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Rheumatology Advance Access originally published online on November 15, 2005
Rheumatology 2006 45(4):405-413; doi:10.1093/rheumatology/kei187
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© The Author 2005. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Inhibition of COX-2 by celecoxib in the canine groove model of osteoarthritis

S. C. Mastbergen, A. C. Marijnissen, M. E. Vianen, B. Zoer, P. M. van Roermund1, J. W. Bijlsma and F. P. Lafeber

Rheumatology and Clinical Immunology, 1 Department of Orthopaedics, University Medical Center Utrecht, The Netherlands.

Correspondence to: S. Mastbergen, University Medical Center Utrecht, PO Box 85500 (F02.127), 3508 GA Utrecht, The Netherlands. E-mail: S.Mastbergen{at}azu.nl


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective. In vitro studies showed a beneficial effect of celecoxib on proteoglycan turnover and content of osteoarthritic cartilage. In the present study we evaluated whether these favourable effects of celecoxib could also be demonstrated in vivo.

Methods. In 24 Beagle dogs, osteoarthritis (OA) was induced in one knee according to the groove model. The animals were divided into three groups and received oral placebo or 100 or 200 mg celecoxib daily, starting directly after surgery. After 15 weeks joint tissue from all dogs was analysed.

Results. Induction of OA resulted in macroscopic and histological damage of cartilage, changes in cartilage proteoglycan turnover, loss of cartilage matrix proteoglycans and slight synovial inflammation, all characteristic of early OA. Surprisingly, none of the parameters was significantly changed upon celecoxib treatment. Synovial fluid prostaglandin E2 levels were dose-dependently diminished by celecoxib, demonstrating that the celecoxib had reached the joint in sufficient amounts. Using an in vitro setup, canine cartilage under degenerative conditions was favourably influenced by celecoxib, demonstrating that canine cartilage is sensitive to celecoxib.

Conclusion. The present study showed a chondroneutral effect of celecoxib on the characteristics of experimentally induced OA in vivo, in contrast to the observed beneficial effect in vitro. It could be that celecoxib had been beneficial to degenerated cartilage in vivo but that these effects were counteracted by increased loading of the affected joint and the associated progression of OA, occurring because of the well-known analgesic effects of celecoxib.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most widely prescribed drugs in the treatment of osteoarthritis (OA) [1]. The clinical efficacy of NSAIDs is primarily related to the inhibition of cyclooxygenase 2 (COX-2), whereas much of the toxicity, particularly in the gastrointestinal system, is related to COX-1 inhibition [2]. This has led to selective COX-2 inhibitors that specifically block COX-2 with similar efficacy and less toxicity [3]. These COX-2-specific drugs have been used clinically for the past few years in the treatment of (osteo)arthritis and other conditions [3, 4]. However, more recently it has appeared that selective COX-2 inhibition also has its dark side and that it might increase the risk of cardiovascular diseases [5–7]. In this respect caution and further study are needed.

Another problem with NSAIDs might be their direct (adverse) effects on cartilage. These effects cannot be studied easily in clinical trials and therefore they are generally ignored in clinical practice because (intrinsic) cartilage changes, catabolic and anabolic, are rather slow processes in OA. In addition, beneficial effects of NSAIDs on inflammation mask possible direct adverse effects on cartilage.

Direct effects of NSAIDs on cartilage may be important specifically in prolonged treatment of joint disease in which inflammation is only mild and secondary, as in OA. Data on direct effects of conventional NSAIDs on cartilage are numerous, but results are far from conclusive [8, 9]. Two frequently used NSAIDs that have been studied regarding their direct effects on cartilage are naproxen and indomethacin. Studies on the influence of naproxen show conflicting results; there are signs that naproxen suppresses cartilage proteoglycan synthesis in vitro [9, 10], but other studies show suppression of proteoglycan degradation [11, 12]. Indomethacin shows principally negative effects on the biochemical parameters of cartilage in in vitro [9, 10] and animal studies [13, 14], but there are also studies that did not find any effect of indomethacin on cartilage [15, 16].

Thus, although NSAIDs may be very useful regarding symptoms in OA, reducing pain and inflammation, they may interfere directly with the processes of cartilage degeneration and regeneration. These direct effects should be considered in addition to gastrointestinal and cardiovascular effects when prescribing NSAIDs in clinical practice.

With respect to the selective COX-2 inhibitors, data are limited. We recently showed a beneficial effect of celecoxib in normal cartilage under the influence of IL-1 and TNF-{alpha}; on normal healthy cartilage no effects were observed [17]. Findings by El Hajjaji et al. [18] showed that celecoxib in vitro was able to increase proteoglycan synthesis and to diminish proteoglycan release of OA cartilage obtained at joint replacement surgery. Recent findings by our group confirmed these data and additionally demonstrated that, in vitro, celecoxib had a favourable effect on proteoglycan synthesis, retention, release and content of both degenerated (preclinical) and (late-stage) human OA cartilage [19]. However, COX-2 has also been reported to be involved in normal physiology, including bone healing [20, 21].

The lack of in vivo data from animal models of OA urged us to study selective COX-2 inhibition in such a model. The groove model of OA [22] has features representative of human OA [23]. The model is distinctive in that the degenerative changes are progressive while synovial inflammation diminishes over time [24]. Because of this, evaluation of direct effects of medication on cartilage is less hampered by a possible anti-inflammatory effect of treatment. Additionally the model is distinctive because there is no permanent trigger causing joint damage, making the model more sensitive to treatment. A permanent trigger for joint damage, such as the joint instability used in the anterior cruciate ligament transection (ACLT) model, will counteract the possible beneficial effects of treatment. Assuming that cartilage repair is possible [25–27], the trigger, intrinsic to the cartilage damage itself, can be eliminated by treatment. Therefore, we chose the canine groove model of OA to study the effect of celecoxib on OA cartilage in vivo.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Animals
Female Beagle dogs, 24 in number, mean age 1.8 ± 0.1 yr, weighing 10–15 kg, were obtained from the animal laboratory of Utrecht University, The Netherlands. They were housed in groups of two or three dogs per pen, and were let out on a patio in large groups for at least 2 h daily. They were fed a standard diet and had water ad libitum. The Utrecht University Medical Ethical Committee for animal studies approved the study.

The groove model
After induction with nesdonal, the dogs were anaesthetized with halothane in a mixture of oxygen and nitrous oxide delivered endotracheally. Surgery was carried out through a 2–2.5 cm medial incision close to the patellar ligament in the right knee. Care was taken to prevent bleeding and soft tissue damage as much as possible. Cartilage of the lateral and medial condyle was damaged with a Kirschner wire (1.5 mm diameter) that was bent 90° 0.5 mm from the tip. This ensured that depth of the grooves was restricted to around 0.5 mm. In utmost flexion, 10 longitudinal and diagonal grooves were made on the weight-bearing parts of the femoral condyles without damaging the subchondral bone [22–24]. The latter was confirmed by histology at the end of the experiment. Macroscopic evaluation after killing of the animals showed similar groove patterns in all affected knees. The tibial plateau was left untouched. After surgery, synovium, fasciae and skin were sutured. The contralateral unoperated knee served as a control. All the animals received analgesics (buprenorphine 0.01 mg/kg) and antibiotics (amoxicyclin 400 mg/kg) during the first 3 days after surgery.

Starting 2 days after surgery, the dogs were let out daily on the patio. To ensure (intensified) mechanical loading of the affected knee, the dogs were forced to load the experimental joint intermittently by fixing the contralateral limb to the trunk for approximately 4 h per day, 3 days per week, for 15 weeks. This is less than 10% of the total loading time of the affected joint. Thus, the control limb was not weight-bearing for less than 10% of the time. We have demonstrated that the control limbs of these animals did not differ from those of animals from which the limbs were not fixed to the trunk [23].

Treatment
The dogs were divided into three groups of eight animals each. Celecoxib (Celebrex; Pfizer, USA) was given once daily orally in two doses; one group received 100 mg/day, the second group received 200 mg/day, and the third group received a placebo. Treatment was started the day after surgery. The choice of these doses was based on previous studies that focused on the plasma kinetics of celecoxib in Beagle dogs [28, 29]. The treatment compliance was 100%.

Evaluations
Severity of OA was evaluated 15 weeks after surgery. At the end of the experiment, the dogs were euthanized with an intravenous injection of euthesate (Na-pentobarbital). Both hind limbs were amputated and synovium and cartilage were collected and processed within 2 h. Procedures were carried out under laminar flow conditions.

Synovial tissue analysis
Macroscopic synovial inflammation was evaluated on digital high-resolution photographs of synovium, by two observers unaware of the source of the photographs. Severity of inflammation was graded from 0 to 2 for colour, angiogenesis and fibrillation: 0 = none, 1 = slightly, 2 = strong. The sum of these three individual scores averaged for the two observers (a maximum of 6) was used as the representative score for each joint and used for statistical analysis.

Three infrapatellar synovial tissue samples for each joint (medial, middle and lateral) were fixed in 4% phosphate-buffered formalin (pH 7.0) and embedded in paraffin. Deparaffined sections were stained with haematoxylin–eosin. Histological sections were examined separately in random order and independently by two observers unaware of the source of the synovium. To determine the degree of inflammation, the slightly modified [30] criteria described by Goldenberg and Cohen [31] was used. For assessing the overall grade, the scores of the three specimens from each knee and of the two observers were averaged (maximum of 10). This score was used as the representative score for each joint and was used for statistical analysis.

Cartilage analysis
Macroscopic cartilage damage was evaluated on digital high-resolution photographs of the tibia and femur by two observers unaware of the source of the photographs. Severity of cartilage damage of the femoral condyle was graded from 0 to 4: 0 = smooth surface, 1 = slightly fibrillated, 2 = fibrillated with shallow grooves, 3 = deep sharp grooves, 4 = deep sharp grooves with surrounding damage. Grading of cartilage damage of the tibial plateau was comparable: 0 = smooth surface, 1 = roughened, 2 = slightly fibrillated, 3 = fibrillated, 4 = damaged. Scores of the two observers were averaged (maximum of 4). This score was used as the representative score for each photograph and was used for statistical analysis.

Cartilage samples for histological and biochemical analyses were obtained from predetermined locations on the weight-bearing areas of the femoral condyles and the tibial plateau of the experimental and control joints. Locations were identically paired with the same location in the contralateral joint [23]. Cartilage was cut as thick as possible while excluding the underlying bone, and subsequently samples were cut into full-thickness cubes weighing 3–10 mg (accuracy 0.1 mg).

For histology, four samples from the tibial plateau and four from the femoral condyles from each knee were fixed in 4% phosphate-buffered formalin containing 2% sucrose (pH 7.0). Cartilage degeneration was evaluated in sections stained with safranin O–fast green iron haematoxylin by light microscopy using the slightly modified [32] criteria of Mankin [33]. Specimens were graded in random order by two observers unaware of the source of the cartilage. For assessing the overall grade, the scores of the four specimens from each knee surface and of the two observers were averaged (maximum of 11). This score of each joint surface was used as the representative score and used for statistical analysis.

For biochemical analysis, cartilage samples were cultured individually in 96-well culture plates (Nunclon®, Denmark) in 200 µl culture medium [Dulbecco's Modified Eagle Medium (DMEM) supplemented with 0.085 mM ascorbic acid, 2 mM glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin and 10% heat-inactivated Beagle serum] according to standard procedures [32]. For femoral condyles and tibial plateau, cartilage proteoglycan content, synthesis, retention and release were determined for six explants per parameter at fixed locations, with identical locations at the contralateral control joint. All six samples were handled individually. The average result of the six samples was taken as representative of that joint surface and was used for statistical analysis [23].

Proteoglycan synthesis
As a measure of proteoglycan synthesis, the rate of sulphate incorporation was determined ex vivo [34]. After 4 h of labelling with Formula, the cartilage samples were washed with cold phosphate-buffered saline and digested with papain for 2 h at 65°C. Glycosaminoglycans (GAGs) were precipitated by addition of cetylpyridium chloride (CPC), and GAGs labelled with Formula were measured by liquid scintillation analysis. Synthetic activity is expressed as nmol of sulphate incorporated per hour per gram wet weight of the cartilage (nmol/h/g).

Proteoglycan retention and release
As a measure of the retention of newly synthesized proteoglycans, the release of Formula-labelled proteoglycans in the medium was determined. After labelling, the cartilage samples were rinsed three times for 45 min in 1.5 ml culture medium and then incubated in 200 µl fresh culture medium without sulphate label for 3 days. GAGs were precipitated from the medium and were stained with Alcian blue dye solution, as described previously [34]. The Formula-labelled GAGs were measured by liquid scintillation analysis and release was normalized to the specific activity of the medium and the wet weight of the explants. The release of newly formed proteoglycans is expressed as percentage release of newly formed proteoglycans in the 3 days (% new proteoglycan release). For the total release of proteoglycans, Alcian blue staining of the medium was quantified photometrically with chondroitin sulphate (Sigma C4384) as a reference. The total amount of GAGs released (blue staining) is expressed as a percentage of the original tissue content (% GAG release).

Proteoglycan content
As a measure of proteoglycan content of the cartilage samples, the amount of GAG was determined as described previously [34]. The GAGs in the papain digest of cartilage samples were precipitated and stained with Alcian blue as described above. Values were normalized to the wet weight of the cartilage explants (mg/g).

Prostaglandin E2 determination
Prostaglandin E2 was determined in thawed (frozen at –80°C) synovial fluid that had been taken after the joints had been opened, by enzyme immunoassay (EIA; Amersham Biosciences) and expressed as pg/ml.

In vitro analyses
In addition, we performed in vitro experiments to see whether ‘human’ celecoxib is able to modify degenerative changes of canine cartilage. Canine synovial tissue, obtained from knees with experimentally induced OA, was cultured for 3 days [35]. Cell-free supernatants were added to healthy canine hip cartilage at a concentration of 50% v/v, resulting in OA-characteristic changes in proteoglycan turnover and content. These cultures were performed in the absence and presence of celecoxib (10 µM) to study whether celecoxib could reverse these effects as previously reported for human degenerated cartilage [17, 19].

Calculations and statistics
Mean values ± S.E.M. (n = 8 animals) of each joint for femoral and tibial cartilage separately are presented. Paired Student's t-test was used to compare data of the experimental and contralateral control joints within each group. The unpaired t-test was used to analyse differences between the three treatment groups (absolute values of the control and experimental joints separately and the change in the experimental compared with the control joints). P values less than 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Induction of OA in the groove model
Cartilage damage
Fifteen weeks after induction of experimental OA, the affected knees clearly showed macroscopic damage of the articular cartilage of femoral condyles in addition to the grooves that were surgically applied and still visible. No damage was found on the condylar cartilage of the control knees (for representative photographs see Fig. 1A vs B). Damage, although less pronounced, was also found on the tibial plateau of the experimental joints compared with the control joints (Fig. 1C vs D). On average, the macroscopic cartilage damage was significantly more severe in the experimental joints compared with that in the control joints (Fig. 1G, placebo; solid lines represent the femoral condyle and dashed lines represent the tibial plateau).


Figure 1
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  		FIG. 1. Macroscopic changes as a result of experimentally induced OA and celecoxib treatment. Representative macroscopic view of articular cartilage surfaces and synovium. Femoral condyles (A and B), tibial plateaus (C and D) and synovium (E and F) of the control (left) and experimental (right) joint of a placebo-treated dog. Average macroscopic score of cartilage damage and synovial inflammation are depicted in graphs (G and H). In G, closed symbols with solid lines represent macroscopic damage of femoral condyles and open symbols with dashed lines represent macroscopic damage of tibial plateaus. Mean values (n = 8, ± S.E.M.) are presented for animals receiving placebo, celecoxib 100 mg and celecoxib 200 mg. Asterisks indicate statistically significant (P<0.05) changes compared with contralateral controls as determined by paired Student's t-test. Unpaired t-test revealed no statistically significant differences between groups.

 
These macroscopic observations were confirmed by histological analysis. The average modified Mankin score of the cartilage degeneration in the experimental femoral condyles was mild but significantly higher compared with that of the contralateral control joints (Fig. 2G, placebo; solid line). The grooves were clearly visible, as depicted by a representative micrograph in Fig. 2A vs B. Although not surgically damaged, mild cartilage degradation of the experimental tibial plateau cartilage was also found in the placebo OA group when compared with the contralateral control joints (representative micrographs are given in Fig. 2C vs D). On average, histological damage of the tibial plateau was statistically significant different from that of control joints (Fig. 2G, placebo, dashed line).


Figure 2
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  		FIG. 2. Histological changes as a result of experimentally induced OA and celecoxib treatment. Representative light micrographs of condylar cartilage (A and B), tibial cartilage (C and D) and synovial tissue (E and F) from the control (left) and experimental (right) joint of a placebo-treated dog. Average scores of histologically determined cartilage damage (modified Mankin grade) for femoral condyles (closed circles, solid lines), tibial plateaus (open circles, dashed lines) and synovial inflammation (modified Goldenberg and Cohen score) are depicted in graphs (G and H). Mean values (n = 8, ±S.E.M.) are presented for animals receiving placebo, celecoxib 100 mg and celecoxib 200 mg. Asterisks indicate statistically significant (P<0.05) changes compared with contralateral controls as determined by paired Student's t-test. Unpaired t-test revealed no statistically significant differences between groups.

 
Synovial inflammation
Macroscopically, the synovial tissue showed mild signs of inflammation in the experimental joints compared with the contralateral control joints. Figure 1E and F depicts representative photographs; Fig. 1H (left) depicts the averages for the placebo group. This was confirmed by light microscopic examination of the synovial tissue (Fig. 2E vs F and H, respectively).

Proteoglycan turnover
Fifteen weeks after surgery, synthesis of proteoglycans of both femoral condyles as well as tibial plateaus was increased in the experimental joints compared with the contralateral control joints (Fig. 3A). This increased proteoglycan synthesis, characteristic of OA, is ineffective as the release of these newly formed proteoglycans was also increased in the experimental femoral condyle and tibial plateau cartilage when compared with the control joints (Fig. 3B). When expressed as a percentage of synthesis, it demonstrates that there is decreased retention of newly formed proteoglycans in the OA joints. Also, the release of the total amount of proteoglycans was enhanced as a result of the experimentally induced OA in both femoral condyle and tibial plateau cartilage (Fig. 3C). As result of the ineffective proteoglycan synthesis and enhanced proteoglycan release, the content of proteoglycans was significantly decreased in cartilage of the femoral condyles and tibial plateau (Fig. 3D). There was no statistically significant change in cartilage DNA content due to induction of OA (OA 0.12 ± 0.01 vs control 0.15 ± 0.01 mg/g and 0.13 ± 0.00 vs 0.12 ± 0.01 mg/g, for femoral condyles and tibial plateau, respectively).


Figure 3
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  		FIG. 3. Biochemical changes as a result of experimentally induced OA and celecoxib treatment. Proteoglycan synthesis rate (A), percentage release of newly formed proteoglycans (B), percentage total release of proteoglycans (C) and proteoglycan content (D) of femoral (closed symbols, solid lines) and tibial (open symbols, dashed lines) cartilage are depicted. Mean values (n = 8, ±S.E.M.) are presented for animals treated with placebo, celecoxib 100 mg and celecoxib 200 mg. Asterisks indicate statistically significant (P<0.05) changes compared with contralateral controls as determined by paired Student's t-test. Unpaired t-test revealed no statistically significant differences between groups.

 
Effect of celecoxib treatment
One day after OA induction, celecoxib treatment was started. There were no clinical signs of discomfort, such as apathy, loss of appetite or weight loss, during the study. Although all dogs were active after the surgery, the celecoxib-treated dogs (100 mg and 200 mg) seemed to walk better and more when compared with the placebo group. Although this difference in walking pattern was noted independently by three observers (S.M., M.V. and F.L.) and discussed, this was not further quantified.

Cartilage damage
In all animals treated with celecoxib, both 100 and 200 mg, macroscopic cartilage damage was not different from that of the OA placebo groups. This applied both to the femoral condyles and to the tibial plateau, and whether considered in terms of absolute values or as changes (Fig. 1G).

Histological examination of the different groups confirmed the macroscopic impression and showed similar results for the celecoxib-treated groups and the placebo group (Fig. 2G).

Synovial inflammation
Synovial inflammation in the celecoxib-treated group when compared with the OA placebo group was not different based on macroscopic evaluation (Fig. 1H) and microscopic evaluation (Fig. 2H).

Proteoglycan turnover under the influence of celecoxib
Biochemical evaluation of proteoglycan turnover and content demonstrated that celecoxib at both concentrations had not resulted in a difference in these parameters of condylar and tibial plateau cartilage (Fig. 3A–D). Also DNA content was not changed by celecoxib (data not shown).

Prostaglandin E2 levels in synovial fluid
Because of the lack of any significant difference in the celecoxib-treated groups compared with the placebo OA group, it was doubtful whether celecoxib had entered the joint in sufficient amounts. Therefore, the synovial fluid samples were analysed for prostaglandin E2. In the synovial fluid of the placebo-treated group, we found increased levels of prostaglandin E2 (Fig. 4). In the group treated with 100 mg celecoxib the levels of prostaglandin E2 were much lower, although still elevated compared with controls. In the animals treated at 200 mg, prostaglandin E2 levels were even lower and not statistically different from those in control joints. Interestingly, a decrease in synovial fluid prostaglandin E2 levels was also observed in the contralateral control joints, although the difference was not statistically significant.


Figure 4
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  		FIG. 4. Synovial fluid prostaglandin E2 levels in experimental OA with or without celecoxib treatment. Synovial fluid prostaglandin E2 levels of control and experimental joints treated with placebo (diamonds), celecoxib 100 mg (squares) or celecoxib 200 mg (triangles). Mean values (n = 8, ±S.E.M.) are presented for animals treated with placebo, celecoxib 100 mg and celecoxib 200 mg. Statistically significant differences in the effects of celecoxib compared with untreated controls, calculated by non-parametric paired analysis (P<0.05) are marked with an asterisk.

 
In vitro effect of celecoxib on canine cartilage
Because celecoxib did not demonstrate an effect on the articular cartilage, although apparently present in sufficient amounts, it was questionable whether celecoxib was able to influence canine cartilage. This was evaluated in vitro. Canine synovium tissue supernatants were used to induce degenerative changes in healthy canine cartilage in the presence or absence of celecoxib. Celecoxib was able to influence the (degenerated) cartilage beneficially (Fig. 5). The enhanced proteoglycan release was decreased to levels even below that of the control cartilage (not treated with synovium culture supernatants; Fig. 5A). Also, the proteoglycan content of canine cartilage was increased by celecoxib to levels of controls (Fig. 5B). Proteoglycan synthesis was only mildly increased, which is in accordance with the effects of celecoxib on proteoglycan synthesis in human degenerated cartilage [17, 19].


Figure 5
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  		FIG. 5. The in vitro effect of celecoxib on canine cartilage; changes in proteoglycan turnover. Percentage total release of proteoglycans (A), proteoglycan content (B) and proteoglycan synthesis rate (C) are depicted. Mean values (n = 6, ±S.E.M.) are presented for cartilage cultured with 50% v/v experimental OA synovial tissue culture supernatants in the absence or presence of celecoxib (10 µM). Dashed lines indicate levels in healthy canine cartilage cultured alone. Asterisks indicate statistically significant (P<0.05) change induced by celecoxib compared with cartilage cultured in the presence of 50% synovium culture supernatant alone, as calculated by paired Student's t-test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
To our knowledge, this study describes for the first time the in vivo effect of celecoxib on experimental OA cartilage, using the canine groove model of OA. Several recent in vitro studies showed that the selective COX-2 inhibitor celecoxib is able to improve and even restore disturbed cartilage turnover, both under inflammatory conditions [17] and under OA conditions [18, 19]. Normal healthy cartilage was not influenced by celecoxib in these studies [17, 19]. However, we could not confirm the positive in vitro results of celecoxib in our in vivo canine model of OA.

Increased synthesis of proteoglycans, decreased retention of these newly formed proteoglycans and increased release of proteoglycans combined with a decreased proteoglycan content, as found in the placebo-treated OA group, characteristic of OA, are comparable to previous studies performed with this model [23, 24] and therefore are not expected to be the cause of the absence of an effect of celecoxib.

There is evidence for polymorphism in the metabolism of celecoxib in Beagle dogs [28]. There are at least two populations of dogs, distinguished by their capacity to eliminate celecoxib from plasma at either a fast or a slow rate after intravenous administration. About half of the 242 tested Beagles were of the fast phenotype and the other half were of the slow phenotype. There was also an equal distribution of the two phenotypes within males and females. The difference between the two populations was due to a difference in the rate of metabolism of celecoxib by the liver cytochrome P-450. We were not able to check our Beagle dogs for this polymorphism. As we used animals with the same genetic background, we assumed that they are either slow or fast metabolizers. Therefore the doses of celecoxib were chosen in such a way that at one of the two doses (100 or 200 mg) an effective dose, resulting in adequate plasma levels, was reached [28, 29]. The other dose may as a consequence have been either too low or too high. Nevertheless, celecoxib treatment did not change proteoglycan turnover of the OA cartilage at either of the two doses.

Because of a lack of effect despite the doses chosen, we questioned whether celecoxib reached the joint in an adequate concentration. Therefore, prostaglandin E2 levels in the synovial fluid at the end of the study were measured. Prostaglandin E2 is one of the main products formed by COX-2 and is an indirect measure of COX-2 activity. In the synovial fluid of the placebo-treated group, we found increased levels of prostaglandin E2. This corroborates the human OA condition, where increased levels of prostaglandin E2 in different joint tissues are also found [19, 36]. More interestingly, we found in the celecoxib-treated groups, dose-dependently, much lower levels of prostaglandin E2. This indirectly proves that celecoxib had entered the joints at a concentration that was effective in inhibiting COX-2 activity. Also, the control joints of the celecoxib-treated animals showed lower prostaglandin E2 levels (although the difference was not statistically significant). This suggests that in the contralateral control joints some COX inhibition had also occurred as a result of the treatment. Nevertheless, despite functional COX(-2) inhibition in the joint, no effects on cartilage were found.

This raised the question of whether canine articular chondrocytes are sensitive to ‘human’ celecoxib. It appeared that canine cartilage under the influence of catabolic mediators released by OA synovial tissue was beneficially influenced by celecoxib. These in vitro effects of celecoxib were comparable to results obtained with human cartilage in vitro exposed to celecoxib [17–19].

Thus, although celecoxib had reached the joint and COX-2 inhibition can be beneficial for canine cartilage under degenerative conditions, in our in vivo study no effects of celecoxib were observed. A possible explanation could be found in the different walking patterns observed in the different treatment groups. The celecoxib-treated dogs (both 100 mg and 200 mg) seemed to walk better and more when compared with the placebo group. This fits well with the analgesic effects of celecoxib. It could be that celecoxib had been beneficial to degenerated cartilage in vivo but that these effects were counteracted by the increased loading of the affected joint and the associated progression of OA, because of the analgesic effects of celecoxib. The efficacy of celecoxib in reducing knee pain in patients with OA has been demonstrated in previous studies [37, 38]. Recently the effect of celecoxib vs placebo treatment was compared on clinical and gait variables in knee OA patients, focusing on the efficiency of the locomotor mechanism [39]. In this study, celecoxib treatment improved the efficiency of the locomotor mechanism significantly. Among the secondary outcome measures assessed, walking cadence was improved by celecoxib treatment and knee pain was significantly reduced.

Improved walking and loading of the animals treated with celecoxib might have resulted in undesired progression of OA in our experiments, counteracting the direct beneficial effects on cartilage. The absence of a net effect on cartilage may also explain the absence of a net effect on histochemically determined synovial inflammation, because, in our model, inflammation is very mild and depends primarily on cartilage damage [22, 23]. Increased loading of a joint is associated with OA pathology [40–43]. In vivo animal experiments indicate that impulsive loading and joint instability results in bone or cartilage changes commonly associated with OA [40, 44, 45]. Severe OA rapidly developed in dogs following anterior cruciate ligament transection combined with dorsal root ganglionectomy [44]. Rapid development of OA was attributed to the increased ground reaction forces, which were 30% higher in these dogs compared with ACLT dogs with an additional sham procedure [46], a difference that may have resulted from the impaired ability of the dogs to experience knee instability and pain. However, the effects of training on cartilage are known to be ambivalent [47, 48]. Moderate exercise seems not to increase the risk of OA, and under certain circumstances even could prevent knee OA [48].

In this perspective, it might have been better to treat the placebo group also with an analgesic. However, this is difficult because gastrointestinal side-effects of conventional NSAIDs are much more severe in dogs than in humans [49]. Furthermore, conventional NSAIDs could have had an adverse direct effect on the cartilage [8, 50]. Opioids are also no option because they will cause interference with walking/joint loading activity. Buprenorphine treatment, which was used as a pain-killer for 3 days after surgery, led to loss of appetite. Paracetamol (acetaminophen) causes toxicity in dogs after repetitive administration of therapeutic doses [51, 52]. In this respect it is remarkable that the prolonged use of celecoxib showed no signs of side-effects. The veterinary market might in this respect offer a new application of this selective COX-2 inhibitor.

All together, this study showed no beneficial effect of celecoxib on the proteoglycan turnover of experimentally induced OA. It could be that celecoxib had been beneficial to degenerated cartilage in vivo but that these effects were counteracted by the increased loading of the affected joint and the associated progression of OA, because of the analgesic effects of celecoxib. To establish whether this concept is plausible further research using, for example, gait analyses is needed.
Figure 6


   Acknowledgments
 
We thank Hans, Nico, Jannie, Hester, Aline and Maurits [Animal Facilities of UMC Utrecht (GDL)] for their support during this study. F.P.L. was supported by the Dutch Arthritis Association.

An unrestricted research grant was received from Pfizer.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Hungin AP, Kean WF. Nonsteroidal anti-inflammatory drugs: overused or underused in osteoarthritis? Am J Med 2001;110(1A):8S–11S.
  2. Lazzaroni M, Bianchi Porro G. Gastrointestinal side-effects of traditional non-steroidal anti-inflammatory drugs and new formulations. Aliment Pharmacol Ther 2004;20(Suppl. 2):48–58.
  3. Hawkey CJ. COX-2 inhibitors. Lancet 1999;353:307–14.[CrossRef][Web of Science][Medline]
  4. Fung HB, Kirschenbaum HL. Selective cyclooxygenase-2 inhibitors for the treatment of arthritis. Clin Ther 1999;21:1131–57.[CrossRef][Medline]
  5. Bombardier C, Laine L, Reicin A et al. Comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in patients with rheumatoid arthritis. VIGOR Study Group. N Engl J Med 2000;343:1520–8.[Abstract/Free Full Text]
  6. Farkouh ME, Kirshner H, Harrington RA et al. Comparison of lumiracoxib with naproxen and ibuprofen in the Therapeutic Arthritis Research and Gastrointestinal Event Trial (TARGET), cardiovascular outcomes: randomised controlled trial. Lancet 2004;364:675–84.[CrossRef][Web of Science][Medline]
  7. Schnitzer TJ, Burmester GR, Mysler E et al. Comparison of lumiracoxib with naproxen and ibuprofen in the Therapeutic Arthritis Research and Gastrointestinal Event Trial (TARGET), reduction in ulcer complications: randomised controlled trial. Lancet 2004;364:665–74.[CrossRef][Web of Science][Medline]
  8. Ding C. Do NSAIDs affect the progression of osteoarthritis? Inflammation 2002;26:139–42.[CrossRef][Web of Science][Medline]
  9. Dingle JT. The effects of NSAID on the matrix of human articular cartilages. Z Rheumatol 1999;58:125.[CrossRef][Medline]
  10. Shield MJ. Anti-inflammatory drugs and their effects on cartilage synthesis and renal function. Eur J Rheumatol Inflamm 1993;13:7–16.[Medline]
  11. Pelletier JP, Martel-Pelletier J. Effects of nimesulide and naproxen on the degradation and metalloprotease synthesis of human osteoarthritic cartilage. Drugs 1993;46(Suppl. 1):34–9.
  12. Ratcliffe A, Azzo W, Saed-Nejad F et al. In vivo effects of naproxen on composition, proteoglycan metabolism, and matrix metalloproteinase activities in canine articular cartilage. J Orthop Res 1993;11:163–71.[CrossRef][Web of Science][Medline]
  13. Gencosmanoglu BE, Eryavuz M, Dervisoglu S. Effects of some nonsteroidal anti-inflammatory drugs on articular cartilage of rats in an experimental model of osteoarthritis. Res Exp Med (Berl) 2001;200:215–26.
  14. Palmoski MJ, Brandt KD. Proteoglycan depletion, rather than fibrillation, determines the effects of salicylate and indomethacin on osteoarthritic cartilage. Arthritis Rheum 1985;28:548–53.[Medline]
  15. Brandt KD. Effects of nonsteroidal anti-inflammatory drugs on chondrocyte metabolism in vitro and in vivo. Am J Med 1987;83(5A):29–34.
  16. Hess EV, Herman JH. Cartilage metabolism and anti-inflammatory drugs in osteoarthritis. Am J Med 1986;81(5B):36–43.
  17. Mastbergen SC, Lafeber FP, Bijlsma JW. Selective COX-2 inhibition prevents proinflammatory cytokine-induced cartilage damage. Rheumatology 2002;41:801–8.[Abstract/Free Full Text]
  18. El Hajjaji H, Marcelis A, Devogelaer JP, Manicourt DH. Celecoxib has a positive effect on the overall metabolism of hyaluronan and proteoglycans in human osteoarthritic cartilage. J Rheumatol 2003;30:2444–51.[Abstract/Free Full Text]
  19. Mastbergen SC, Bijlsma JW, Lafeber FP. Selective COX-2 inhibition is favorable to human early and late-stage osteoarthritic cartilage: a human in vitro study. Osteoarthritis Cartilage 2005;13:519–26.[CrossRef][Medline]
  20. Crofford LJ, Lipsky PE, Brooks P, Abramson SB, Simon LS, van de Putte LB. Basic biology and clinical application of specific cyclooxygenase-2 inhibitors. Arthritis Rheum 2000;43:4–13.[CrossRef][Web of Science][Medline]
  21. Goodman S, Ma T, Trindade M et al. COX-2 selective NSAID decreases bone ingrowth in vivo. J Orthop Res 2002;20:1164–9.[CrossRef][Web of Science][Medline]
  22. Mastbergen SC, Marijnissen AC, Vianen ME, van Roermund PM, Bijlsma JW, Lafeber FP. The canine ‘groove’ model of osteoarthritis is more than simply the expression of surgically applied damage. Osteoarthritis Cartilage 2005, Sep 23; [Epub ahead of print].
  23. Marijnissen AC, van Roermund PM, TeKoppele JM, Bijlsma JW, Lafeber FP. The canine ‘groove’ model, compared with the ACLT model of osteoarthritis. Osteoarthritis Cartilage 2002;10:145–55.[Medline]
  24. Marijnissen AC, van Roermund PM, Verzijl N, Tekoppele JM, Bijlsma JW, Lafeber FP. Steady progression of osteoarthritic features in the canine groove model. Osteoarthritis Cartilage 2002;10:282–9.[Medline]
  25. Hunziker EB. Articular cartilage repair: basic science and clinical progress. A review of the current status and prospects. Osteoarthritis Cartilage 2002;10:432–63.[CrossRef][Web of Science][Medline]
  26. Marijnissen AC, Lafeber FP. Re: E. B. Hunziker. Articular cartilage repair: basic science and clinical progress. A review of the current status and prospects. Osteoarthritis Cartilage 2002; 10:432–63. Osteoarthritis Cartilage 2003;11:300–1; author reply 302–4.[CrossRef]
  27. Marijnissen AC, Van Roermund PM, Van Melkebeek J et al. Clinical benefit of joint distraction in the treatment of severe osteoarthritis of the ankle: proof of concept in an open prospective study and in a randomized controlled study. Arthritis Rheum 2002;46:2893–902.[CrossRef][Medline]
  28. Paulson SK, Engel L, Reitz B et al. Evidence for polymorphism in the canine metabolism of the cyclooxygenase 2 inhibitor, celecoxib. Drug Metab Dispos 1999;27:1133–42.[Abstract/Free Full Text]
  29. Paulson SK, Vaughn MB, Jessen SM et al. Pharmacokinetics of celecoxib after oral administration in dogs and humans: effect of food and site of absorption. J Pharmacol Exp Ther 2001;297:638–45.[Abstract/Free Full Text]
  30. Pelletier JP, Martel-Pelletier J, Ghandur-Mnaymneh L, Howell DS, Woessner JF Jr. Role of synovial membrane inflammation in cartilage matrix breakdown in the Pond-Nuki dog model of osteoarthritis. Arthritis Rheum 1985;28:554–61.[Web of Science][Medline]
  31. Goldenberg DL, Cohen AS. Synovial membrane histopathology in the differential diagnosis of rheumatoid arthritis, gout, pseudogout, systemic lupus erythematosus, infectious arthritis and degenerative joint disease. Medicine (Baltimore) 1978;57:239–52.[Medline]
  32. Lafeber FP, Vander Kraan PM, Huber-Bruning O, Vanden Berg WB, Bijlsma JW. Osteoarthritic human cartilage is more sensitive to transforming growth factor beta than is normal cartilage. Br J Rheumatol 1993;32:281–6.[Abstract/Free Full Text]
  33. Mankin HJ, Dorfman H, Lippiello L, Zarins A. Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. II. Correlation of morphology with biochemical and metabolic data. J Bone Joint Surg Am 1971;53:523–37.[Abstract/Free Full Text]
  34. Lafeber FP, Vander Kraan PM, Van Roy JL, Huber-Bruning O, Bijlsma JW. Articular cartilage explant culture; an appropriate in vitro system to compare osteoarthritic and normal human cartilage. Connect Tissue Res 1993;29:287–99.[Medline]
  35. Hooiveld M, Roosendaal G, Vianen M, van den Berg M, Bijlsma J, Lafeber F. Blood-induced joint damage: longterm effects in vitro and in vivo. J Rheumatol 2003;30:339–44.[Abstract/Free Full Text]
  36. Hardy MM, Seibert K, Manning PT et al. Cyclooxygenase 2-dependent prostaglandin E2 modulates cartilage proteoglycan degradation in human osteoarthritis explants. Arthritis Rheum 2002;46:1789–803.[CrossRef][Web of Science][Medline]
  37. Geba GP, Weaver AL, Polis AB, Dixon ME, Schnitzer TJ. Efficacy of rofecoxib, celecoxib, and acetaminophen in osteoarthritis of the knee: a randomized trial. JAMA 2002;287:64–71.[Abstract/Free Full Text]
  38. Goldenberg MM. Celecoxib, a selective cyclooxygenase-2 inhibitor for the treatment of rheumatoid arthritis and osteoarthritis. Clin Ther 1999;21:1497–513; discussion 1427–8.[CrossRef][Web of Science][Medline]
  39. Detrembleur C, De Nayer J, van den Hecke A. Celecoxib improves the efficiency of the locomotor mechanism in patients with knee osteoarthritis. A randomised, placebo, double-blind and cross-over trial. Osteoarthritis Cartilage 2005;13:206–10.[CrossRef][Medline]
  40. Lukoschek M, Schaffler MB, Burr DB, Boyd RD, Radin EL. Synovial membrane and cartilage changes in experimental osteoarthrosis. J Orthop Res 1988;6:475–92.[CrossRef][Web of Science][Medline]
  41. Mow V, Setton L, Guilak F, Ratcliffe A. Mechanical factors in articular cartilage and their role in osteoarthritis. In Kuettner KE and Goldberg VM, eds. Osteoarthritic disorders. Rosemont, IL: American Academy of Orthopaedic Surgeons, 1995;147–173.
  42. Pelletier JP, Martel-Pelletier J, Howell DS. Etiopathogenesis of osteoarthritis. Baltimore: Williams & Wilkins; 1997.
  43. Radin EL, Schaffler MB, Gibson G, Tashman S. Osteoarthriitis as a result of repetitive trauma. In Kuettner KE and Goldberg VM, eds. Osteoarthritic disorders. Rosemont, IL: American Academy of Orthopaedic Surgeons, 1995;197–203.
  44. O’Connor BL, Visco DM, Brandt KD, Albrecht M, O’Connor AB. Sensory nerves only temporarily protect the unstable canine knee joint from osteoarthritis. Evidence that sensory nerves reprogram the central nervous system after cruciate ligament transection. Arthritis Rheum 1993;36:1154–63.[Medline]
  45. Radin EL, Martin RB, Burr DB, Caterson B, Boyd RD, Goodwin C. Effects of mechanical loading on the tissues of the rabbit knee. J Orthop Res 1984;2:221–34.[CrossRef][Medline]
  46. O’Connor BL, Visco DM, Brandt KD, Myers SL, Kalasinski LA. Neurogenic acceleration of osteoarthrosis. The effects of previous neurectomy of the articular nerves on the development of osteoarthrosis after transection of the anterior cruciate ligament in dogs. J Bone Joint Surg Am 1992;74:367–76.[Abstract/Free Full Text]
  47. Felson DT, Zhang Y. An update on the epidemiology of knee and hip osteoarthritis with a view to prevention. Arthritis Rheum 1998;41:1343–55.[CrossRef][Web of Science][Medline]
  48. Galois L, Etienne S, Grossin L, Watrin-Pinzano A et al. Dose-response relationship for exercise on severity of experimental osteoarthritis in rats: a pilot study. Osteoarthritis Cartilage 2004;12:779–86.[CrossRef][Medline]
  49. Isaacs J. Adverse effects of non-steroidal anti-inflammatory drugs in the dog and cat. Aust Vet Pract 1996;26:180–6.
  50. Dingle JT. The effect of nonsteroidal antiinflammatory drugs on human articular cartilage glycosaminoglycan synthesis. Osteoarthritis Cartilage 1999;7:313–4.[CrossRef][Medline]
  51. Richardson J. Management of acetaminophen and ibuprofen toxicoses in dogs and cats. J Vet Emerg Crit Care 2002;July-Sept.
  52. Villar D, Buck WB, Gonzalez JM. Ibuprofen, aspirin and acetaminophen toxicosis and treatment in dogs and cats. Vet Hum Toxicol 1998;40:156–62.[Medline]
Submitted 7 July 2005; revised version accepted 5 October 2005.
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R. O. Sanderson, C. Beata, R-M. Flipo, J-P. Genevois, C. Macias, S. Tacke, A. Vezzoni, and J. F. Innes
Systematic review of the management of canine osteoarthritis
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