometer was connected to a computer (486 SX; Packard Bell) for data analysis. The results were expressed as hydroxyproline equivalent per milligram of cartilage.

Statistical Evaluation
Data were recorded as median or mean ± standard error of the mean for continuous variables and as percentages for categorical variables. To account for moderate sample size and skewness of the data distribution, the study of the effect of age and/or the degree of degradation was conducted after rank transformation of continuous data by using rank analysis of variance. Associations between continuous variables were assessed with the Spearman rank correlation test. Results were interpreted at an {alpha} level of .05. All statistical analyses were performed by using software (SAS; SAS Institute, Cary, NC).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Histologic Findings in Immature Cartilage
Undegraded cartilage.—At polarized light microscopy, the cartilage appeared to be composed of three zones: superficial (5% of the cartilage thickness), transitional (35% of the cartilage thickness), and hypertrophic (60% of the cartilage thickness) (Fig 3). The superficial zone was characterized by ellipsoid cells and orange collagen fibers parallel to the cartilage surface. The transitional zone was characterized by a typical alveolar structure of spherical cells, surrounded by green collagen fibers that were mostly parallel to the articular surface. In the hypertrophic zone, the fibers were oriented perpendicular to the articular surface and appeared as a green area surrounding the hypertrophic cells. Regarding matrix content, normal immature cartilage appeared deep blue after toluidine blue staining of the matrix (proteoglycan content), without spatial variations according to cartilage depth. Stain uptake with picrosirius red was poor (type II collagen content).



View larger version (134K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. Photomicrographs show histologic specimens of immature patellar cartilage without hyaluronidase degradation (Control) or after degradation (Hyalu) for 1 hour or 6 hours. A, Control slice shows normal proteoglycan content, with intense staining of matrix. B, Cartilage degraded for 1 hour shows slight loss of proteoglycans. C, Cartilage degraded for 6 hours shows severe loss, with decreased intensity of staining from superficial to deep cartilage zone. D, Matrix shows normal collagen content in control cartilage, with only slight staining. E, Slice from cartilage degraded for 1 hour shows intensified staining in superficial and transitional zones of cartilage, indicating collagen depletion. F, Slice from cartilage degraded for 6 hours shows intense staining from superficial to deep zone, indicating severe depletion. G-I, Same slices as D-F, viewed with polarized light microscopy to characterize collagen network organization. G, Control slice shows undegraded immature cartilage organization, with thick transitional and hypertrophic zones. H, I, Slices degraded with hyaluronidase show modifications of cartilage color (enhancement of birefringence of collagen fibers) without structural alteration in collagen network. (A-C, toluidine blue stain; D-I, picrosirius red stain; original magnification, x10.)

 
Degraded cartilage.—The digestion of proteoglycans was clearly demonstrated by decreased intensity of toluidine blue staining, from the superficial cartilage to the deep zone, without zonal variations. This loss of proteoglycans was more marked in severely depleted cartilage and was associated with increased intensity of picrosirius red staining in the most severely depleted part of cartilage, the superficial zone. In polarized light, collagen fibers depicted in green in normal cartilage became orange after depletion, with this change being more pronounced in the severely depleted group of patellae.

No concomitant modification of the collagen network was observed. As demonstrated at biochemical analysis (Table 1), the proteoglycan content in immature cartilage decreased significantly (P < .001) after degradation (19% and 45% of proteoglycan was lost after 1 and 6 hours of degradation, respectively), while no significant variation of the hydroxyproline content was measured.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Glycosaminoglycan and Hydroxyproline Contents of Rat Patellar Cartilage by Age and Hyaluronidase Degradation

 
Histologic Findings in Mature Cartilage
Undegraded cartilage.—At polarized light microscopy, the collagen network in mature cartilage showed an organization similar to that described by Benninghoff (23), with four zones: superficial (6% of the cartilage thickness), transitional (10% of the cartilage thickness), radial (68% of the cartilage thickness), and hypertrophic (16% of the cartilage thickness). The superficial zone was characterized by ellipsoid cells and dense orange collagen fibers parallel to the articular surface. Collagen fibers in the transitional zone were arched in several directions and appeared dark, with no birefringence (Fig 4). Finally, the radial zone was characterized by green collagen fibers oriented perpendicular to the articular surface. Regarding matrix content, undegraded mature cartilage showed uniformly poor toluidine blue staining, with no variation in intensity according to cartilage depth, whereas picrosirius red staining was globally more intense than that in undegraded immature cartilage.



View larger version (135K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. Photomicrographs show histologic specimens of mature patellar cartilage without hyaluronidase degradation (Control) or after degradation (Hyalu) for 1 hour or 6 hours. A, Control slice shows normal proteoglycan content, with intense staining of matrix. B, Cartilage depleted for 1 hour shows slight loss of proteoglycans. C, Cartilage depleted for 6 hours shows severe proteoglycan loss, with decreased intensity of staining from superficial to deep cartilage zone. D-F, Slices show intensive staining in the same zones that showed light staining in A-C. G-I, Same slices as D-F, viewed with polarized light microscopy to characterize collagen network organization. G, Mature undegraded cartilage shows classic organization, with superficial, transitional, radial, and hypertrophic zones. H, I, Specimens degraded with hyaluronidase show modifications of cartilage color (enhancement of birefringence of collagen fibers) without structural alteration of collagen network. (A-C, toluidine blue stain; D-I, picrosirius red stain; original magnification, x10.)

 
Degraded cartilage.—Proteoglycan losses were similar in mature and immature cartilage and involved the entire cartilage thickness, as indicated by globally intense picrosirius red staining. These modifications were more pronounced in severely depleted cartilage. At polarized light microscopy, color changes of collagen fibers in degraded mature cartilage were similar to those observed in degraded immature cartilage, without concomitant modification of the collagen network organization.

At biochemical analysis (Table 1), significant decreases in proteoglycan content were found in mature cartilage after 1 and 6 hours of degradation (proteoglycan losses of 13% and 53%, respectively), while no significant variation was found in hydroxyproline content between slightly and severely depleted mature cartilage. In addition, a comparison of immature and mature rat patellar cartilage confirmed previous findings regarding maturation, with significantly decreased proteoglycan content (P = .001) and significantly increased hydroxyproline content (P = .002) in the cartilage of 10-week-old rats compared with that of 5-week-old rats.

Histologic Measurement of Cartilage Thickness
As was previously reported (11), cartilage thickness decreased with maturation (P < .001). The degree of proteoglycan depletion, however, did not influence cartilage thickness in either immature or mature rats (Fig 5).



View larger version (16K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. Bar graph shows mean thickness of rat patellar cartilage measured at histologic analysis and T2 mapping (MRI), according to age, without hyaluronidase degradation (white bars) or after 1 hour (gray bars) or 6 hours (black bars) of degradation. Thickness of both immature and mature cartilage was not affected by proteoglycan depletion. Good correlation was observed between thickness measured at histologic analysis and on T2 maps (r = 0.754; P < .001). Error bars indicate standard error of the mean.

 
Quantitative T2 Mapping
Undegraded cartilage.—T2 mapping in normal patellae permitted clear differentiation between structures: The cartilage, a thin and smooth tissue layer 340–200 µm thick, appeared blue-green, and subchondral bone was depicted in black (Fig 2). As previously described (11), we observed a decrease in global T2 values as a result of the maturation process, with a decrease from 12.60 msec ± 0.36 in the immature group to 9.28 msec ± 0.20 in the mature group (P < .001). We also confirmed a significant decrease in T2 values in the superficial region, as well as in the deep region, related to the maturation process, with a gradient of T2 values between these two regions (high T2 values in the superficial region and low T2 values in the deep region).

T2 values in depleted immature cartilage.—A slight proteoglycan depletion induced a significant increase (P < .001) by 10% in global T2 (13.86 msec ± 0.26 vs 12.60 msec ± 0.36 in control cartilage) and deep T2 values. A severe proteoglycan depletion was characterized by a significant increase (P < .001) by 17% in global T2 (14.79 msec ± 0.79 vs 12.60 msec ± 0.36 in control cartilage), as well as in superficial and deep T2 values (Fig 6, Table 2).



View larger version (15K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6. Bar graph shows variation in mean global T2 between undegraded cartilage (white bars) and cartilage degraded with hyaluronidase for 1 hour (gray bars) or 6 hours (black bars). Significantly increased global T2 values were observed in both immature and mature cartilage after either slight or severe proteoglycan depletion. P < .001 for all comparisons within the two age groups (*) and for mature controls compared with immature controls ({dagger}). Error bars indicate standard error of the mean.

 

View this table:
[in this window]
[in a new window]

  		TABLE 2. T2 of Superficial and Deep Cartilage in Rats by Age and Hyaluronidase Degradation

 
T2 values in depleted mature cartilage.—In mature cartilage, both slight and severe depletion caused a significant increase in global T2 values, from 9.28 msec ± 0.20 in control cartilage to 10.02 msec ± 0.31 (+8%) and 13.17 msec ± 0.86 (+41%) in slightly depleted and severely depleted cartilage, respectively. The zonal variations in T2 values were not affected by proteoglycan depletion; the difference between superficial and deep zones persisted, although superficial and deep T2 values both increased (P < .001) (Fig 6, Table 2). Additionally, the increase in global T2 values seems more marked in mature cartilage with severe depletion when compared with immature cartilage, although this trend appears nonsignificant (Mann-Whitney U test).

MR measurement of cartilage thickness.—Cartilage thickness as measured on T2 maps decreased between 5-week-old rats and 10-week-old rats (P < .001) as a result of the maturation process (Fig 4). The cartilage thickness was not affected by either slight or severe proteoglycan depletion. A comparison between thickness measurements on MR images and at histologic analysis showed a good correlation (r = 0.754, P < .001).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
 
T2-weighted MR imaging sequences can be used to depict the biochemical composition of bovine patellar cartilage structure, as well as changes in its composition with aging (24). T2 mapping, a quantitative approach derived from T2-weighted pulse sequences, has been developed with both experimental and clinical scanners. Both human cartilage (25,26) and animal cartilage (11) show variations in T2 values, from the superficial to the deepest zones, which could be linked to the structure of the cartilage. Previously, we reported a maturation-related decrease in global T2 values concomitant with a decrease in proteoglycan content and an increase in type II collagen content, as well as a modification of the collagen network organization (11). We concluded that collagen content was a stronger determinant of MR signal intensity than was proteoglycan content during maturation. To assess the specific effect of variations in proteoglycan content on T2 values, we developed an ex vivo model of patellar chondropathy in the form of selective proteoglycan depletion. It is well established that testicular hyaluronidase degrades hyaluronic acid and chondroitin, as well as the chondroitin sulfates (27,28). Two different levels of proteoglycan depletion were used to compare the degree of depletion with variations in T2. In this model, degradation affected the entire cartilage thickness, and the accuracy of T2 measurements was greater than it would have been if the entire cartilage thickness had not been affected.

Cartilage Thickness Measurement
The good correlation between cartilage thickness measurements on T2 maps and those at histologic analysis shows the usefulness of MR imaging for determining changes in the volume and thickness of cartilage (29). In addition, the lack of modification in cartilage thickness that we observed shows that this model does not alter cartilage thickness.

T2 Mapping
The observations we made in the control groups confirm our previous results regarding the decrease in global T2 values with aging and the zonal variation of these values with depth. In the present study, we found an increase of global, superficial, and deep T2 values in both groups after either slight or severe proteoglycan depletion. This depletion leads to an increase of free space inside the matrix that is then filled by an influx of water. As demonstrated by Lusse et al (30) and Liess et al (31), the increase in water content is responsible for increased T2 values. As the pool of free water increases, T2 values increase. Slight depletion (proteoglycan loss of 20%) induced an 8% increase in T2 in both immature and mature cartilage, while severe depletion (proteoglycan loss of 50%) induced global T2 increases of 17% and 41% in immature and mature cartilage, respectively. These results suggest that the T2 increase depends directly on the severity of proteoglycan depletion, especially in mature cartilage. The persistent spatial variations in T2 after proteoglycan depletion also suggest a central contribution of collagen fibers in determining this gradient. Different age-related spatial organizations of type II collagen in the extracellular matrix may account for these different effects of severe depletion on global T2 values. Thus, these data suggest that, in addition to its usefulness for assessing maturation or aging processes (decrease in global T2 values), T2 mapping also depicts proteoglycan loss and concurrent changes in the extracellular matrix (increase in global T2 values) in depleted cartilage compared with normal cartilage.

Beyond correlations observed between variations in global T2 values and variations in global proteoglycan content (biochemical analysis), we noted a good correlation between T2 variations in the superficial and deep regions and variations in the intensity of toluidine blue staining observed in the same regions. Thus, T2 mapping seems able to characterize not only the global proteoglycan content of the patellar cartilage but also the proteoglycan content in a given region of this cartilage.

Two other MR imaging studies have been performed to evaluate the effect of selective proteoglycan depletion on T2 mapping. In a quantitative MR imaging study of enzymatically degraded bovine cartilage samples, Nieminen et al (32) found no modification of T2 values in the upper part of the proteoglycan-depleted cartilage. In contrast with the rat patellae used in our study, the bovine cartilage samples were heterogeneous in stage of maturation (1–3 years old), and no biochemical data were available to determine the degree of proteoglycan depletion. Borthakur et al (33) used proton MR imaging and did not find any variation of T1 and T2 values in proteoglycan-depleted calf patellar cartilage. In contrast with our study design, however, theirs did not take into account the potential effect of maturation. In addition, a lower degree of depletion was used, and proteoglycan depletion was limited to the upper part of the cartilage (2 or 3 pixels deep). Concerning this latter point, it should be emphasized that the small region thus characterized and the partial volume effect both limited the accuracy of T2 measurements in that study. Variations between species also should probably be taken into account.

T2 mapping in articular cartilage at high magnetic field strengths also has been reported in other studies (34,35), and T2 values have been measured that are greater than those found in our experiments. There are, however, a number of differences between these studies and our work: First, different species were investigated, with different physical characteristics (eg, rats are much smaller than dogs, pigs, or humans). Second, because of the small size of the animals in our study, the full patellae could be investigated, whereas in other studies the specimens were plugs cut from anatomic sites other than the patellae. Third, because T2 measurements are affected by specimen orientation with regard to the magnetic field (33), care was taken in our study so that the articular surface would be positioned perpendicular to the magnetic field, to minimize orientation effects; in the study of Xia et al (35), by contrast, the articular surface was parallel to the magnetic field. The most likely reason for the shorter T2 values observed in our study is the difference in the animal model.

Study Limitations
Several limitations of this pilot experimental study should be mentioned. The first limitation is the technical impossibility of precisely measuring the influence of water content in rat patellae, because of the thinness of rat patellar cartilage (approximately 300 µm). To minimize the effect of this parameter, we rehydrated patellae for 16 hours so as to avoid the putative effect of dehydration ancillary to freezing. Moreover, there is potential for error as a result of the freezing and thawing of cartilage. Previous studies, however, have demonstrated a lack of influence of freezing on T2 in human cartilage (17), as well as in cartilage of animal species (18), including that of rats (12). Tissular characterization has been performed only in experimental or cadaveric studies, however, and caution should be exercised in extrapolating these results to the clinical setting. The major advantage with experimental modeling of osteoarthritis is the possibility of modulating the severity of the disease and monitoring the disease course. The clinical relevance of experimental modeling, however, remains to be established, because human osteoarthritis is multifactorial and involves both collagen and proteoglycans. Although this experimental study was performed at 8.5 T in the rat, our preliminary T2 maps of the patella performed with a clinical MR imager (Signa EchoSpeed; GE Medical Systems, Milwaukee, Wis) at 1.5 T in healthy human volunteers and in patients with osteoarthritis demonstrated age- and disease-related changes in global T2 of patellar cartilage (11). Standardization of this method will be important for performing adequate in vivo T2 mapping in humans, as was recently suggested by Maier et al (36).

A potential bias in calculated T2 values might have resulted from the TE values we selected. Concerning the choice of TE, however, the results of our previous study (12) suggest that a TE longer than 30 msec would result in noisy images. It was not necessary to use a longer TE at image acquisition because, with the fitting algorithm used in this study, the noise level was assessed from a background (noisy) region of interest on the short TE image.

T2 mapping provides data about cartilage structure and enables evaluation of proteoglycan and collagen content and collagen network organization by means of variations in global and zonal T2. Both global and zonal T2 measurements permit characterization of the structural lesions that are usually observed in the early stages of osteoarthritis. The quantitative characterization of internal abnormalities of cartilage tissue, such as proteoglycan depletion or collagen network alteration, would be possible (and useful) in clinical practice, if T2 maps obtained in patients suspected of having osteoarthritis could be compared with T2 maps obtained in age-matched healthy patients.

Because of the high spatial resolution in this study, molecular diffusion during imaging with these imaging gradients may have resulted in undervaluation of T2. At quantitative evaluation, if the diffusion coefficient is that of free water, it could cause an undervaluation of T2 by no more than 5%. Since this variation is much smaller than the T2 variations measured in our study, the apparent T2 maps that we obtained should be representative of the pure T2 maps.

Practical application: Although these results indicate that proteoglycan depletion is correlated with a small increase in T2 of cartilage, it is unlikely that increased T2 is specific for decreased proteoglycan content. Treatment of cartilage with hyaluronidase leads to concurrent changes in the extracellular cartilage matrix that previously were shown to lead to increased T2 of cartilage (37). These changes include increase in cartilage water content, relative decrease in collagen concentration, and alteration in collagen fiber anisotropy. Further work is needed in older rats or with experimentally induced osteoarthritis (concomitant damage to collagen matrix and proteoglycan depletion) to determine the optimal conditions for clinical use of T2 mapping in patients with osteoarthritis, since comparisons between clinical settings, biochemical data, and MR findings in cartilage lesions are rare. In this direction, an interesting approach to T2 mapping was developed by Dardzinski et al (26), using normalized T2 profiles defined for normal versus osteoarthritic knees on the basis of arthroscopy. The different effects of proteoglycan depletion in immature and mature cartilages also suggest that the further definition of age-adjusted T2 values for normal subjects versus patients with osteoarthritis would be clinically relevant. Finally, the combination of global T2 values and T2 profiles could be an innovative approach for T2 mapping to assess osteoarthritis qualitatively and quantitatively.


   ACKNOWLEDGMENTS
 
We thank Francis Guillemin, MD, PhD, Center of Clinical Epidemiology INSERM (National Institute of Health and Medical Research)–Ministry of Health, CHU, Nancy, France, for statistical support; Stephanie Etienne, MSc, for technical assistance; and Michel Thiery for his good care of the animals.


   FOOTNOTES
 
Abbreviation: TE = echo time

Authors stated no financial relationship to disclose.

Author contributions: Guarantors of integrity of entire study, D.L., A.W.P., P. Gillet, P.O.; study concepts, D.L., A.B., G.G., A.W.P.; study design, D.L., J.P.R., P. Gonord, A.W.P.; literature research, D.L., A.W.P., P.O.; experimental studies, A.W.P., J.P.R., P. Gonord; data acquisition, A.W.P., J.P.R., P. Gonord; data analysis/interpretation, D.L., A.W.P., P.O., L.G.; statistical analysis, D.L., A.W.P., P. Gonord; manuscript preparation, P. Gillet, A.W.P., L.G.; manuscript definition of intellectual content, D.L., G.G., A.W.P., L.G.; manuscript editing, P.O., A.W.P., D.L., L.G.; manuscript revision/review, D.L., P.N., A.B., A.W.P.; manuscript final version approval, D.L., A.W.P., P.N., P. Gillet


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 

  1. Mankin HJ, Treadwell BV. Osteoarthritis: a 1987 update. Bull Rheum Dis 1986; 36:1-10.
  2. Wachsmuth L, Juretschke HP, Raiss RX. Can magnetization transfer magnetic resonance imaging follow proteoglycan depletion in articular cartilage? MAGMA 1997; 5:71-78.
  3. 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.[Abstract/Free Full Text]
  4. Bashir A, Gray ML, Hartke J, Burstein D. Nondestructive imaging of human cartilage glycosaminoglycans concentration by MRI. Magn Reson Med 1999; 41:857-865.[CrossRef][Medline]
  5. Trattnig S, Mlynarik V, Breitenseher M, et al. MRI visualization of proteoglycan depletion in articular cartilage via intravenous administration of Gd-DTPA. Magn Reson Imaging 1999; 17:577-583.[CrossRef][Medline]
  6. Bacic G, Liu KJ, Goda F, Hoopes PJ, Rosen GM, Swartz HM. MRI contrast enhanced study of cartilage proteoglycan degradation in the rabbit knee. Magn Reson Med 1997; 37:764-768.[Medline]
  7. Mlynarik V, Trattnig S, Huber M, Zembsch A, Imhof H. The role of relaxation times in monitoring proteoglycan depletion in articular cartilage. J Magn Reson Imaging 1999; 10:497-502.[CrossRef][Medline]
  8. Akella SV, Regatte RR, Gougoutas AJ, et al. Proteoglycan-induced changes in T1rho-relaxation of articular cartilage at 4T. Magn Reson Med 2001; 46:419-423.[CrossRef][Medline]
  9. Frank LR, Wong EC, Luh WM, Ahn JM, Resnick D. Articular cartilage in the knee: mapping of the physiologic parameters at MR imaging with a local gradient coil—preliminary results. Radiology 1999; 210:241-246.[Abstract/Free Full Text]
  10. Mosher TJ, Dardzinski BJ, Smith MB. Human articular cartilage: influence of aging and early symptomatic degeneration on the spatial variation of T2—preliminary findings at 3 T. Radiology 2000; 214:259-266.[Abstract/Free Full Text]
  11. Loeuille D, Olivier P, Watrin A, et al. The biochemical content of articular cartilage: an original MRI approach. Biorheology 2002; 39:269-276.[Medline]
  12. Watrin A, Ruaud JP, Olivier PT, et al. T2 mapping of rat patellar cartilage. Radiology 2001; 219:395-402.[Abstract/Free Full Text]
  13. Dardzinski BJ, Laor T, Schmithorst VJ, Klosterman L, Graham T. Mapping T2 relaxation time in the pediatric knee: feasibility with a clinical 1.5-T MR imaging system. Radiology 2002; 225:233-239.[Abstract/Free Full Text]
  14. Pellaumail B, Loeuille D, Watrin A, Netter P, Berger G, Saied A. Correlation of high frequency ultrasound backscatter with cartilage matrix constituents. IEEE Int Ultrasonics Symp Proc 1998; 2:1463-1466.
  15. Xia Y, Farquhar T, Burton-Wurster N, Vernier-Singer M, Lust G, Jelinski LW. Self-diffusion monitors degraded cartilage. Arch Biochem Biophys 1995; 323:323-328.[CrossRef][Medline]
  16. Vilim V, Fosang AJ. Proteoglycans isolated from dissociative extracts of differently aged human articular cartilage: characterization of naturally occurring hyaluronan-binding fragments of agrecan. Biochem J 1994; 304:887-894.
  17. Grushko G, Schneiderman R, Maroudas A. Some biochemical and biophysical parameters for the study of the pathogenesis of osteoarthritis: a comparison between the processes of aging and degeneration in human hip cartilage. Connect Tissue Res 1989; 19:149-176.[Medline]
  18. Rubenstein JD, Kim JK, Morava-Protzner I, Stanchev PL, Henkelman RM. Effects of collagen orientation on MR imaging characteristics of bovine articular cartilage. Radiology 1993; 188:219-226.[Abstract/Free Full Text]
  19. Ruaud JP. Etude et réalisation d’un imageur RMN à très haute résolution. PhD dissertation University of Paris XI: Orsay, France, 1991.
  20. Gonord P, Kan S. Multigap parallel-plate bracelet resonator frequency determination and applications. Rev Sci Instrum 1994; 65:3363-3366.[CrossRef]
  21. Goldberg RL, Kolibas LM. An improved method for determining proteoglycans synthesized by chondrocytes in culture. Connect Tissue Res 1990; 24:265-275.[Medline]
  22. Woessner JF, Jr. The determination of hydroxyproline in tissue and protein samples containing small proportions of the amino acid. Arch Biochem Biophys 1961; 93:440-447.[CrossRef][Medline]
  23. Benninghoff A. Über den funktionellen Bau des Knorpels. Anat Anz 1922; 55:250-267.
  24. Olivier P, Loeuille D, Watrin A, et al. Structural evaluation of articular cartilage: potential contribution of magnetic resonance techniques used in clinical practice. Arthritis Rheum 2001; 44:2285-2295.[CrossRef][Medline]
  25. Smith HE, Mosher TJ, Dardzinski BJ, et al. Spatial variation in cartilage T2 of the knee. J Magn Reson Imaging 2001; 14:50-55.[CrossRef][Medline]
  26. Dardzinski BJ, Mosher TJ, Li S, Van Slyke MA, Smith MB. Spatial variation of T2 in human articular cartilage. Radiology 1997; 205:546-550.[Abstract/Free Full Text]
  27. Takagi M. Ultrastructural cytochemistry of cartilage proteoglycans and their relation to the calcification process In: Ultrastructure of skeletal tissues. New York, NY: Kluwer Academic, 1990; 111-127.
  28. Yamada K. Effects of Streptomyces hyaluronidase digestion on the histochemical reactions of proteoglycans in cartilage compared with its effects on certain non-cartilaginous tissues. Histochem J 1982; 14:149-160.[CrossRef][Medline]
  29. Loeuille D, Olivier P, Mainard D, Gillet P, Netter P, Blum A. Magnetic resonance imaging of normal and osteoarthritic cartilage. Arthritis Rheum 1998; 41:963-975.[CrossRef][Medline]
  30. Lusse S, Claassen H, Gehrke T, et al. Evaluation of water content by spatially resolved transverse relaxation times of human articular cartilage. Magn Reson Imaging 2000; 18:423-430.[CrossRef][Medline]
  31. Liess C, Lusse S, Karger N, Heller M, Gluer CC. Detection of changes in cartilage water content using MRI T2-mapping in vivo. Osteoarthritis Cartilage 2002; 10:907-913.[CrossRef][Medline]
  32. Nieminen MT, Toyras J, Rieppo J, et al. Quantitative MR microscopy of enzymatically degraded articular cartilage. Magn Reson Med 2000; 43:676-681.[CrossRef][Medline]
  33. Borthakur A, Shapiro EM, Beers J, Kudchodkar S, Kneeland JB, Reddy R. Sensitivity of MRI to proteoglycan depletion in cartilage: comparison of sodium and proton MRI. Osteoarthritis Cartilage 2000; 8:288-293.[CrossRef][Medline]
  34. Mlynarik V, Degrassi A, Toffanin R, Vittur F, Cova M, Pozzi-Mucelli RS. Investigation of laminar appearance of articular cartilage by means of magnetic resonance microscopy. Magn Reson Imaging 1996; 14:435-442.[CrossRef][Medline]
  35. Xia Y, Farquhar T, Burton-Wurster N, Ray E, Jelinski LW. Diffusion and relaxation mapping of cartilage-bone plugs and excised disks using microscopic magnetic resonance imaging. Magn Reson Med 1994; 31:273-282.[Medline]
  36. Maier CF, Tan SG, Hariharan H, Potter HG. T2 quantitation of articular cartilage at 1.5 T. J Magn Reson Imaging 2003; 17:358-364.[CrossRef][Medline]
  37. Wayne JS, Kraft KA, Shields KJ, Yin C, Owen JR, Disler DG. MR imaging of normal and matrix-depleted cartilage: correlation with biomechanical function and biochemical composition. Radiology 2003; 228:493-499.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Figures Only
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2341030394v1
234/1/162    most recent
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Watrin-Pinzano, A.
Right arrow Articles by Loeuille, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Watrin-Pinzano, A.
Right arrow Articles by Loeuille, D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
RADIOLOGY RADIOGRAPHICS RSNA JOURNALS ONLINE