To examine the glycosaminoglycan (GAG) content in cartilage and that in synovial fluid and determine whether they are associated, in patients with an acute anterior cruciate ligament (ACL) injury.
To examine the glycosaminoglycan (GAG) content in cartilage and that in synovial fluid and determine whether they are associated, in patients with an acute anterior cruciate ligament (ACL) injury.
Twenty-four patients (14 of whom were male) with a mean age of 27 years (range 17–40 years) were assessed with delayed gadolinium-enhanced magnetic resonance imaging (MRI) of cartilage an average of 3 weeks after an ACL rupture and compared with 24 healthy volunteers. Two hours after an intravenous injection of Gd-DTPA2− (0.3 mmoles/kg body weight), quantitative measurements of the T1 relaxation time (T1Gd [T1 relaxation time in the presence of Gd-DTPA]) were made in lateral and medial femoral weight-bearing cartilage. In the patients, synovial fluid was aspirated immediately before the MRI, and GAG was analyzed using dye precipitation with Alcian blue.
Fifteen of the 24 patients had an isolated bone bruise in the lateral femoral condyle, where the cartilage T1Gd was shorter than that in the controls (mean ± SD 385 ± 83 msec and 445 ± 41 msec, respectively; P = 0.004), consistent with decreased GAG content. However, the T1Gd was also decreased in the medial femoral cartilage, where bone bruises were rare (376 ± 76 msec in patients versus 428 ± 38 msec in controls; P = 0.006). The mean ± SD synovial fluid GAG concentration in patients was 157 ± 86 μg/ml and showed a positive correlation with the T1Gd (r = 0.49, P = 0.02).
This study indicates that an ACL injury causes posttraumatic edema of the lateral femoral cartilage but initializes a generalized biochemical change within the knee that leads to GAG loss from both lateral and medial femoral cartilage. In cartilage with a high GAG content (long T1Gd), more GAG is released into the synovial fluid, suggesting that cartilage quality is a factor to consider when interpreting cartilage biomarkers of metabolism.
Rupture to the anterior cruciate ligament (ACL) is a common sports-related injury, with an annual incidence of 2–3 per 10,000 (1–3). Patients with ACL-deficient knees, with or without a concomitant meniscus injury, are at high risk for posttraumatic osteoarthritis (OA) and may serve as a model for the development of OA. Radiographic changes of OA occur in 60–90% of ACL-injured patients 10–20 years after the injury (4–9). Furthermore, Roos et al showed that ACL-injured patients with posttraumatic OA are, on average, 15–20 years younger than patients with primary OA (2).
The mechanisms responsible for cartilage degeneration in the ACL-deficient knee are poorly understood. Mechanical instability does not seem to be the only cause. More than 50% of patients undergoing surgical ACL reconstruction show radiographic signs of OA 5 years after injury (3, 10). Moreover, using Fairbank's criteria, no correlation between the severity of knee laxity and radiographic changes of OA has been observed (10). It can therefore be assumed that in addition to a biomechanical disturbance, biochemical factors are important in the development of posttraumatic OA.
The most important matrix molecule with regard to viscoelastic properties of articular cartilage is the large aggregating proteoglycan, aggrecan. Aggrecan consists of a protein core to which a large number of highly negatively charged glycosaminoglycans (GAGs) are attached. By attracting counterions to GAG, the aggrecan creates a swelling pressure that is counteracted by the rigid framework of the collagen network. Immediately after an ACL injury, an increase of aggrecan fragments in the synovial fluid occurs, generated by one or more of the ADAM-TS aggrecanases (11, 12). Also released are other cartilage-related molecules such as cartilage oligomeric matrix protein (COMP) and stromelysin (13–16). Such molecules are known as molecular biomarkers of cartilage metabolism and have been suggested to reflect alterations in cartilage matrix homeostasis that may be prognostic for disease progression in OA (17, 18). In addition, during the chronic phase (>3 months) after an ACL injury, the level of proteoglycan fragments remains higher than that in controls, indicating a biochemical disturbance within the joint. (11, 13–16).
Due to the impact of the trauma, an ACL rupture is associated with damage to the subchondral bone. Magnetic resonance imaging (MRI) within the first weeks after trauma reveals subchondral edema, known as “bone bruise,” in up to 92% of ACL-injured patients, mainly in the lateral compartment (19–23). Histologic examination of cartilage/bone plugs adjacent to bone bruises has shown substantial cartilage damage, including cell death, loss of proteoglycans, and altered COMP distribution (24, 25). A noninvasive technique to estimate joint cartilage GAG content is known as delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) (26–28).
According to the dGEMRIC technique, the negatively charged contrast medium (Gd-DTPA2−) is injected intravenously and distributes in the cartilage by diffusion. The diffusion time depends on the cartilage thickness and is ∼2 hours in femoral weight-bearing cartilage (27). After saturation, the T1 relaxation time in the presence of Gd-DTPA2− (T1Gd) correlates to the cartilage GAG content (29–31). The T1Gd is obtained in the cartilage from inversion recovery images with different inversion times. When using the dGEMRIC technique, a long T1Gd is consistent with high cartilage GAG content. Analysis of the T1Gd in weight-bearing femoral cartilage by the use of a standardized region of interest (ROI) has shown intraobserver and interobserver variability of <3% (32). Using the triple dose of 0.3 mmoles/kg body weight, dGEMRIC has identified T1Gd differences in the order of 5% (27). Recently, it was shown that individuals who engage in regular physical exercise have a longer T1Gd compared with sedentary individuals, indicating a relationship between GAG content and activity level (33). It was suggested that exercise level is a factor to consider in dGEMRIC (e.g., in the selection of controls for clinical studies).
The objectives of the present study were to examine femoral cartilage GAG content by dGEMRIC in patients with an acute ACL rupture and to compare the results with those in healthy controls, and to determine the synovial fluid GAG concentration in these patients and establish whether the T1Gd and synovial fluid GAG concentration are associated.
The study included 24 patients, ages 40 years and younger, with closed physes and an MRI-verified ACL rupture in a previously uninjured knee. MRI was performed an average of 3 weeks after the trauma (range 11–38 days). Immediately before the MRI, an orthopedic surgeon aspirated synovial fluid, and the amount was measured. The synovial fluid samples were centrifuged at 3,500 revolutions per minute for 20 minutes and then stored frozen at −20°C until used for GAG analysis. The age, height, body mass index (BMI; kg/m2), and activity level of the patients were registered. Activity level was categorized into 3 groups according to the level of regular physical activity in which the patient engaged during the year preceding the ACL injury, as follows: no regular physical exercise; moderate physical exercise, averaging twice weekly; and training at an elite level (33).
The patients were compared with 24 healthy volunteers who were matched for exercise level and had previously been examined by members of our group (33). The reference group comprised 14 men and 10 women with a mean age of 25 years and a mean ± SD BMI (22.5 ± 2.3 kg/m2) similar to that of the group with ACL injuries. Before subjects were included, the study was explained to all of them, and written informed consent was obtained. The study was approved by the ethics review board.
All investigations were made with a standard 1.5T MRI system with a dedicated knee coil (Magnetom Vision; Siemens Medical Solutions, Erlangen, Germany). Initially, a diagnostic series was performed to verify the ACL rupture and concomitant injuries, such as meniscus tears and subchondral bone bruises. Following the diagnostic series, precontrast T1 analysis (described in detail below) was performed in weight-bearing lateral and medial femoral knee cartilage to estimate the level of cartilage edema (28, 34). After the precontrast analyses, Gd-DTPA2− (Magnevist; Schering, Berlin, Germany) was injected into an antecubital vein at a dose of 0.3 mmoles/kg body weight (triple dose) during 2–3 minutes, with the patient in the supine position. In order to optimize the distribution of Gd-DTPA2− into the cartilage, the patients exercised by walking up and down stairs for ∼7 minutes, starting 5 minutes after receiving the injection (27). Postcontrast imaging with subsequent T1 analysis (T1Gd) was performed 2 hours after the injection was administered.
Pre- and postcontrast T1 analyses were performed according to our previous protocol (27). Central parts of the weight-bearing lateral and medial femoral cartilage were identified using the diagnostic series. Quantitative relaxation time calculations were made in a 3-mm–thick sagittal slice on each condyle, using sets of 6 turbo inversion recovery images with different inversion times (repetition time [TR] 3,000 msec, time to echo [TE] 15 msec, turbo factor 7, field of view [FOV] 120 × 120 mm2, matrix 256 × 256, time from inversion [TI] 100, 200, 400, 800, 1,600, and 2,800 msec precontrast; TR 2,000 msec, TE 15 msec, turbo factor 7, FOV 120 × 120 mm2, matrix 256 × 256, TI 50, 100, 200, 400, 800, and 1,600 msec postcontrast). In the lateral and medial slices, a full-thickness ROI was drawn in the cartilage between the center of the tibial plateau and the rear insertion of the meniscus. In these standardized and validated ROIs, T1 was calculated using the mean signal intensity from each ROI as input to a 3-parameter fit (32, 35).
Synovial fluid GAG was analyzed by a commercially available kit, using dye precipitation of chondroitin and keratan sulfate with Alcian blue (36).
All data, including those for joint fluid GAG content, were tested for normality using the Kolmogorov-Smirnov test and matched the pattern of normal distribution. For this reason, parametric statistics, i.e., t-test and Pearson's product, were used.
Table 1 shows the characteristics of the patients in the study (14 men and 10 women, mean age 27 years). Seventeen of the 24 patients had injured the right knee. The diagnostic MRI revealed subchondral bone bruising in all but 1 patient, most frequently involving the lateral femorotibial compartment (in 21 of 23 patients). In a majority of the patients (15 of 24), an isolated bone bruise in the lateral femoral condyle was observed. The diagnostic MRI detected a meniscus injury in one-third of the patients and almost exclusively in men (7 of 8). The mean amount of synovial fluid that was aspirated immediately before the MRI was 19 ml (range 1–65 ml). In 1 patient, the amount aspirated was too small for analysis. The mean time from injury to MRI was 22 days (range 11–38 days).
|Patient/ sex/age||BMI, kg/m2||Injured knee||No. of days postinjury||SF in knee, ml||Bone bruises†||Concomitant meniscus injury||GAG content in SF, μg/ml||Precontrast T1||T1Gd|
Individual precontrast T1 values are shown in Table 1. In the patients with ACL injuries, the mean ± SD precontrast T1 relaxation time was longer in lateral femoral cartilage (where bone bruises were common) than in medial femoral cartilage (1,119 ± 218 msec and 982 ± 94 msec, respectively; P = 0.005). In contrast, in the reference group the T1 relaxation times in lateral and medial femoral cartilage were similar (955 ± 56 msec and 944 ± 62 msec, respectively; P = 0.35) (Figure 1).
The precontrast T1 relaxation time was longer in patients than in controls in the lateral femoral cartilage (P = 0.001) but not in the medial femoral cartilage (Figure 1).
In the patients with ACL injuries, the mean ± SD T1Gd was similar in lateral and medial femoral cartilage (385 ± 83 msec and 376 ± 76 msec, respectively; P = 0.62) (Table 1 and Figure 2). In comparison, in the group of healthy volunteers, the T1Gd was slightly longer in lateral femoral cartilage compared with medial femoral cartilage (445 ± 41 msec and 428 ± 38 msec, respectively; P = 0.01) (33).
The T1Gd in both lateral and medial femoral cartilage in ACL-injured patients was shorter (consistent with decreased GAG content) than that in healthy volunteers (P = 0.004 and P = 0.006, respectively) (Figure 2). The difference in the T1Gd between patients and controls was 16% in lateral femoral cartilage and 14% in medial femoral cartilage (Figure 2).
The mean ± SD GAG concentration in the 23 samples of synovial fluid that were examined was 157 ± 86 μg/ml. Individual values are shown in Figure 3. There was a positive correlation between synovial fluid GAG concentration and the T1Gd (mean of lateral and medial femoral cartilage) (r = 0.49, P = 0.02) (Figure 3). Similarly, there was a positive correlation with synovial fluid GAG content (amount of synovial fluid × GAG concentration) (r = 0.60, P < 0.01).
No sex difference was observed; in both men and women, the mean ± SD T1Gd in lateral and medial femoral cartilage was 380 ± 73 msec and 380 ± 63 msec, respectively. The T1Gd did not differ between patients with and those without a meniscus injury (373 ± 85 msec and 384 ± 60 msec, respectively; P = 0.71). In this rather homogeneous material, no correlation was observed between the T1Gd and age (R = 0.12, P = 0.58) or BMI (R = 0.15, P = 0.50). There was also no correlation between the T1Gd and the amount of synovial fluid (R = 0.25, P = 0.25) nor with the time from injury (R = −0.18, P = 0.41).
In the present study, we showed decreased T1Gd consistent with GAG loss in femoral weight-bearing cartilage in the early phase after an ACL injury. Furthermore, the results indicate a positive relationship between the GAG content in cartilage and that in synovial fluid.
Several studies have shown elevated levels of GAG and proteoglycan fragments in synovial fluid during the early phase after an ACL injury (11, 14, 37). The concentration increases to a maximum ∼1 week after the trauma and remains high for at least 1 month after the trauma (14, 37). An average of 3 weeks posttrauma, we observed a mean synovial fluid GAG concentration of 157 μg/ml. Dahlberg et al (14) observed a median synovial fluid GAG concentration of 234 μg/ml within 5 weeks posttrauma in patients with an ACL injury. In comparison, in healthy athletes the GAG concentration was 35 ± 27 μg/ml (38).
It is generally agreed that the release of primarily cartilage-derived matrix molecules, or fragments thereof (biomarkers), to synovial fluid reflects ongoing metabolic processes in the cartilage. However, several issues need to be considered in order to establish the prognostic value of such measurements, e.g., which factors contribute to the marker release at the time of an acute knee injury, and what relationship exists between the release of a biomarker and the content of that marker in the cartilage matrix? How is the level of synthesis of a particular marker molecule within the cartilage related to its concentration in synovial fluid? What constitutes release of the marker into synovial fluid with respect to the ratio of resident and newly synthesized molecules? The present study deals with 2 of these issues: whether the increased GAG concentration in synovial fluid in ACL-injured knees relates only to the areas of cartilage impact (bone bruises), or whether it also involves other cartilage regions within the joint. Furthermore, it examines the relationship between GAG concentration in synovial fluid and that in cartilage.
In patients with radiographic OA and rheumatoid arthritis (i.e., those in whom macroscopic loss of cartilage has occurred), the concentration of cartilage degradation products in synovial fluid correlates inversely to disease stage (38, 39). Hence, the level of proteoglycan fragments in synovial fluid depends on the quantity of cartilage remaining in the joint (38, 39). The ACL-injured patients in the present study were young and had no knee symptoms prior to the injury. Because cartilage loss in their knees is unlikely, the positive correlation between synovial fluid GAG concentration and the T1Gd that we observed relates to factors other than cartilage quantity. We suggest that cartilage GAG content is one such factor. In support of this suggestion, it was recently concluded that compared with sedentary individuals, persons who engage in regular physical exercise have a higher content of GAG in their cartilage (33). These new data indicate that the amount of GAG that is released to the synovial fluid depends not only on cartilage quantity but also on cartilage quality. In other words, a high GAG concentration in synovial fluid could be explained by a matrix that has more GAG to release.
It may be argued that increased synthesis of new molecules after the trauma may have influenced the concentration of GAG in the synovial fluid. However, results of a recent in vitro study showed that the GAG loss that occurs after cartilage injury is attributable to leakage of resident molecules and not to increased synthesis (<1%) (40). The positive correlation between the T1Gd and the synovial fluid GAG concentration indicates that cartilage matrix molecular content, for example due to different levels of physical activity, may be a factor to consider when interpreting synovial fluid molecular biomarkers of cartilage metabolism (33).
In both the lateral and the medial femoral cartilage of patients, the T1Gd was significantly shorter than that in healthy volunteers. The short T1Gd is consistent with a low cartilage GAG content (29–31). The difference between patients and controls was in the order of 15% in both compartments. Use of the dGEMRIC technique does not provide information about whether the lower GAG content is attributable to an actual GAG loss from the matrix, dilution of existing GAG molecules by increased water content, or both. In this regard, the precontrast T1 relaxation time can be used to estimate hydration of the cartilage. A long T1 relaxation time precontrast is consistent with a decreased protein-to-water ratio (i.e., increased water content) (34). In normal cartilage, the T1 relaxation time precontrast is slightly less than 1 second (27, 41). Accordingly, the mean precontrast T1 relaxation time in the medial femoral cartilage in the ACL-injured patients was 982 msec and did not differ from that in the healthy volunteers (Figure 1). However, in patients the precontrast T1 relaxation time in lateral cartilage was longer than that in medial cartilage and also was longer than that in the healthy volunteers, indicating increased hydration (Figure 1).
It cannot be excluded that the low GAG content that we observed in the lateral area is, to some extent, attributable to an increased water content per se. However, in patients who underwent surgical ACL reconstruction 7 weeks after the trauma, biopsy specimens obtained from cartilage overlying bone bruises (all of which were in the lateral femoral condyle) showed a marked loss of toluidine blue staining, consistent with actual GAG loss (25). With regard to the medial femoral cartilage, the short T1Gd in combination with a normal T1 relaxation time precontrast strongly suggests a net loss of GAG without increased hydration. We therefore argue that an ACL injury results in a global biochemical disturbance within the joint, an idea first suggested by metabolic analysis of ACL injury in animal models (42).
Biomechanics is another issue to consider in OA development in patients with ACL injuries, as shown by their different knee kinematics and increased anterior tibial translation during gait (43, 44). In the current study, patients were examined early after the injury (an average of 3 weeks). It seems less likely that such altered gait patterns would be established at this time point. Although no correlation was found between the T1Gd and the time from injury, it cannot be ruled out that inactivity of the patient between the time of trauma and the time of the examination contributed, to some extent, to the short T1Gd that we observed (33).
In the present study, as in other studies, the vast majority of bone bruises involved the lateral compartment, suggesting that the adjacent cartilage was also damaged (21, 22, 45–47). It may be argued that the absence of a bone bruise does not automatically exclude cartilage damage in the medial compartment. However, the precontrast analysis also indicated normal signal in the medial femoral cartilage, whereas it was increased in the lateral cartilage. As discussed previously, an increased T1 relaxation time precontrast indicates increased water content of the cartilage (34). We interpret this increased hydration to be a direct effect of the blunt trauma to the cartilage that occurred at the time of the injury—the same trauma that caused the bone bruise. We do not believe that such posttraumatic edema resembles the cartilage swelling that has been demonstrated after ACL transection in dogs (48). In the dog model, no blunt trauma exists, and the cartilage swelling that eventually occurs is more likely attributable to a degenerative process within the joint that finally leads to OA (48). In contrast, in humans with ACL injuries it has been shown that most bone bruises resolve spontaneously within 2 years after the trauma (49). Furthermore, posttraumatic OA after an ACL injury most frequently involves the medial rather than the lateral compartment (3, 7). The significance of the GAG loss demonstrated in the present study with regard to future OA is not known. It is possible that the cartilage GAG content can be regained by increased synthesis both laterally and medially. Longitudinal followup of these patients with dGEMRIC could provide a means by which to study the relationship between cartilage GAG concentration and OA development.
In conclusion, the results of this study indicate that an ACL injury causes posttraumatic edema of the lateral femoral cartilage but initializes a generalized biochemical change within the knee that leads to GAG loss from both the lateral and medial femoral cartilage. In cartilage with a high GAG content (long T1Gd), more GAG is released to the synovial fluid, suggesting that cartilage quality is a factor to consider when interpreting cartilage biomarkers of metabolism.