International Cartilage Repair Society

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1 OsteoArthritis and Cartilage (2004) 12, 818e825 ª 2004 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved. doi: /j.joca The potential and limitations of cartilage-specific (VDC) K fibronectin and cartilage oligomeric matrix protein as osteoarthritis biomarkers in canine synovial fluid 1 Michele A. Steffey D.V.M.y, Naoki Miura D.V.M., Ph.D.z 2, Rory J. Todhunter B.V.Sc., Ph.D.y, Stephanie G. Nykamp D.V.M.y 3, Kathleen P. Freeman D.V.M., Ph.D.x, Virginia Scarpino M.A.z, Margaret A. Vernier-Singer B.S.y, Hollis N. Erb D.V.M., Ph.D.k, James N. MacLeod V.M.D., Ph.D.z 2, George Lust Ph.D.z and Nancy Burton-Wurster Ph.D.z* y Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA z Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA x Idexx Laboratories, Wetherby, West Yorkshire, UK k Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA Summary International Cartilage Repair Society Objective: To determine if levels of the cartilage-specific (V C C) ÿ fibronectin isoform in the synovial fluid is associated with cartilage change during osteoarthritis. Design: Synovial fluid was collected from 26 healthy dogs presenting to the Orthopedic Surgery Clinic with unilateral cranial cruciate rupture, 22 control dogs, and 13 dogs from a colony maintained for the study of canine hip dysplasia. Total fibronectin, (V C C) ÿ fibronectin, and cartilage oligomeric matrix protein (COMP) were quantitated by ELISA assays. Statistical analysis used Wilcoxon s signed-rank and rank-sum tests and Spearman s rank correlation. Results: The concentration of total fibronectin was increased in affected (P! ) and contralateral (P Z ) knees of the clinic population (compared to unaffected knees in colony controls). Both (V C C) ÿ fibronectin and COMP concentrations were elevated in the contralateral knees in clinical patients relative to unaffected knees in the colony controls (P Z 0.03 and P Z 0.04, respectively), and relative to the affected knees (P Z 0.003); however, corrections for joint effusions suggest elevated totals in the affected knees. (V C C) ÿ fibronectin and COMP concentrations were correlated (r sp Z 0.74; P! ) in 30 unaffected knees of patients and colony controls. Total fibronectin was correlated negatively with months since the initial injury (r sp Z ÿ0.44; P Z 0.03) in the affected joints. The intraoperative lesion severity score did not correlate with total fibronectin or (V C C) ÿ fibronectin (P R 0.35). Conclusions: Concentration of total fibronectin in synovial fluid might be a useful biomarker for cross-sectional studies in osteoarthritis, but only (V C C) ÿ fibronectin provides information specifically about cartilage damage. Elevated concentrations of (V C C) ÿ fibronectin and COMP seen in the contralateral knees of patients with cranial cruciate rupture might indicate cartilage changes early in the disease process ( pre-clinical). However, the wide range of values obtained limits the diagnostic value for any one individual. Joint effusions obscure the total amount of biomarkers in affected synovial joints. ª 2004 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved. Key words: (VC C) ÿ fibronectin, COMP, Synovial fluid, Biomarker, Osteoarthritis. 1 Supported by a grant from the Arthritis Foundation, and by the Dean s Fund for Clinical Excellence, College of Veterinary Medicine, Cornell University. 2 Current address: University of Kentucky, Department of Veterinary Science, 108 Gluck Equine Research Center, Lexington, KY , USA. 3 Current address: University of Guelph, Guelph, ON NIG 2W1, Canada. * Address correspondence and reprint requests to: Dr Nancy Burton-Wurster, Ph.D., Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA. Tel: ; Fax: ; niw1@cornell.edu Received 3 March 2004; revision accepted 7 July Introduction Osteoarthritis is a common and major cause of pain and disability in human and animal patients. Currently there is a lack of routine diagnostic methods to detect early osteoarthritis before radiographic changes have occurred. Non- or minimally-invasive ways to identify individuals in the early stages of osteoarthritis, to follow its progression or determine the severity of disease, and to test the efficacy of various treatment regimens are needed in both human and veterinary medicine. In addition to imaging modalities, products of cartilage matrix degradation or turnover which appear in the blood, urine or synovial fluid have received much attention since they could serve as biomarkers for osteoarthritis. However, while there are differences 818

2 Osteoarthritis and Cartilage Vol. 12, No. 10 between average values of these markers between normal and osteoarthritic populations, the variation among individuals has been large. The extensive overlap of values between affected and unaffected patients limits their use as a diagnostic tool for any one individual. Existing biomarkers have shown more promise in longitudinal studies, especially when synovial fluid is available 1. Another approach is to use combinations of markers, and this does offer more discrimination 2. One of the more promising markers to date in both longitudinal and cross-sectional studies is cartilage oligomeric matrix protein (COMP), a pentameric matrix protein in the thrombospondin family. COMP is located preferentially in the territorial matrix, but also in the interterritorial matrix of articular cartilage in adults. Its function is not known, but it may have a role in chondrogenesis and in the interaction of the chondrocyte with its surrounding matrix 3,4. Overall significant increases in synovial fluid and serum COMP have been reported in human and canine populations with osteoarthritis 5e7, but variation among individuals remains high. Furthermore, although COMP is found predominantly in cartilage, it is also present in ligament, tendon, meniscus, and synovium 3,7e9. Furthermore, it also has been found in mouse skeletal muscle, heart and placenta 10. As such, its elevation in the blood cannot be considered specific for articular cartilage degeneration. Additional and better biomarkers still need to be found. Fibronectins are important extracellular molecules through which cells interact with their surrounding matrix. The (V C C) ÿ fibronectin isoform, which lacks the V, III-15 and I-10 segments, is restricted to cartilage in mammals and constitutes from 50% to 80% of the total fibronectin present in articular cartilage 11. Preliminary studies show this isoform to be elevated 10-fold in osteoarthritic cartilage and its loss appears to be a very early and sensitive marker of chondrocyte dedifferentiation 12e14. A monoclonal antibody has been developed that makes it possible to detect this isoform without cross-recognition of other fibronectin isoforms present in blood and synovial fluid 13. Since (V C C) ÿ fibronectin is specific to cartilage, its presence in bodily fluids infers a cartilage origin 12. Thus (V C C) ÿ fibronectin could be an excellent biomarker to monitor cartilage changes during osteoarthritis progression, response to therapy, and the quality of cartilage repair. Our hypothesis is that (V C C) ÿ fibronectin in the synovial fluid will be associated with the presence of osteoarthritis. We tested this hypothesis in a canine model of spontaneous osteoarthritis consequent to hip dysplasia, and in dogs which presented at the Cornell University Hospital for Animals with rupture of the cranial (anterior) cruciate ligament (CCL). For comparison, analysis of COMP was performed on the same samples. Materials and methods COLLECTION OF BIOLOGICAL SAMPLES Clinic dogs Twenty-six otherwise healthy dogs presenting to the Orthopedic Surgery Service for surgical stabilization of naturally occurring, unilateral CCL rupture were included in this study. Clinical patients exhibiting bilateral CCL rupture, or other important orthopedic diseases (manifested by lameness, palpable musculoskeletal abnormality, or pain on joint manipulation not associated with the CCL rupture) were excluded. The left side was affected in 21 dogs. Previous therapy included non-steroidal anti-inflammatory drugs (16/26), corticosteroids (3/26) and chondroprotective agents (6/26). At presentation, dogs had been lame for 2 weeks to 2 years (median, 5 months). For each patient, lateral and cranio-caudal (antero-posterior) radiographs of both stifles were obtained preoperatively. Synovial fluid from both stifles was collected aseptically and percutaneously prior to surgical intervention in the affected stifle. The joint was held in slight flexion and stifle landmarks were palpated. A 1 and 1/2-inch, 22-gauge needle was inserted in a cranio-caudal direction at right angles to the joint at a location just lateral to the patellar tendon and midway between the patella and the most proximal aspect of the tibial tuberosity. Synovial fluid was aspirated from the joint with a 6-cc syringe. Protease inhibitors [0.3 mm benzamidene/20 mm ethylenediamine tetraacetic acid (EDTA)/ 10 mm N-ethylmaleimide/0.4 mm phenylmethylsulfonyl fluoride (PMSF), final concentrations; ph 7.0] were added to the synovial fluid at one-tenth the volume. Synovial fluid was stored at ÿ80(c until the individual assays were performed. An open arthrotomy was performed on the affected stifle and the joint was evaluated for gross evidence of osteoarthritis prior to surgical stabilization. The breeds of dogs seen as clinical patients included Labrador Retriever (5), Rottweiler (3), Airdale Terrier (1), Australian Shepherd (1), Beagle (1), German Shepherd (1), Golden Retriever (1), Siberian Husky (1), and mixed breed (12). Of the clinical patients, 12 were castrated males, and 14 were spayed females. The median age for the clinical patients was 4.5 years (range 2e10 years). Colony dogs 819 Synovial fluid was obtained at necropsy from the stifles of 22 laboratory dogs (4 Labrador RetrievereGreyhound cross-breeds and 18 beagles) that did not exhibit evidence of clinical lameness or stifle arthropathy. These animals served as normal controls for the clinic dogs with unilateral CCL rupture. The beagles were from a colony maintained for unrelated research at the Cornell College of Veterinary Medicine. The Labrador RetrievereGreyhound crosses were from a colony for the study of canine hip dysplasia and osteoarthritis maintained at the Baker Institute for Animal Health. Synovial fluid sample volume was noted, protease inhibitors were added, and samples were frozen at ÿ80(c until further analysis. Of the colony dogs, 8 were intact males, and 14 were intact females. The median age was 3 years (range 1e8 years). An additional four Labrador Retrievers and nine Labrador RetrievereGreyhound cross-breeds were examined for the presence of visible cartilage lesions on hips, shoulders or stifles at necropsy. Dogs with no visible lesions on any joint were classified as normal. Dogs with a visible cartilage lesion in a hip joint were classified as dysplastic. A joint with no cartilage lesion from a dysplastic dog or from a normal dog was classified as unaffected. Joints with cartilage lesions were classified as affected. Dogs with hip lesions often have cartilage lesions in multiple joints 15. Synovial fluid was collected, volume noted, and frozen at ÿ80(c with protease inhibitors. The age at necropsy ranged from 8 months to 11 years. The median age at necropsy was 28 months. This project was approved by the Cornell University Animal Care and Use Committee. Figure 1 depicts schematically the dog populations used in this study.

3 820 M. A. Steffey et al.: (VD C) K fibronectin and COMP in canine OA Fig. 1. Schema of dog populations used in this study. The shaded boxes correspond to the groups identified in Tables I and II. SYNOVIAL FLUID, CYTOLOGY AND HISTOLOGY An aliquot of synovial fluid was placed on gelatin-coated slides, fixed and stained as previously described 16,17. A piece of synovial membrane was excised from the craniolateral aspect of the stifle joint and fixed in 10% formalin. Microscopic examination determined the infiltration and distribution of inflammatory cells, the presence of synovial hyperplasia, and the morphology of synovial lining cells. All cytologic and histopathologic preparations were evaluated, blinded, by one author (Freeman). QUANTITATION OF FIBRONECTINS AND COMP BY ELISA Fibronectins Concentration of total fibronectin was quantitated by ELISA as described previously 18. The concentration of (V C C) ÿ fibronectin was quantitated in a sandwich ELISA using the monoclonal antibody Mab5D10 anti (V C C) ÿ as the capture antibody. The assay, as well as characterization of the antibody as specific for (V C C) ÿ fibronectin with no cross-recognition of V C/ÿ C C isoforms, has been described 13. Further modifications have increased assay sensitivity. Specifically, 72 wells of a 96-well NUNC (Rochester, NY) plate were coated with 1 mg of purified Mab5D10 IgG diluted in 10 mm TRIS, ph 9.4 and kept at 37(C for 12e18 h. Wells were then blocked with 4% non-fat dry milk in 50 mm NaHCO 3, ph 8.0. Synovial fluid was thawed and digested with bovine testicular hyaluronidase (Calbiochem, La Jolla, CA) at 1 ml of hyaluronidase per 100 ml of fluid (1 unit/ml, final concentration) at 37(C for 45 min 7. Following hyaluronidase treatment, the synovial fluid was diluted into 4% milk in 50 mm NaHCO 3, ph 8.0 and added to the wells. Plates were incubated at 37(C for 3 h. The primary antibody was rabbit anti-human fibronectin (Cappel, Durham, NC) diluted 1:1000 into 4% milk in 0.05 M phosphate buffer, ph 7.0 containing 0.15 M NaCl. The secondary antibody was peroxidase-linked mouse antirabbit IgG (Sigma, St. Louis, MO) at a 1:10,000 dilution. Detection used a Quanta-Blu Kit with fluorogenic peroxidase substrate (Pierce, Rockford, IL) and fluorescence was read on an ELISA plate reader (Tecan, Safire with Magellan software; Triangle Research Park, NC). Wells with no capture antibody and wells with no first-stage antibody served as negative controls. Cartilage oligomeric matrix protein Prior to the COMP assay, all synovial fluid samples were treated with hyaluronidase as described above with incubation at 37(C for 45 min. The concentration of COMP was quantitated by competitive inhibition ELISA with monoclonal antibody 17-C10 as previously described, with minor modifications 6,19. Purified canine COMP antigen was used as both a COMP standard and as a plate-coating antigen in the ELISA. A 96-well NUNC-Immuno plate (Rochester, NY) was coated with 50 ml of canine COMP antigen (0.3 mg/ml) in 20 mm sodium carbonate, 20 mm sodium bicarbonate, % sodium azide, ph 10, and incubated for 2 h at room temperature, and then kept at 4(C for 12e18 h. The initial dilution of monoclonal antibody 17-C10 was 1:40,000 in PBS-0.05% Tween 20 (PBS-Tween), 1% bovine serum albumin (BSA), ph 7.0. Canine COMP standards were prepared to 0.8 mg/ml, 0.6 mg/ml, 0.4 mg/ ml, 0.3 mg/ml, 0.2 mg/ml, 0.15 mg/ml, 0.1 mg/ml, and mg/ml in PBS-Tween, ph 7.0. Synovial fluids were diluted 1/25, 1/37.5, 1/50, 1/75, 1/100, 1/200, and 1/300 in the same buffer. COMP standard or unknown synovial fluid was mixed with the antibody in a NUNC-Microwell plate and maintained at 4(C for 12e18 h. The plates previously coated with canine COMP antigen were washed three times with PBS-Tween and blocked prior to use by incubating for 60 min on an orbital shaker with 100 ml of PBS, 5% BSA, ph

4 Osteoarthritis and Cartilage Vol. 12, No Blocked, antigen-coated wells were incubated with 100 ml of the monoclonal antibody-comp inhibition mixture for 2 h at room temperature. The secondary antibody was peroxidase-linked goat anti-mouse IgG (A4416, Sigma), diluted 1:5000 in PBS-Tween, 1% BSA, ph 7.0. Detection was with o-phenylenediamine plus H 2 O 2 and color was quantitated with an ELISA plate reader (EL340, Bio-Tek Instruments) using KC junior software (Bio-Tek Instruments). SCORING SYSTEMS Radiographic scores Lateral and cranio-caudal radiographs of the affected and unaffected stifles were performed on all clinical patients. All radiographs were evaluated, blinded, by one author (Nykamp) and each joint was scored separately. Each joint was evaluated for radiographic evidence of joint effusion, subchondral sclerosis, subchondral cysts, periarticular osteophytes, enthesiophytes, intra-articular calcified bodies, and periarticular calcified bodies. Each of these categories were scored with 0 Z absent, 1 Z mild, 2 Z moderate, 3 Z severe. The total of all subscores on the scoring sheet was considered the radiographic score for a given joint (range 0e21). Intraoperative scores The affected joints of the clinical patients and all joints of the colony dogs were evaluated via open arthrotomy at surgery or necropsy, respectively. Each joint was evaluated for thickening of the joint capsule, fibrillation of the articular cartilage, enthesiophytes, osteophytes associated with the patella, and osteophytes on the trochlear ridges. Each of these categories were scored with 0 Z absent, 1 Z mild, 2 Z moderate, 3 Z severe as described previously 20. The total of all subscores on the scoring sheet was considered the intraoperative score for a given joint (range 0e15). STATISTICAL ANALYSIS Clinic dogs with colony controls The Wilcoxon signed-rank test was used to make withingroup comparisons (affected vs unaffected joints in clinical patients) and the Wilcoxon rank-sum test was used to make between-group comparisons (clinic dogs vs colony dogs). Within each group, Spearman s rank test was used to detect correlations among total fibronectin, (V C C) ÿ fibronectin and COMP. For all tests P! 0.05 (two-tailed) was considered significant. Colony dogs For statistical analysis, at most two joints from a dysplastic dog, one affected and one unaffected, were allowed. From normal dogs, only one joint, which was unaffected, was allowed. Since synovial fluid samples from multiple joints in the same dog had been collected, selection of the joint for analysis was made following a predetermined sequence of rules. First, from among affected joints, the one with the highest necropsy score based on size of lesion and, if a hip joint, synovial fluid volumes O0.3 ml and round ligament volumes O0.9 ml were selected. Then, if multiple affected or unaffected joints per dog still remained, the highest total fibronectin concentration was the final selection criterion. This yielded three groups: (1) affected joint, dysplastic dog, (2) unaffected joint, dysplastic dog, and (3) unaffected joint, normal dog, with any single dog represented no more than once in each group. Wilcoxon s rank-sum test was used to test for differences between unaffected joints in normal dogs and either unaffected or affected joints in dysplastic (osteoarthritic) dogs. Wilcoxon s signed-rank test was used for paired comparisons between affected and unaffected joints in the dysplastic (osteoarthritic) dogs. Within each group, Spearman s rank correlation test was used to detect correlations among total fibronectin, (V C C) ÿ fibronectin, and COMP. For all tests, P! 0.05 (two-tailed) was considered significant. Results TOTAL FIBRONECTIN The concentration of total fibronectin was increased significantly in synovial fluid from both affected (P! ) and contralateral, clinically unaffected (P Z ) knees of the clinic population with CCL deficiency relative to unaffected knees of colony control dogs (Table I). The concentration of total fibronectin seemed higher by inspection in the synovial fluid in those joints which had cartilage lesions concomitant with canine hip dysplasia but this increase was not statistically significant (P Z 0.08) (Table II). Table I Fibronectin, (V C C) ÿ fibronectin, COMP and fluid volume for synovial fluid from clinic patients with cranial cruciate ligament rupture Clinical patient affected stifle Clinical patient contralateral stifle Colony control dog unaffected stifle Fluid volume (ml) 1450y (110e4500)z (0e1500) (0e400) [26]x [26] [22] P Z k P! ; P Z 0.08{ Total fibronectin (mg/ml) (V C C) ÿ Fibronectin (mg/ml) (253e846) (64e392) (54e139) [26] [19] [14] P Z P! ; P Z (3.5e21) (4.4e29) (3.2e32) [26] [19] [14] P Z P Z 0.36; P Z 0.03 COMP (mg/ml) (17e49) (13e63) (19e45) [26] [16] [15] P Z P Z 0.49; P Z Synovial fluid was collected and analyzed as described in Materials and methods. ymedian. zminimumemaximum. xnumber of dogs. ktwo-tailed P compared to clinical patient unaffected stifle (signed-rank test). {Two-tailed P compared to clinical patient affected and unaffected stifles, respectively (rank-sum test).

5 822 M. A. Steffey et al.: (VD C) K fibronectin and COMP in canine OA Table II Fibronectin, (V C C) ÿ fibronectin, COMP and fluid volume for synovial fluid from colony dogs for the study of canine hip dysplasia and spontaneous osteoarthritis Dysplastic dog affected joint Dysplastic dog unaffected joint Control dog unaffected joint Fluid volume (ml) 250y (100e400)z (100e500) (100e250) [5, 9]x [5, 9] [4] Total fibronectin (mg/ml) (V C C) ÿ fibronectin (mg/ml) (58e304) (28e118) (34e107) [8, 9] [8, 9] [4] (5e28) (2e45) (2e22) [8, 9] [8, 9] [4] COMP (mg/ml) (40e58) (24e63) n.d. [3, 3] [3, 4] [1] Synovial fluid was collected and analyzed as described in Materials and methods. ymedian. zminimumemaximum. xnumber of dogs in each group for the paired and unpaired comparisons (respectively) for the dysplastic dogs, and for the unpaired comparison for the normal dog. (V C C) ÿ FIBRONECTIN AND COMP In the clinic dogs, (V C C) ÿ fibronectin and COMP concentrations were not significantly different in synovial fluid from knees with CCL deficiency compared to synovial fluid from knees of control animals. However, both (V C C) ÿ fibronectin and COMP concentrations were elevated in the synovial fluid from the contralateral knees in the dogs with CCL deficiency compared to synovial fluid from the control dogs (P Z 0.03 and P Z 0.04, respectively) and from the affected knees (P Z 0.003) (Fig. 2 and Table I). Readers concerned about a Bonferroni-like correction for the multiple tests in Table I will note that a decision would change only for the two tests with P Z 0.04 and P Z (V C C) ÿ fibronectin exhibited a wide range of values in the clinic dogs. (V C C) ÿ fibronectin and COMP concentrations were correlated significantly in synovial fluid from the control and contralateral knees (r sp Z 0.74; P! , n Z 30), but not from the control and affected knees (r sp Z 0.21, P Z 0.19, n Z 40). In the dogs from the colony with canine hip dysplasia, the concentration of the cartilage-specific (V C C) ÿ fibronectin isoform in the synovial fluid varied as much as 20-fold and was not significantly different among groups (Table II). COMP values were not significantly different between affected and unaffected joints, although the number of samples available for COMP analysis was limited. Nevertheless, COMP values and (V C C) ÿ fibronectin concentrations in the synovial fluid of the unaffected joint from the dysplastic dogs were significantly correlated (r sp Z 1.00; P! 0.01, n Z 4 dogs). CORRELATIONS WITH CLINICAL MEASURES The radiographic scores in the CCL deficient knees were significantly greater (P! ) than in the contralateral knees (Fig. 3). The intraoperative scores ranged widely from 3 to 15 (median Z 7) and were not correlated with the radiographic score (r sp Z 0.38, P Z 0.07) or with either the concentration of total fibronectin or with (V C C) ÿ Fig. 2. COMP and (V C C) ÿ fibronectin in synovial fluid in response to CCL deficiency. Synovial fluid samples were collected and analyzed for COMP (A) and for (V C C) ÿ fibronectin (B) as described in Materials and methods. Although the data show considerable overlap among individuals (as can be visualized in these box and whisker plots, indicating medians, 25th and 75th percentiles) both COMP and (V C C) ÿ fibronectin concentrations were significantly elevated in synovial fluid from the contralateral knees relative to affected knees (P Z 0.003) and to knees from control dogs (P Z 0.04 and P Z 0.03, respectively). * indicates possible outliers. fibronectin concentrations in the synovial fluid (both P O 0.35). Total fibronectin concentration, but not (V C C) ÿ fibronectin concentration, was correlated negatively with months since the initial injury (r sp Z ÿ0.44; P Z 0.03). Microscopic examination of synovial membrane and exfoliated synovial lining cells confirmed slight to marked, focally extensive synovial hyperplasia and hypertrophy, infiltration of lymphocytes, neutrophils and eosinophils, and other evidence of active and chronic inflammation. Therefore, it is not surprising that the affected stifle in the clinical patients was marked by joint effusions and the synovial fluid volume was significantly elevated compared to synovial fluid volume from knees of control dogs (P! ). Some of the contralateral stifles had elevated synovial fluid volumes as well (Table I). Multiplication of fibronectin and COMP values in Table I by total synovial fluid volume reveal that total amounts of these proteins in the affected joint were greater than in the control and contralateral joints. Discussion The concentration of total fibronectin is increased in cartilage of osteoarthritic hip joints secondary to hip

6 Osteoarthritis and Cartilage Vol. 12, No Fig. 3. Radiographic scores for clinic patients. Lateral and craniocaudal radiographs were performed on all clinical patients and scored as described in Materials and methods. Scores were higher in the affected stifles in almost all cases; nevertheless, the highest score was less than 8 out of a possible total of 21. dysplasia in dogs 18. We recently reported that the (V C C) ÿ fibronectin isoform also is elevated in lesioned cartilage of osteoarthritic hip joints 13. Thus, it was our hypothesis that the appearance of (V C C) ÿ fibronectin in the synovial fluid would be a biomarker for the development of cartilage lesions. There is precedent for the use of fibronectin isoforms to serve as a biomarker for the rheumatic diseases. Bases encoding two type III homologous protein segments named Extra Domain-A (ED-A) and Extra Domain-B (ED-B) can be spliced in or out in their entirety. The correlation of the concentration of ED-AC fibronectin in the synovial fluid with the progression of joint destruction in rheumatoid arthritis was reported by Shiozawa et al. 21. ED- BC fibronectin levels in plasma were reported to be elevated in patients with spondyloarthropathy and might be useful in distinguishing this group of patients from those with rheumatoid arthritis 22. The elevation of the concentration of total fibronectin in synovial fluid from the affected joints of the clinic patients was not surprising because an increase in the concentration of total fibronectin was observed previously in dogs with osteoarthritis secondary to canine hip dysplasia 18. Elevation in the concentration of total fibronectin also has been reported in synovial fluid of human patients with osteoarthritis 23. Failure to reach significance for the increased concentration of total fibronectin in synovial fluid in the hip dysplasia dogs compared to control dogs in this study was probably due to limited numbers. It is interesting to us, however, that the concentration of total fibronectin was correlated inversely with the duration of clinical signs in the dogs with knee osteoarthritis, and that a significant elevation also was found in the contralateral knee without clinically detectable laxity. This suggests that total fibronectin in synovial fluid is a marker of an early repair phase post-injury, i.e., an anabolic marker. The canine clinical patients in our study showed signs of lameness for a median of 5 months. The radiographic scores all were less than 8 out of a possible total score of 21, with a median score of 4. Similarly, the median intraoperative score was 7 out of a possible 15. These numbers confirm that a mild to moderate form of the disease, and not end-stage osteoarthritis, was characteristic of this canine clinical population. Of clinical interest was the significant elevation of (V C C) ÿ fibronectin and COMP concentration noted in the contralateral knee of the clinically affected dogs. In these knees, (V C C) ÿ fibronectin and COMP concentrations also were significantly correlated. These contralateral knees were not yet clinically affected (as judged on physical examination), but three had mild radiographic evidence of osteoarthritis. The median radiographic score for these contralateral knees was 0, but ranged as high as 3. The significant elevation of (V C C) ÿ fibronectin and COMP concentration in these knees suggests that the contralateral knees are not normal on a population basis; however, they were not unstable as detected by the anterior cruciate drawer test which evaluates cranialecaudal (anterioreposterior) instability. The pathogenesis of CCL rupture in the canine knee has been the subject of debate. Does the ligament rupture precede the development of osteoarthritis? Or is there prior osteoarthritis that subsequently results in ligamentous degeneration and secondary rupture? Indeed, it is known that knees contralateral to the osteoarthritic knee are at higher-than-usual risk for CCL tears 24. Our data in the contralateral knee suggest that, prior to palpable effusion, and prior to any evidence of ligament rupture, (V C C) ÿ fibronectin and COMP concentration might be a marker of early articular cartilage injury and degeneration. Their elevation supports the concept of an anabolic response of the chondrocyte in the early phase of osteoarthritis 25,26. However, longitudinal studies would be required to prove that this early anabolic phase would lead invariably to a subsequent catabolic phase. It is perhaps surprising that no significant difference was found between (V C C) ÿ fibronectin concentration in synovial fluid from the affected joint of the clinical patients and from the knee joints of the colony dogs which served as normal controls. However, effusion can affect the concentration of a marker in synovial fluid. Investigators have attempted to deal with this problem in acute joint injury by normalizing to the urea concentration expected in a normal joint 27. Because there is a linear relationship between the concentration of urea in serum and in synovial fluid of disease-free joints, a normalization factor can be calculated to account for the dilutional effect of lavage, if lavage is used to collect synovial fluid. Kraus et al. 27 also used this method to correct for a chymopapain-induced joint effusion. However, in joints with chronic effusions, urea concentrations may not differ from serum concentrations because urea rapidly equilibrates across the synovium. Another factor that affects the interpretation of synovial fluid concentrations of proteins is the clearance rate of synovial fluid by drainage into the lymphatics. If the clearance rate stays the same, the synovial fluid volume is irrelevant to the concentration. However, this is not likely. There are some reports of significant increases in lymphatic clearance rates in osteoarthritic cartilage in human knees and in dogs with mild synovitis 28,29. Recently, Myers et al. 30 measured clearance in dogs prior to and 16 and 32 weeks after anterior cruciate ligament transection (ACLT) and documented, on average, a three-fold increase. Prior to correction for clearance, synovial fluid GAG concentrations showed no significant increase 16 weeks after surgery (20 G 6 mg/ml vs 18 G 4 mg/ml). After correction, GAG concentration in the ACLT knee was raised to 57 G 36 mg/ ml. Since we did not measure clearance rate, we cannot be sure, but we think it likely that concentrations of (V C C) ÿ fibronectin and COMP in the synovial fluid from the affected knee are underestimated. Our results with COMP are consistent with those of Vilim et al. 31 who reported significantly lower COMP concentrations in synovial fluid from knees of human patients with osteoarthritis and rheumatoid arthritis compared to knees of normal volunteers. The authors point to the 10-fold

7 824 M. A. Steffey et al.: (VD C) K fibronectin and COMP in canine OA increases in synovial fluid volumes in the patients as a possible explanation. Dilutional effects on biomarker concentrations in effusive joints would not be a problem for useful biomarkers in serum or plasma. However, the search for combinations of serum biomarkers to assist in the early diagnosis of osteoarthritis and to monitor therapy response and progression has been difficult 32e34 especially in cross-sectional studies 35. Further, ethnicity and sex affect serum concentrations of COMP in humans 36.(VCC) ÿ fibronectin is present in plasma at concentrations that are 20-fold or more less than in synovial fluid, but are quantifiable. Work is in progress to assess the potential of (V C C) ÿ fibronectin as a plasma biomarker. In contrast to the recent report by Vilim et al. 31, COMP concentrations have been reported as elevated in the synovial fluid and serum of both human and canine patients with osteoarthritis 2,6,7. COMP generally has been considered a marker of articular cartilage catabolism, but elevated levels in synovial fluid or serum could indicate elevated synthesis and lack of retention in the matrix 35. As with other cross-sectional studies, in our study, there was much overlap in the ranges of values from control and osteoarthritic populations. Similar to the data obtained for (V C C) ÿ fibronectin in the synovial fluid, only the contralateral joint of the clinic patients showed a statistically significant increase. Because of the categorization of COMP as a catabolic biomarker, and the presumed anabolic nature of fibronectin (increased fibronectin in osteoarthritis cartilage results from both increased synthesis and increased retention 18 ) and (V C C) ÿ fibronectin, the hypothesis was tested that the combination of the two might prove better at distinguishing osteoarthritis in hip and stifle joints in a cross-sectional study than either biomarker alone. However, logistic regression modeling (data not shown) failed to demonstrate additional discriminatory power with the addition of COMP to (V C C) ÿ fibronectin concentrations and total fibronectin concentrations in synovial fluid. In fact, this failure to demonstrate additional discriminatory power is consistent with the finding that (V C C) ÿ fibronectin and COMP concentrations in synovial fluid were significantly correlated in clinical patients and in colony dogs with spontaneous osteoarthritis. The strong correlation of (V C C) ÿ fibronectin and COMP concentrations suggests that both markers measure a similar cartilage response process, whether anabolic or catabolic. In summary, the elevated concentrations of total fibronectin, (V C C) ÿ fibronectin and COMP seen in the contralateral knees of unilaterally affected dogs with CCL deficiency might be indicative of cartilage changes early in the disease process ( pre-clinical). Indeed, the contralateral knees in dogs with CCL deficiency are at high risk to become affected. However, the wide range of values obtained in these cross-sectional studies would limit the diagnostic value for any one individual. Others have also noted that the contralateral joint is not unaffected. Dahlberg et al. 37 reported elevations in COMP fragments in the contralateral knee in human patients after an unilateral knee injury. Although the synovial fluid concentrations of (V C C) ÿ fibronectin and COMP were not increased in the affected knee of dogs with CCL deficiency, the large effusions in those knees probably obscured the fact that much more (V C C) ÿ fibronectin and COMP were released into the affected joints than into the control joints. Thus, the ability to monitor cartilage changes by following the appearance of cartilage matrix components in the synovial fluid might be masked by inflammatory and other changes within the joint. Consistent with this, increases in total fibronectin concentration in synovial fluid from affected joints in both colony and clinic dogs exceeded changes in cartilage-specific (V C C) ÿ fibronectin concentration and may be derived, in part, from non-cartilage sources such as plasma and synovium. Total fibronectin should be an easyto-assay synovial fluid marker to add to biomarker panels for cross-sectional studies in osteoarthritis. (V C C) ÿ fibronectin can provide information specifically related to cartilage damage. However, this study points out that even for a cartilage-specific marker like (V C C) ÿ fibronectin, synovial fluid concentrations may not reflect the severity of cartilage degeneration during osteoarthritis. Acknowledgments The authors thank Dr Kazuhiro Misumi, Department of Veterinary Surgery, Kagoshima University, Kagoshima, Japan, for the gift of canine COMP antigen which was used in the ELISA to measure cartilage oligomeric matrix protein. The authors thank Dr Vladimir Vilim, Institute of Rheumatology, Prague, Czech Republic, for providing anti-comp monoclonal antibody 17-C10. We appreciate the assistance of Ms Dorothy Scorelle in preparation of the manuscript and Ms Alma Jo Williams for her work with the hip dysplasia colony. This work was supported by grants from the Arthritis Foundation, and Grant #RO1 AR44340 from the National Institutes of Health. References 1. 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