Evaluation of intramuscularly administered sodium pentosan polysulfate for treatment of experimentally induced osteoarthritis in horses

C. Wayne McIlwraith, BVSc, PhD, DSc; David D. Frisbie, DVM, PhD; Christopher E. Kawcak, DVM, PhD

ObjectiveTo assess clinical, radiographic, histologic, and biochemical effects of sodium pentosan polysulfate (NaPPS) administered IM for treatment of experimentally induced osteoarthritis in horses. Animals18 horses. ProceduresOsteoarthritis was induced arthroscopically in 1 middle carpal joint of all horses. Nine horses received NaPPS (3 mg/kg, IM) on study days 15, 22, 29, and 36. Nine control horses received the same volume of saline (0.9% NaCl) solution IM on study days 15, 22, 29, and 36. Clinical, radiographic, gross, histologic, histochemical, and biochemical findings as well as findings of synovial fluid analysis were evaluated. ResultsNo adverse treatment-related events were detected. Induced osteoarthritis caused a substantial increase in lameness, response to flexion, joint effusion, radiographic findings, synovial membrane inflammation, and articular cartilage fibrillation. Articular cartilage fibrillation was substantially reduced by NaPPS treatment, and concentrations of chondroitin sulfate 846 epitope were significantly increased in the synovial fluid of osteoarthritic and nonosteoarthritic joints of treated horses. Conclusions and Clinical RelevanceResults indicated that NaPPS has some beneficial disease-modifying effects and may be a therapeutic option for osteoarthritis in horses. (Am J Vet Res (2012;73:628633)

oint disease, specifically osteoarthritis, is one of the most prevalent and debilitating diseases affecting horses and has a notable economic impact on the equine industry.14 Various medications have been evaluated or used for treatment of horses with osteoarthritis, including NSAIDs, corticosteroids, PSGAG, and hyaluronan, and controlled studies512 have been reported. More recently, PPS has been used IM in equine joint disease, and this use has been reviewed by Little and Ghosh.13 Although NaPPS has been used in Europe for > 30 years as an antithrombotic-antilipidemic agent, its potential as a disease-modifying antiarthritic agent has been realized more recently. The backbone of PPS, which consists of repeating units of (1-4)-linked D-xylano-pyranoses, is isolated from beech-wood hemicellulose. An anabolic effect on chondrocytes has been found in sheep with osteoarthritis induced via unilateral meniscectomy.a Also, in experimental joint disease in rabbits, oral administration of calcium PPS (10 mg/ kg, q 7 d) maintained the normal articular cartilage ratio of aggrecan to dermatan sulfate (interpreted by the
Received October 27, 2010. Accepted March 1, 2011. From the Gail Holmes Equine Orthopaedic Research Center, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523. Supported by Nutramax Laboratories Incorporated. Address correspondence to Dr. McIlwraith (wayne.mcilwraith@ colostate.edu). 628

DMMB GAG IGF NaPPS PPS PSGAG SOFG TP

1,9-dimethyl-methylene blue Glycosaminoglycan Insulin-like growth factor Sodium pentosan polysulfate Pentosan polysulfate Polysulfated glycosaminoglycan Safranin O fast green Total protein

AbbreviAtions

authors as chondrocyte phenotype).14 Sodium PPS also stimulates hyaluronan synthesis by cultured synoviocytes obtained from both rheumatoid and osteoarthritic joints.15 The in vitro effects of NaPPS on hyaluronan synthesis were confirmed in a rat air pouch model of inflammation, and increased synthesis of hyaluronan was not stimulated by PSGAG.16 A number of in vivo studies reveal that PPS inhibits various processes that induce degeneration of the articular cartilage matrix. For example, PPS inhibits metalloproteinase 3.17 There is a suggestion that PPS may modulate receptor-mediated binding of cytokines.13 In sheep with experimental osteoarthritis (induced via medial meniscectomy), weekly intra-articular injections of PPS for 4 weeks improved joint function and reduced mean radiographic scores and Mankin histologic scores of articular cartilage damage in the femoral condyle.18 The simultaneous adAJVR, Vol 73, No. 5, May 2012

ministration of NaPPS and IGF-1 significantly reduced the severity of lesions in experimentally induced osteoarthritis in dogs, whereas IGF-1 alone had little effect.19 The presence of PPS appeared to decrease the amount of total and active matrix metalloproteinases in the cartilage. The authors also suggested that PPS reduced enzymatic breakdown of IGF-1binding protein or receptor, thus allowing IGF-1 to exert its influence. There are no published reports describing the use of PPS for equine joint disease, but the drug has been used in Australia. When administered to racing Thoroughbreds with chronic osteoarthritis (2 to 3 mg/kg, IM, once weekly for 4 weeks, then as required), PPS treatment improved but did not eliminate clinical signs of joint disease.13 It has also been proposed that because of the vascular effects of the drug, it could decrease the rate of subchondral bone necrosis and sclerosis.13 A comparison of the lipolytic and anticoagulative properties of heparin and PPS in Thoroughbreds revealed that although there was a lipolytic effect similar to that of heparin, including a significant increase of plasmafree fatty acids, PPS had much less of an effect on clotting function.20 A study21 in which single injections of NaPPS at doses of 0, 3, 6, and 10 mg/kg were compared revealed a dose-dependent increase in partial prothrombin time, including at the dosage presently recommended for treatment of joint problems in horses. The authors found that the increase was small and that partial prothrombin time remained increased from baseline for 24 hours. They concluded that on the basis of these findings, doses of PPS up to 3 mg/kg should not be administered to horses within 24 hours after highstress activities or when there is a risk of physical injury. Some in vitro studies have been performed in horses, including one that revealed that PPS and PSGAG stimulate proteoglycan synthesis in chondrocyte monolayer cultures in a concentration-related manner, with maximal effects at a concentration of 10 g/mL, but neither PSGAG nor PPS exerted significant effects on proteoglycan synthesis in cartilage explants.22 In another study,23 it was concluded that PPS probably had no substantial effect on gelatinase matrix metalloproteinase activity. A method for experimental induction of osteoarthritis has been used in horses for > 10 years for assessment of the pathophysiologic processes as well as controlled evaluation of the efficacy of treatments.612,24,25 The purpose of the study reported here was to evaluate the effects of NaPPS, administered IM, in experimentally induced osteoarthritis by evaluation of clinical (joint lameness, range of motion, response to flexion, and synovial effusion), radiographic, gross, macroscopic, histologic, immunologic, and biochemical outcome measures. Our hypothesis was that the outcome of horses treated with PPS would be more favorable than that of control horses. Materials and Methods Experimental design and induction of osteoarthritisThe Colorado State University Animal Care and Use Committee approved the study protocol for this experiment, which included the use of 18 healthy 2- to 5-year-old horses. Prior to inclusion in the study, horses underwent a lameness examination, and body condition, radiographs of the carpal joints, range of
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motion (flexion) of the carpal joints, and evidence of joint effusion were assessed to ensure all variables were within normal limits. Seven days after induction of osteoarthritis, horses were evaluated for degree of lameness. The horses were ranked by lameness and assigned to treatment groups, with each horse assigned alternatively by rank to receive NaPPSb or placebo treatment. All evaluators were unaware of treatment assignment. The study was terminated at day 70. As described,25 on day 0, following anesthesia and routine preparation for surgery, each horse underwent bilateral arthroscopic surgery of the middle carpal joints to ensure that there were no preexisting abnormalities. During this procedure, an osteochondral fragment was created in 1 randomly selected middle carpal joint. The fragment was created with an 8-mm curved osteotome directed perpendicular to the articular surface of the radial carpal bone at the level of the medial synovial plica. The fragment was allowed to remain adhered to the joint capsule proximally. A motorized cutting bladec was used to debride the exposed subchondral bone between the fragment and parent bone. A 15-mm-wide defect bed for the 8-mm-wide fragment was created, and the debris was not actively flushed from the joint, thereby inducing osteoarthritis. This joint was designated as the osteoarthritic joint; the sham-operated joint was used as the control joint. The arthroscopic portals were closed with 2-0 nylon suture in a simple interrupted pattern. The forelimbs were bandaged, and the horses were allowed to recover from anesthesia and surgery. All study horses received ceftiofurd (2.2 mg/ kg, IM, once) prior to surgery and phenylbutazonee (2 g, PO, q 24 h for 5 days) after surgery. Bandages were changed every 3 to 5 days and maintained until suture removal at 10 days after surgery. ExerciseHorses were housed in stalls (3.65 X 3.65 m). Beginning on day 15, horses were exercised on a high-speed treadmill 5 days each week until the end of the study. Each day, the horses were trotted (16 to 19 km/h) for 2 minutes, galloped (approx 32 km/h) for 2 minutes, and trotted again (16 to 19 km/h) for 2 minutes to simulate the strenuous exercise of race training. Treatment groupsTreatment began on day 15. The 9 treatment horses were administered NaPPS (3 mg/kg, IM) on study days 15, 22, 29, and 36. The 9 placebo horses received saline (0.9% NaCl) solution (at the same volume as their pair-ranked horse). Assessment of clinical outcomesAnimal care personnel assessed horses daily. Clinical examinations of both forelimbs were performed weekly from day 0 (baseline) throughout the study period. Lameness was graded on a standardized scale of 0 to 5.26 All other clinical outcomes were graded on a scale of 0 to 4 (0 represented normal, and 4 represented severe change). As an indication of joint pain, carpal flexion was performed after lameness grading. Joint effusion was also graded after carpal flexion. All clinical outcome variables were assessed by a board-certified large animal surgeon who focuses on equine lameness. Radiographic evaluationRadiographic evaluation of both carpi was performed prior to inclusion in
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the study (day 7), following the induction of osteoarthritis (day 14), and at termination of the study (day 70). A board-certified radiologist assessed the images. The radiographic images were evaluated categorically for bony proliferation at the joint capsule attachment, subchondral bone lysis, and osteophyte formation on a scale of 0 to 4 (0 represented normal, and 4 represented severe change). Synovial fluidColor, clarity, TP concentration, and WBC count were determined for synovial fluid in triplicate samples via routine methods. The concentration of hyaluronate in synovial fluid was determined in triplicate samples via a modified Alcian blue technique.22 Glycosaminoglycan concentration in synovial fluid was determined in duplicate samples with a modified DMMB dyebinding assay.23 The synovial fluid biomarkers chondroitin sulfate 846 epitope and CPII propeptide were assessed in synovial fluid and serum with an ELISA.f These competitive immunoassays have been validated for use in equine biological fluids in the investigators laboratory.27 Prothrombin and activated prothrombin times The prothrombin time and activated prothrombin time were measured by the clinical pathology laboratory, which used routine techniques. Blood samples were collected for analysis 7 days after each injection of NaPPS. Gross evaluation of jointsAt the end of the study, all horses were euthanized by administration of pentobarbital sodium. For each horse, a necropsy examination was performed during which both middle carpal joints were specifically examined for degree and location of articular cartilage fibrillation or erosion. A subjective grade (scale of 0 to 4) was assigned for partial- and full-thickness cartilage erosion as well as synovial membrane hemorrhage. A total erosion score was assigned, also with a scale of 0 to 4. For each of the 3 variables, grade 0 represented no pathological change and 4 represented a severe change. Each joint was also assessed for kissing lesions and the presence of synovial adhesions. Histologic examinationsAt necropsy, specimens of synovial membrane and joint capsule were harvested from the region dorsal to the osteochondral fragment and placed in neutral-buffered 10% formalin for H&E staining. Five-micrometer sections of the tissue samples were prepared. An evaluator who was unaware of treatment assignments assessed the sections for cellular infiltration, synovial intimal hyperplasia, subintimal edema, subintimal fibrosis, and subintimal vascularity.25 Each variable was graded on a scale of 0 to 4 (0 represented no abnormal change, and 4 represented the most severe change). Articular cartilage pieces (5 mm2) were obtained from each middle carpal joint (Figure 1). Samples were stored in neutral-buffered 10% formalin for 7 days and then processed routinely for histologic examination by an evaluator who was unaware of treatment assignment. The 5-m sections were stained with H&E and SOFG. Collection locations were chosen to represent an area directly adjacent to the osteochondral fragment, a por630

tion of the opposing articulating surface (third carpal bone), and a remote location (fourth carpal bone). The H&E-stained sections were evaluated for articular cartilage fibrillation, chondrocyte necrosis, chondrone formation (chondrocyte division within a lacuna), and focal cell loss.25 Numeric values ranging from 0 to 4 were assigned to each variable (0 represented no abnormal change, and 4 represented the most severe change). A cumulative pathology score (modified Mankin score) for each articular cartilage sample was determined by adding the values for each of the 4 assigned variables. Without knowledge of treatment assignments, articular cartilage sections stained with SOFG were evaluated for intensity of staining in the tangential, intermediate, radiate territorial, and radiate interterritorial zones of the third carpal, fourth carpal, and radial carpal bones.25 Numeric values ranging from 0 to 4 were assigned to each variable (0 indicated no stain uptake, and 4 indicated normal stain uptake), and a cumulative score for each articular cartilage specimen was calculated by summation of the zonal scores. Articular cartilage matrix evaluationTo estimate articular cartilage proteoglycan content, the total articular cartilage GAG content was measured by use of a DMMB technique.28 Articular cartilage pieces were obtained from the radial and intermediate carpal bones (Figure 1). Each piece was stored at 80C prior to processing and analysis. Samples were processed in duplicate (cartilage was digested prior to analysis [10 mg of cartilage {wet weight}/mL of papain]).

Figure 1Schematic drawing of an equine middle carpal joint (dorsal surface) indicating an area of osteochondral fragmentation (darkly shaded region) on the radial carpal bone (CR) and sample collection sites (outlined boxes). The outlined areas include the cartilage collection site for a proteoglycan synthesis assay (a), the collection site for an articular cartilage GAG content assay (b), and the collection site for histologic and histochemical analyses (c). C2 = Second carpal bone. C3 = Third carpal bone. C4 = Fourth carpal bone. CI = Intermediate carpal bone. CU = Ulnar carpal bone. (Adapted from McIlwraith CW, Wright I, Nixon AJ, et al. Chapter 4. In: Diagnostic and surgical arthroscopy in the horse. 3rd ed. Edinburgh: Mosby Elsevier, 2005;49. Reprinted with permission.) AJVR, Vol 73, No. 5, May 2012

Table 1Mean values of variables that were measured over time in 9 control horses with experimentally induced osteoarthritis and 9 horses with experimentally induced osteoarthritis that were treated with NaPPS. Mean lameness score Day 0 14 21 28 35 42 49 56 63 70 Control 0.2 1.4 1.4 1.6 1.0 1.2 1.1 1.2 1.1 1.2 NaPPS 0.2 1.1 1.1 1.7 1.2 1.1 0.9 0.9 0.6 0.9 Carpal flexion score Control 0.1 1.9 1.4 1.6 1.4 1.0 1.0 1.2 1.0 0.7 NaPPS 0.3 1.3 1.4 1.2 1.0 0.8 0.9 0.8 1.0 0.7 Effusion score Control 0.3 2.0 1.8 1.7 1.7 1.4 1.4 1.6 1.3 1.3 NaPPS 0.2 2.3 1.9 1.8 2.1 1.7 1.7 1.7 1.7 1.8 Synovial fluid TP (mg/dL) Control 2.8 3.5 3.0 2.9 3.0 2.9 2.9 2.7 2.8 2.8 NaPPS 3.1 3.1 2.9 3.1 2.8 2.9 2.8 2.9 2.8 2.6

For analysis of articular cartilage GAG synthesis, articular cartilage samples were aseptically collected from the weight-bearing surface that was remote from the osteochondral fragment in each joint (Figure 1), and incorporation of sulfur 35 radiolabeled SO4 was measured via reported methods.25 Samples were processed in duplicate. Statistical analysisData collected over time (subjective scores and synovial fluid variables) were analyzed via repeated-measures ANOVA. Scored data collected at a single time point (or at most twice) were compared for treatment effect by use of a Fisher exact test. Comparisons between osteoarthritic and nonosteoarthritic joints were performed with the Wilcoxon signed rank test. Cartilage histologic and GAG data were analyzed via ANOVA and general linear models with statistical software.g All tests were 2 sided and conducted at the 0.05 level. Values of P < 0.05 were considered significant. Results Clinical assessmentsThere was a significant difference in lameness scores between osteoarthritic and nonosteoarthritic limbs in both treated and control groups. Lameness scores peaked between days 14 and 28 of the study and had not returned to baseline by day 70 in all osteoarthritic limbs. Lameness scores in the osteoarthritic limbs at the end of the study were not significantly different between control and NaPPS-treated groups; mean SEM lameness score was 1.2 0.5 for the control group and 0.9 0.3 for the NaPPS-treated group (P = 0.451). Flexion and effusion scores were not significantly affected by NaPPS treatment (Table 1). The flexion score on day 35 in the NaPPS-treated osteoarthritic joints was (mean SEM) 1.0 0.4 and in nonosteoarthritic joints was 1.4 0.4 (P = 0.422). Mean SEM effusion scores at day 35 were 1.7 0.3 in the untreated osteoarthritic joints and 2.1 0.3 in NaPPS-treated osteoarthritic joints (Table 1). Synovial fluidTotal protein concentration in the synovial fluid was significantly increased in the osteoarthritic joints in both groups, but was not different between treatment groups (Table 1). Mean SEM TP concentration was 3.0 0.3 g/dL in the control osteoAJVR, Vol 73, No. 5, May 2012

Figure 2Photomicrographs of articular cartilage from a placebotreated middle carpal joint in a horse with experimentally induced osteoarthritis, compared with an NAPPS-treated middle carpal joint in a horse with experimentally induced osteoarthritis. H&E stain; bar = 100 m.

arthritic joints and 2.8 0.01 g/dL in NaPPS-treated osteoarthritic joints at day 35 (P = 0.642) and 3.0 0.1 g/dL in the control group and 2.6 0.1 g/dL in the control nonosteoarthritic joints at day 70 (P = 0.642). Synovial fluid cytology variables were similarly unaffected by NaPPS treatment, and prothrombin time and activated prothrombin time were unaffected by NaPPS treatment. The original analysis revealed that there were higher synovial fluid concentrations of CPII in osteoarthritic joints, compared with nonosteoarthritic joints, although this difference was not significant (P = 0.056). Residual plots of this data suggested a transformation of the data should be performed. Following natural logarithmic transformation of the mean synovial fluid CPII concentrations, no significant difference was detected between osteoarthritic and nonosteoarthritic joints. Similarly, a natural logarithmic transformation was performed on synovial fluid chondroitin sulfate 846 epitope data, and analysis revealed a significantly greater mean concentration of synovial fluid chondroitin sulfate 846 epitope from the osteoarthritic versus nonosteoarthritic joints (both groups combined). The synovial fluid concentrations of chondroitin sulfate 846 epitope were significantly greater
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in osteoarthritic and nonosteoarthritic joints of PPS-treated horses, compared with those of controls. Radiographic scoresAnalyzing the categorical data in a continuous fashion revealed significantly higher enthesophyte and lysis scores in osteoarthritic versus nonosteoarthritic limbs (both groups combined), although there was no significant effect of treatment on radiographic scores in osteoarthritic limbs at day 70 between treated and control horses. Gross evaluationBoth groups of osteoarthritic joints had low to moderate total erosion scores, moderate hemorrhagic scores, low full- and partial-thickness erosion scores, low kissing lesion scores, and no synovial adhesion. When the categorical data were analyzed, significantly higher hemorrhage and erosion scores were recorded in the osteoarthritic joints, compared with the nonosteoarthritic joints, but no significant treatment effect was found. Histologic examinationThe synovial membrane variable cellular infiltration had increased frequency of low scores in the nonosteoarthritic joints, compared with that of the osteoarthritic joints, although this difference was not significant (P = 0.094). There was no difference between osteoarthritic joints in the treated group, compared with osteoarthritic joints in the untreated group. The mean score (sum of the variables, articular cartilage fibrillation, chondrocyte necrosis, chondrone formation [chondrocyte division within a lacuna], and focal cell loss [each ranging from 0 to 4]) for histologic examination of the articular cartilage in the 3 regions of the carpal joint sampled (radial carpal bone [CR], third carpal bone [C3], and fourth carpal bone [C4]) was 3.2 and 1.9 for the untreated and NaPPS-treated osteoarthritic joints, respectively (P = 0.062). There was a significant (P = 0.049) difference between osteoarthritic and nonosteoarthritic joints for this variable. The C3 component had the highest overall mean score, compared with Cr and C4 sections. As determined on the basis of the mean score for C3, there was decreased cartilage damage with NaPPS treatment, although this difference was not significant (P = 0.093). There was significantly (P = 0.022) less articular cartilage fibrillation in osteoarthritic joints from treated horses, compared with that of osteoarthritic joints from untreated horses (Figure 2). There was less chondrone formation, although this difference was not significant (P = 0.071). Safranin Ofast green scores were not different between treatment groups or between osteoarthritic and nonosteoarthritic joints. Articular cartilage matrix evaluationThe GAG concentrations determined by means of the DMMB analytic technique and GAG synthesis measured by means of sulfur 35 radiolabeled SO4 were not different between osteoarthritic and nonosteoarthritic joints or treatment groups. Discussion Biomarkers were used in this study, and synovial fluid markers of osteoarthritis, specifically type II col632

lagen degradation (CPII propeptide) and aggrecan synthesis (chondroitin sulfate 846 epitope), were significantly different between the osteoarthritic and nonosteoarthritic joints but did not change with treatment (data not presented). Also, unlike previous reports, GAG concentrations evaluated both histologically and biochemically (SOFG staining, DMMB assay, or Alcian blue methodology) were not different between osteoarthritic and nonosteoarthritic joints. Treatment with NaPPS resulted in significantly less articular cartilage fibrillation and overall, albeit nonsignificant, improvement in cartilage histologic scores. Several variables (lameness, joint flexion, synovial fluid TP concentration, and chondroitin sulfate 846 epitope concentration) improved, but not significantly. Treatment with NaPPS significantly increased synovial fluid concentrations of chondroitin sulfate 846 epitope. Historically, increases in serum and synovial fluid chondroitin sulfate 846 epitope have been interpreted as a reparative response in horses, and this would also be supported by an increase in synovial fluid chondroitin sulfate 846 epitope concentrations in osteoarthritic versus nonosteoarthritic joints of untreated animals in the present study. The fact that the synovial fluid from both the osteoarthritic and nonosteoarthritic joints of the NaPPS-treated horses had higher chondroitin sulfate 846 epitope concentrations potentially suggests a systemic upregulation of aggrecan synthesis, rather than a simple response to pathological change. Pentosan polysulfate modulates proteolytic enzymes in vitro and protects cartilage from proteoglycan loss from the extracellular matrix.13 This anticatabolic effect, combined with the antithrombotic effect, may result in improved cellular nutrition and matrix homeostasis. Hence, NaPPS might be classified as a disease-modifying osteoarthritis drug and might have usefulness in slowing the progression of osteoarthritis. This classification would be supported by the significant decrease in articular cartilage fibrillation with NAPPS treatment observed in the present study. Clinically, NaPPS has been used to treat horses with mild or early-stage osteoarthritis, particularly with multiple joint involvement, because it is a systemic treatment rather than an intra-articular one. The osteochondral fragment model used in the present study creates mild osteoarthritis and should be ideal to evaluate the efficacy of NaPPS. It is possible that the dosage administered (3 mg/kg, once weekly for 4 weeks) was minimally effective and too low to elicit a therapeutic response in all outcome variables in this model. Alternatively, the treatment effect of NaPPS may have been beyond detection with the experimental design (n = 9/group). In any event, most of the variables evaluated in the present study were different between osteoarthritic and nonosteoarthritic joints, and therefore the technique performed as reported.8,9,24,29 The authors believe that there was evidence that NaPPS had a beneficial therapeutic effect on many variables, but differences were only significant for articular cartilage. Because NaPPS resulted in significant improvement in reducing articular cartilage fibrillation, nearsignificant improvement in other variables was found, and no adverse effects were detected, the continued
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study of this therapeutic agent is justified. It is particularly notable that there was significant reduction of articular cartilage fibrillation with treatment with NaPPS, confirming that NaPPS has disease-modifying properties. Also, Little and Ghosh13 suggested that rather than a reduction in signs of lameness, the main effects of the drug were disease modifying. In addition, improvement with this treatment compares favorably with that in an experiment with an injectable PSGAGh administered IM.10 In that study, there was no improvement in any outcome variable except for serum GAG concentrations, which were lower at 1 time point in the PSGAG group, compared with the placebo group.
a. Little C, Ghosh P, Bellinger C. Meniscectomy increases aggrecan, decorin (PGS2) and biglycan (PG-S1) metabolism in cartilage (abstr), in Proceedings. 39th Annu Meet Orthop Res Soc 1993;18:707. AUPEN5000, Nutramax Laboratories Inc, Edgewood, Md. Arthroburr, Richard Wolf Medical Instruments Corp, Vernon Hills, Ill. Naxcel, Pfizer Inc, New York, NY. Equi-Phar, Schering-Plough Animal Health Corp, Union, NJ. Ibex Diagnostics, Montreal, QC, Canada. PROC GLM, SAS, version 9.1, SAS Institute Inc, Cary, NC. Adequan, Novartis Animal Health US Inc, Greensboro, NC.

b. c. d. e. f. g. h.

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