Glycosaminoglycan Content Of Cartilage Research Articles

Positive effects of moderate exercise on glycosaminoglycan content in knee cartilage: A four‐month, randomized, controlled trial in patients at risk of osteoarthritis

To evaluate the effects of moderate exercise on glycosaminoglycan (GAG) content in knee cartilage in subjects at high risk of knee osteoarthritis (OA). Forty-five subjects (16 women, mean age 46 years, mean body mass index 26.6 kg/m(2)) who underwent partial medial meniscus resection 3-5 years previously were randomized to undergo a regimen of supervised exercise 3 times weekly for 4 months or to a nonintervention control group. Cartilage GAG content, an important aspect of the biomechanical properties of cartilage, was estimated by delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC), with results expressed as the change in the T1 relaxation time in the presence of Gd-DTPA (T1[Gd]). Thirty of 45 patients were examined by dGEMRIC at baseline and followup. The exercise group (n = 16) showed an improvement in the T1(Gd) compared with the control group (n = 14) (15 msec versus -15 msec; P = 0.036). To study the dose response, change in the T1(Gd) was assessed for correlation with self-reported change in physical activity level, and a strong correlation was found in the exercise group (n = 16, r(S) = 0.70, 95% confidence interval [95% CI] 0.31-0.89) and in the pooled group of all subjects (n = 30, r(S) = 0.74, 95% CI 0.52-0.87). This in vivo cartilage monitoring study in patients at risk of knee OA who begin exercising indicates that adult human articular cartilage has a potential to adapt to loading change. Moderate exercise may be a good treatment not only to improve joint symptoms and function, but also to improve the knee cartilage GAG content in patients at high risk of developing OA.

Role of interleukin‐1 and tumor necrosis factor α in matrix degradation of human osteoarthritic cartilage

To determine whether interleukin-1 (IL-1) or tumor necrosis factor alpha (TNFalpha), or both, plays a role in the excessive degradation that is observed in cultured osteoarthritic (OA) articular cartilage. Antagonists of IL-1 and TNFalpha, namely, IL-1 receptor antagonist and the PEGylated soluble TNFalpha receptor I, respectively, were added at different concentrations to explant cultures of nonarthritic (5 obtained at autopsy) and OA (15 obtained at arthroplasty) articular cartilage. The cleavage of type II collagen (CII) by collagenase was measured by an immunoassay in cartilage and culture media. Proteoglycan (mainly aggrecan) content and degradation were measured by a colorimetric assay for glycosaminoglycan (GAG) content in cartilage and culture media. Reverse transcriptase-polymerase chain reaction was used to analyze gene expression of matrix metalloproteases (MMPs) 1, 3, and 13, CII, aggrecan, IL-1, and TNFalpha. Antagonists of IL-1 and TNFalpha inhibited the increase in CII cleavage by collagenase as well as the increase in GAG release observed in OA cartilage compared with normal cartilage. Inhibition was significant in tissue from some patients but not from others, although significant inhibition was observed when all the results were analyzed together. An increase in the GAG content in cartilage was seen in 4 of 15 cases. However, this increase was not significant when all the data were combined. Preliminary results indicated no effect of these antagonists on nonarthritic cartilage from 3 different donors. Independent analyses of gene expression in cultured cartilage from 9 other OA patients revealed that IL-1 or TNFalpha blockade, either alone and/or in combination, frequently down-regulated MMP-1, MMP-3, and MMP-13 expression. Expression of IL-1 and TNFalpha was inhibited by either antagonist or by the combination in essentially half the cases. The combined blockade up-regulated aggrecan and CII gene expression in approximately half the cases. These results suggest that the autocrine/paracrine activities of TNFalpha and IL-1 in articular cartilage may play important roles in cartilage matrix degradation in OA patients but not in all patients. Inhibition of either or both of these cytokines may offer a useful therapeutic approach to the management of OA by reducing gene expression of MMPs involved in cartilage matrix degradation and favoring its repair.

Cartilage glycosaminoglycan loss in the acute phase after an anterior cruciate ligament injury: Delayed gadolinium‐enhanced magnetic resonance imaging of cartilage and synovial fluid analysis

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-DTPA(2-) (0.3 mmoles/kg body weight), quantitative measurements of the T1 relaxation time (T1(Gd) [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 T1(Gd) 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 T1(Gd) 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 mug/ml and showed a positive correlation with the T1(Gd) (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 T1(Gd)), more GAG is released into the synovial fluid, suggesting that cartilage quality is a factor to consider when interpreting cartilage biomarkers of metabolism.

Effects of sodium hyaluronate and methylprednisolone acetate on proteoglycan synthesis in equine articular cartilage explants

To determine effects of sodium hyaluronate (HA) on corticosteroid-induced cartilage matrix catabolism in equine articular cartilage explants. 30 articular cartilage explants from fetlock joints of 5 adult horses without joint disease. Articular cartilage explants were treated with control medium or medium containing methylprednisolone acetate (MPA; 0.05, 0.5, or 5.0 mg/mL), HA (0.1, 1.0, or 1.5 mg/mL), or both. Proteoglycan (PG) synthesis was measured by incorporation of sulfur 35-labeled sodium sulphate into PGs, and PG degradation was measured by release of radiolabeled PGs into the medium. Total glycosaminoglycan (GAG) content in media and explants and total explant DNA were determined. Methylprednisolone acetate caused a decrease in PG synthesis, whereas HA had no effect. Only the combination of MPA at a concentration of 0.05 mg/mL and HA at a concentration of 1.0 mg/mL increased PG synthesis, compared with control explants. Methylprednisolone acetate increased degradation of newly synthesized PGs into the medium, compared with control explants, and HA alone had no effect. Hyaluronate had no effect on MPA-induced PG degradation and release into media. Neither MPA alone nor HA alone had an effect on total cartilage GAG content. Methylprednisolone acetate caused an increase in release of GAG into the medium at 48 and 72 hours after treatment. In combination, HA had no protective effect on MPA-induced GAG release into the medium. Total cartilage DNA content was not affected by treatments. Our results indicate that HA addition has little effect on corticosteroid-induced cartilage matrix PG catabolism in articular cartilage explants.

Gene expression and matrix proteinase activity in osteochondrosis dessicans

Introduction Osteochondrosis dessicans (OCD) is a disorder of unknown aetiology where often a fragment of cartilage and subchondral bone separates from the articular surface. Previous studies have shown histological changes in glycosaminoglycan content in OCD cartilage compared to normal cartilage (Koch et al. 1997). It has also been shown in equine OCD cartilage that there is excessive degradation of type‐II collagen compared to normal cartilage (Laverty et al. 2002). The present study was undertaken to examine the gene expression in human OCD cartilage compared to its normal autologous articular cartilage and human osteoarthritic (OA) cartilage.Methods Cartilage from five OCD patients (18–34 years) was obtained at the time of surgery. Pieces of cartilage were either snap‐frozen (in preparation for RNA isolation) or the proteoglycans extracted with 4 m GuHCl. Total RNA was isolated from the cartilage using RNeasy minicolumns and reagents (Qiagen) according to the manufacturer's protocol. RT‐PCR was performed using an RNA PCR kit (Perkin‐Elmer) using a number of oligonucleotide primers. GuHCl‐extracted proteoglycan fragments were analysed using Western blotting with a number of antibodies to aggrecan metabolites, collagen metabolites and the small leucine‐rich proteoglycans.Results and discussion When OCD cartilage was compared to normal and human OA cartilage, there was an increase in aggrecan, collagen type‐II and collagen type‐X RNA expression. There was no change in RNA expression of link protein or type‐I collagen. The RNA expression of the aggrecanases (ADAMTS enzymes) was also different in the three different cartilage samples. Neither ADAMTS‐1, ‐4 or ‐5 was present in the normal cartilage. In contrast, in the OCD cartilage, there was expression of both ADAMTS‐1 and ‐4, whereas in the OA cartilage, there was expression of ADAMTS‐4 and ‐5. In the case of MMP RNA expression, MMP‐3 was decreased and MMP‐13 increased in OCD cartilage compared to both normal and OA samples. In addition, the expression of all three TIMP isoforms was increased in the OCD cartilage. Although inflammatory components are not expected in OCD pathology, expressions of inflammatory mediators such as COX‐2, IL‐1‐α and TNF‐α were all increased in the OCD cartilage when compared to normal, but expression of these mRNAs in the OA cartilage was higher. Analysis of proteoglycan fragments in the OCD cartilage by Western blotting showed the presence of aggrecan fragments containing the G1 domain, interglobular domain and the C‐terminal neoepitope generated by aggrecanase cleavage. There was also immunoreactivity for biglycan and link protein.Conclusion These results suggest that the phenotypic expression of chondrocytes at the site of the OCD lesion are markedly different from ‘normal’ articular cartilage and also pathological OA cartilage. Interestingly, the expression patterns of matrix proteinases and their natural inhibitors were also markedly different in OCD cartilage, again suggesting that there are specific biochemical expression patterns in OCD pathology, which may potentially be biomarkers of the disease process. Further studies are necessary to elucidate how the differences in gene expression and matrix protease activity may be involved in the aetiology of OCD.

DGEMRIC (delayed gadolinium-enhanced MRI of cartilage) indicates adaptive capacity of human knee cartilage.

Delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) is a new imaging technique to estimate joint cartilage glycosaminoglycan content by T1-relaxation time measurements after penetration of the hydrophilic contrast agent Gd-DTPA(2-). This study compares dGEMRIC in age-matched healthy volunteers with different levels of physical activity: Group 1 (n = 12): nonexercising individuals; Group 2 (n = 16): individuals with physical exercise averaging twice weekly; Group 3 (n = 9): male elite runners. dGEMRIC was performed 2 hr after an intravenous injection of Gd-DTPA(2-) at 0.3 mmol/kg body weight. T1 differed significantly between the three different levels of physical exercise. T1 values (mean of medial and lateral femoral cartilage) for Groups 1, 2, and 3 were: 382 +/- 33, 424 +/- 22 and 476 +/- 36, respectively (ms, mean +/- SD) (P = 0.0004, 1 vs. 2 and 0.0002, 2 vs. 3). Irrespective of the exercise level, T1 was longer in lateral compared to medial femoral cartilage (P = 0.00005; n = 37). In conclusion, this cross-sectional study indicates that human knee cartilage adapts to exercise by increasing the glycosaminoglycan content. Furthermore, results suggest a compartmental difference within the knee with a higher glycosaminoglycan content in lateral compared to medial femoral cartilage. A higher proportion of extracellular water, i.e., larger distribution volume, may to some extent explain the high T1 in the elite runners.

Ultrasound indentation of bovine knee articular cartilage in situ

We have earlier developed a handheld ultrasound indentation instrument for the diagnosis of articular cartilage degeneration. In ultrasound indentation, cartilage is compressed with the ultrasound transducer. Tissue thickness and deformation are calculated from the A-mode ultrasound signal and the stress applied is registered with the strain gauges . In this study, the applicability of the ultrasound indentation instrument to quantify site-dependent variation in the mechano-acoustic properties of bovine knee cartilage was investigated. Osteochondral blocks ( n=6 per site) were prepared from the femoral medial condyle (FMC), the lateral facet of the patello-femoral groove (LPG) and the medial tibial plateau (MTP). Cartilage stiffness (dynamic modulus, E dyn), as obtained with the ultrasound indentation instrument in situ, correlated highly linearly ( r=0.913, p<0.01) with the values obtained using the reference material-testing device in vitro. Reproducibility (standardized coefficient of variation) of the ultrasound indentation measurements was 5.2%, 1.7% and 3.1% for E dyn, ultrasound reflection coefficient of articular surface ( R) and thickness, respectively. E dyn and R were site dependent ( p<0.05, Kruskall–Wallis H test). E dyn was significantly higher ( p<0.05, Kruskall–Wallis Post Hoc test) in LPG (mean±SD: 10.1±3.1 MPa) than in MTP (2.9±1.4 MPa). In FMC, E dyn was 4.6±1.3 MPa. R was significantly ( p<0.05) lower at MTP (2.0±0.7%) than at other sites (FMC: 4.2±0.9%; LPG: 4.4±0.8%). Cartilage glycosaminoglycan concentration, as quantified with the digital densitometry, correlated positively with E dyn ( r=0.678, p<0.01) and especially with the equilibrium Young's modulus (reference device, r=0.874, p<0.01) but it was not associated with R ( r=0.294, p=0.24). We conclude that manual measurements are reproducible and the instrument may be used for detection of cartilage quality in situ. Especially, combined measurement of thickness, E dyn and R provides valuable diagnostic information on cartilage status.

Exercise: medicine for knee cartilage?

Many of the 10–60% of the working-age population with knee pain will develop osteoarthritis (OA), a progressive joint disease with cartilage deterioration and increased disability. In knee OA, exercise decreases joint pain and improves function. Lack of human in vivo monitoring methods has made studies of influence of exercise on cartilage composition impossible. Delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) can estimate joint cartilage glycosaminoglycan (GAG) content. It is based on the principle that the negatively charged Gd-DTPA2- ions distribute in the cartilage in an inverse relationship to the GAG content. A high GAG content results in a low contrast medium distribution and a long T1 relaxation time that can be measured by magnetic resonance imaging. We used dGEMRIC in a cross-sectional study in healthy subjects with different exercising levels, and in a longitudinal exercise study in patients at risk for OA.

Effect of oral glucosamine on cartilage and meniscus in normal and chymopapain-injected knees of young rabbits.

To determine if oral glucosamine (GlcN) improves joint biology after acute damage by a protease. The effect of 8 weeks of dietary GlcN (20 or 100 mg/kg/day) on knee joint cartilage was evaluated in 2.2-kg male NZW rabbits with and without damage introduced by intraarticular injection of chymopapain (CP). Cartilage was evaluated histologically and scored according to the Mankin scale. Analyses of total hydroxyproline and glycosaminoglycan (GAG) contents and reverse transcription-polymerase chain reaction (RT-PCR) analysis of selected genes were performed. After 8 weeks, there was no effect of GlcN on the GAG content of normal cartilage. Both levels of GlcN treatment significantly increased the sulfated GAG content in the cartilage of the medial femoral condyle in damaged and contralateral knees, but did not change the collagen content. In CP-injected knees, there was still some loss of surface proteoglycan (PG) that was not completely corrected by dietary GlcN. Even after 8 weeks, levels of messenger RNA (mRNA) detected by RT-PCR showed changes indicative of damage and repair, such as elevated type II collagen mRNA, and these levels were not influenced by GlcN treatment. Meniscal GAG content was increased in the contralateral knee of rabbits receiving high-dose GlcN, but was decreased in those receiving no GlcN or low-dose GlcN. Neither diet nor treatment affected the meniscal collagen content. These results suggest that oral GlcN treatment might be useful in a situation where GlcN is limiting, such as where there is a rapid replacement of cartilage PG.

Cultured chondrocytes produce injectable tissue-engineered cartilage in hydrogel polymer.

The purpose of this study was to determine if chondrocytes cultured through several subcultures at very low plating density would produce new cartilage matrix after being reimplanted in vivo with or without a hydrogel polymer scaffold. Chondrocytes were initially plated in low-density monolayer culture, grown to confluence, and passaged four times. After each passage cells were suspended in purified porcine fibrinogen and injected into the subcutaneous space of nude mice while simultaneously polymerizing with thrombin to reach a final concentration of 40 million cells/cc. Controls were made by injecting fresh, uncultured cells with fibrin polymer and by injecting the cultured cells in saline (without polymer). All samples were harvested at 6 weeks. When injected in polymer, both fresh cells and cells that had undergone only one passage in culture produced cartilaginous nodules. Cultured cells did not produce cartilage, regardless of length of time spent in culture, when injected without polymer. Cartilage was also not recovered from samples with cells kept in culture for longer than one passage, even when provided with a polymer matrix. All samples harvested were subjected to histological analysis and assayed for total DNA, glycosaminoglycan (GAG), and type II collagen. There was histological evidence of cartilage in the groups that used fresh cells and cultured cells suspended in fibrin polymer that only underwent one passage. No other group contained areas that would be consistent with cartilage histologically. All experimental samples had a higher percent of DNA than native swine cartilage, and there was no statistical difference between the DNA content of the groups containing cultured or fresh cells in fibrin polymer. Whereas the GAG content of native cartilage was 8.39% of dry weight and fresh cells in fibrin polymer was 12.85%, cultured cells in fibrin polymer never exceded the 2.48% noted from first passage cells. In conclusion, this study demonstrates that porcine chondrocytes that have been cultured in monolayer for one passage will produce cartilage in vivo when suspended in fibrin polymer.

Effects of intra-articular injections of bufexamac suspension in healthy horses

To evaluate the effects of intra-articular (IA) injections of bufexamac in horses, focusing particularly on the effects of bufexamac on articular cartilage. 20 Standardbreds. Horses were randomly allocated into 4 groups consisting of 5 horses each, and 20, 60, or 100 mg of bufexamac or 1 ml of sterile saline (0.9% NaCl) solution (control) was injected into 1 intercarpal joint at weekly intervals for 6 treatments (days 0, 7, 14, 21, 28, and 35). Clinical signs and results of hematologic, serum biochemical, and synovial fluid (SF) analyses and radiography were used to evaluate treatment effects. On day 49, all horses were euthanatized; gross necropsy and histologic examinations of internal organs and articular tissues were performed. Glycosaminoglycan concentration of the articular cartilage was evaluated in safranin O-stained sections by use of a semiquantitative microspectrophotometric method. No systemic signs were observed. Temporary mild to moderate heat and effusion were the only clinical signs observed in a number of joints after IA injections and more often only in the 100 mg group, compared with controls. The 100 mg dose resulted in significant increases in SF WBC counts, with relative neutrophilia and SF total protein concentration 24 hours after injection (day 1). No lesions suggestive of toxic effects were detected at necropsy or on histologic examination. No changes in articular cartilage glycosaminoglycan concentration were detected. Six injections of 20, 60, or 100 mg of bufexamac at weekly intervals did not cause any untoward systemic or local effects. These data suggest that bufexamac is a safe nonsteroidal anti-inflammatory drug for IA administration in horses.

Intra-articular hyaluronic acid following knee immobilisation for 6 weeks in rabbits.

Thirty-two mature female New Zealand White rabbits were immobilised in an aluminium splint on one knee, using the contralateral non-treated knee as control. After 6 weeks the splints were removed and the rabbits allowed unrestricted movement. On a random basis, 16 rabbits were given an intra-articular injection of 5 mg in 0.5 ml of hyaluronic acid (HA) in the knee of the immobilised hindlimb at weekly intervals for 6 weeks, starting 1 week after the joint had been remobilised, for a total of six injections. In the other 16 rabbits no further intervention was conducted. At the end of the experiment the rabbits were killed, the area of degenerated joint surface of the distal femur, and water and proteoglycan content were measured, and the articular cartilage stained with haematoxylin and eosin and safranin O. Remobilisation without HA administration resulted in a significantly larger degenerated joint surface area. By the end of the experiment both remobilisation and remobilisation and intra-articular HA injections had produced a greater but non-significant water cartilage content compared to the control side. The average cartilage glycosaminoglycan content of the remobilisation and intra-articular HA injection group was significantly greater than in the remobilisation group. In conclusion, in the rabbits with one knee immobilised for 6 weeks, 6 weeks of remobilisation alone are not sufficient to recover from the moderate articular surface changes produced, and the intra-articular administration of HA may produce a morphologically and biochemically more normal cartilage. More extensive animal and human studies should be performed before the routine use of intra-articular administration of HA following musculoskeletal injuries that required immobilisation can be recommended.

Nondestructive imaging of human cartilage glycosaminoglycan concentration by MRI

Despite the compelling need mandated by the prevalence and morbidity of degenerative cartilage diseases, it is extremely difficult to study disease progression and therapeutic efficacy, either in vitro or in vivo (clinically). This is partly because no techniques have been available for nondestructively visualizing the distribution of functionally important macromolecules in living cartilage. Here we describe and validate a technique to image the glycosaminoglycan concentration ([GAG]) of human cartilage nondestructively by magnetic resonance imaging (MRI). The technique is based on the premise that the negatively charged contrast agent gadolinium diethylene triamine pentaacetic acid (Gd(DTPA)2-) will distribute in cartilage in inverse relation to the negatively charged GAG concentration. Nuclear magnetic resonance spectroscopy studies of cartilage explants demonstrated that there was an approximately linear relationship between T1 (in the presence of Gd(DTPA)2-) and [GAG] over a large range of [GAG]. Furthermore, there was a strong agreement between the [GAG] calculated from [Gd(DTPA)2-] and the actual [GAG] determined from the validated methods of calculations from [Na+] and the biochemical DMMB assay. Spatial distributions of GAG were easily observed in T1-weighted and T1-calculated MRI studies of intact human joints, with good histological correlation. Furthermore, in vivo clinical images of T1 in the presence of Gd(DTPA)2- (i.e., GAG distribution) correlated well with the validated ex vivo results after total knee replacement surgery, showing that it is feasible to monitor GAG distribution in vivo. This approach gives us the opportunity to image directly the concentration of GAG, a major and critically important macromolecule in human cartilage. Magn Reson Med 41:857–865, 1999. © 1999 Wiley-Liss, Inc.

Nondestructive imaging of human cartilage glycosaminoglycan concentration by MRI.

Despite the compelling need mandated by the prevalence and morbidity of degenerative cartilage diseases, it is extremely difficult to study disease progression and therapeutic efficacy, either in vitro or in vivo (clinically). This is partly because no techniques have been available for nondestructively visualizing the distribution of functionally important macromolecules in living cartilage. Here we describe and validate a technique to image the glycosaminoglycan concentration ([GAG]) of human cartilage nondestructively by magnetic resonance imaging (MRI). The technique is based on the premise that the negatively charged contrast agent gadolinium diethylene triamine pentaacetic acid (Gd(DTPA)2-) will distribute in cartilage in inverse relation to the negatively charged GAG concentration. Nuclear magnetic resonance spectroscopy studies of cartilage explants demonstrated that there was an approximately linear relationship between T1 (in the presence of Gd(DTPA)2-) and [GAG] over a large range of [GAG]. Furthermore, there was a strong agreement between the [GAG] calculated from [Gd(DTPA)2-] and the actual [GAG] determined from the validated methods of calculations from [Na+] and the biochemical DMMB assay. Spatial distributions of GAG were easily observed in T1-weighted and T1-calculated MRI studies of intact human joints, with good histological correlation. Furthermore, in vivo clinical images of T1 in the presence of Gd(DTPA)2- (i.e., GAG distribution) correlated well with the validated ex vivo results after total knee replacement surgery, showing that it is feasible to monitor GAG distribution in vivo. This approach gives us the opportunity to image directly the concentration of GAG, a major and critically important macromolecule in human cartilage.

Remobilization Does Not Fully Restore Immobilization Induced Articular Cartilage Atrophy

The recovery of articular cartilage from immobilization induced atrophy was studied. The right hind limbs of 29-week-old beagle dogs were immobilized for 11 weeks and then remobilized for 50 weeks. Cartilage from the immobilized knee was compared with tissue from age matched control animals. After the immobilization period, uncalcified articular cartilage glycosaminoglycan concentration was reduced by 20% to 23%, the reduction being largest (44%) in the superficial zone. The collagen fibril network showed no significant changes, but the amount of collagen crosslinks was reduced (13.5%) during immobilization. After remobilization, glycosaminoglycan concentration was restored at most sites, except for in the upper parts of uncalcified cartilage in the medial femoral and tibial condyles (9% to 17% less glycosaminoglycans than in controls). The incorporation of 35SO4 was not changed, and remobilization also did not alter the birefringence of collagen fibrils. Remobilization restored the proportion of collagen crosslinks to the control level. The changes induced by joint unloading were reversible at most sites investigated, but full restoration of articular cartilage glycosaminoglycan concentration was not obtained in all sites, even after remobilization for 50 weeks. This suggests that lengthy immobilization of a joint can cause long lasting articular cartilage proteoglycan alterations at the same time as collagen organization remains largely unchanged. Because proteoglycans exert strong influence on the biomechanical properties of cartilage, lengthy immobilization may jeopardize the well being of articular cartilage.

Ageing of equine articular cartilage: structure and composition of aggrecan and decorin.

In order to identify the pathological processes involved in the destruction of articular cartilage in arthritic diseases, it is first necessary to characterise the normal homeostasis of cartilage in a healthy joint. In particular, normal age-related changes in the biochemistry of cartilage complicate any comparisons that are made between diseased and healthy tissue. There are, however, no reports in the literature detailing the influence of ageing on the biochemistry of proteoglycans in equine articular cartilage. This study addresses the absence of such information by investigating the structure of aggrecan and decorin extracted from a wide age-range of full thickness equine tissue. The total glycosaminoglycan content of articular cartilage from the metacarpophalangeal joint remained relatively constant throughout life. In contrast, specific components such as hyaluronan increased in concentration with advancing age as did the content of a structural epitope present on keratan sulphate chains. There were also significant age-related changes in the sulphation pattern of chondroitin sulphate chains. The structure of the large aggregating proteoglycan (aggrecan) became more heterogeneous in size with increasing age and each of the subspecies of aggrecan identified in the extracts was shown to carry a hyaluronan binding region (G1) domain. All subspecies of aggrecan also expressed specific epitopes to keratan sulphate, chondroitin-4-sulphate and chondroitin-6-sulphate glycosaminoglycan chains. The structure of the small nonaggregating proteoglycan decorin and the aggrecan stabilising molecule link protein were demonstrated to be similar in size and charge to that reported for other species.

Glycosaminoglycan in articular cartilage: in vivo assessment with delayed Gd(DTPA)(2-)-enhanced MR imaging.

To investigate the feasibility of applying magnetic resonance (MR) imaging with use of an anionic compound, Gd(DTPA)2- (gadolinium diethylenetriamine-pentaacetic acid), for measuring glycosaminoglycan concentration in human cartilage in clinical studies. Penetration of Gd(DTPA)2- into cartilage was monitored through sequential T1-calculated images obtained after intraarticular (n = 2) or intravenous (n = 2) injection. T1-weighted and T1-calculated image series were then obtained in seven volunteers (nine knees) after penetration of Gd-(DTPA)2- into cartilage. If T1 was heterogeneous on Gd(DTPA)(2-)-enhanced images, images were also obtained after penetration of the cartilage with the nonionic contrast agent, gadoteridol. Gd(DTPA)2- penetrated cartilage from the articular surface after intraarticular injection and from both the articular surface and the subchondral bone after intravenous injection. The latter resulted in shorter overall penetration time. T1 values on Gd(DTPA)(2-)-enhanced images were homogeneous in four knees, but in five knees T1 differences of up to 30% were observed. These T1 differences were not seen in the presence of gadoteridol. These variations in T1 reflected about 50% variations in glycosaminoglycan. The data suggest that Gd(DTPA)(2-)-enhanced MR imaging has potential for monitoring glycosaminoglycan content of cartilage in vivo.

Effects of intravenous administration of sodium hyaluronate on carpal joints in exercising horses after arthroscopic surgery and osteochondral fragmentation

Abstract Objective To evaluate the effects of arthroscopic surgery, osteochondral fragmentation, and treatment with IV administered hyaluronate on histologic, histochemical, and biochemical measurements within the carpal joints of horses. Animals 12 clinically normal horses, 2 to 7 years of age. Procedure Horses had an osteochondral fragment created at the distal aspect of the radiocarpal bone of 1 randomly chosen middle carpal joint to simulate osteochondral fragmentation. Horses were treated with 40 mg of hyaluronate or saline solution (placebo) intravenously once a week for 3 consecutive weeks (days 13, 20, and 27 after surgery). Treadmill exercise proceeded 5 days per week beginning 15 days, and ending 72 days, after surgery. Clinical evaluations were performed at the beginning and end of the study. Synovial fluid samples were obtained aseptically from both middle carpal joints on days 0, 13, 20, 27, 34, and 72 after surgery, and total protein, inflammatory cell, hyaluronate, glycosaminoglycan, and prostaglandin E2 concentrations were measured in each sample. All horses were euthanatized on day 72. Synovial membrane and articular cartilage were obtained for histologic evaluation. Articular cartilage samples were also obtained aseptically for determining glycosaminoglycan content and chondrocyte synthetic rate for glycosaminoglycans. Results Horses treated with hyaluronate intravenously had lower lameness scores (were less lame), significantly better synovial membrane histologic scores, and significantly lower concentrations of total protein and prostaglandin E2 within synovial fluid 72 days after surgery, compared with placebo-treated horses. Treatment with intravenously administered hyaluronate had no significant effects on glycosaminoglycan content, synthetic rate or morphologic scoring in articular cartilage, or other synovial fluid measurements. Conclusion Intravenously administered hyaluronate appears to alleviate signs of lameness by interacting with synoviocytes, and by decreasing production and release of inflammatory mediators. (Am J Vet Res 1997;58:1132–1140)

Maintenance of equine articular cartilage explants in serum-free and serum-supplemented media, compared with that in a commercial supplemented medium

Abstract Objective To evaluate the effects of a commercially defined, serum-free medium additive on equine articular cartilage explants, compared with effects of serum-free and serum-supplemented media. Animals Articular cartilage from a 3-year-old, mixed breed horse euthanatized for reasons other than musculoskeletal disease or sepsis. Procedure Media were changed every 48 hours, and the glycosaminoglycan (GAG) content was determined in media collected at each time point. Glycosaminoglycan synthesis by explant chondrocytes, and residual GAG content of articular cartilage (as a measure of explant GAG loss) were determined at the end of the study (day 8). Results Articular cartilage explants in serum-free medium and the commercial supplemented medium had significantly lower GAG synthesis and GAG content than did those incubated in serum-supplemented medium. There were no significant differences in GAG synthesis and content between serum-free and commercial supplemented medium groups. When comparing medium GAG content for all treatment groups, the GAG content in serum-free medium on day 8 was significantly greater than that in commercial supplemented medium, but significant differences were not evident in percentage of release of GAG (as an indicator of GAG degradation) among all 3 treatment groups. Conclusions Commercial supplemented medium had effects on articular cartilage matrix GAG loss into medium equal to those of serum-supplemented medium (eg, both lost articular cartilage explant GAG to a similar degree). However, residual articular cartilage GAG content was higher in serum-supplemented medium, as was GAG synthesis. Commercial supplemented medium appears to either lack the proper ingredients to maintain steadystate GAG synthesis, or lacks proper concentrations of these ingredients. (Am J Vet Res 1996;57:1261–1265)

Local and remote matrix responses to chondrocyte-laden collagen scaffold implantation in extensive articular cartilage defects

Chondrocyte-laden collagen scaffolds were evaluated in extensive cartilage defects in an equine model. Arthroscopic techniques were used to implant a chondrocyte-collagen culture product in 15-mm defects in the lateral trochlear ridge of the femoropatellar joint of 12 horses. Ungrafted control defects were formed in the opposite joint. Groups of six horses were terminated at 4 and 8 months after implantation and the repair sites, adjacent cartilage, and remote cartilage within each femoropatellar joint examined biochemically. Eight months following surgery the relative proportions of type II collagen in grafted and ungrafted defects, determined using the ratio of cyanogen bromide cleavage products alpha 1(II)CB10/alpha 2(I)CB3,5, were not significantly different (31.57 +/- 2.76% and 26.88 +/- 2.76%, respectively). Aggrecan content was significantly improved in grafted defects (85.61 +/- 6.51 and 74.91 +/- 10.31 micrograms/mg dry weight). Cartilage surrounding grafted defects also showed improved maintenance of cartilage glycosaminoglycan content. Thus, chondrocyte grafting in collagen scaffold vehicles improved the aggrecan content in extensive cartilage defects and surrounding normal cartilage. However, given the continued disparity between repair tissue and normal cartilage aggrecan content, and the low proportion of type II collagen in grafted defects, the utility of collagen scaffolds for chondrocyte grafting of large cartilage defects seems limited.