Effects of intra-articular 2.5% polyacrylamide hydrogel in an experimental model of equine osteoarthritis

Effects of intra-articular 2.5% polyacrylamide hydrogel in an experimental model of equine osteoarthritis | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 4 March 2026 V1 Latest version Share on Effects of intra-articular 2.5% polyacrylamide hydrogel in an experimental model of equine osteoarthritis Authors : Erin Contino 0000-0002-1756-510X [email protected] , AbuAlia M , Brad Nelson 0000-0002-0205-418X , Laurie Goodrich , Myra Barrett 0000-0001-5060-6009 , Melissa King 0000-0002-1203-3059 , Lynn Pezzanite 0000-0003-4990-5006 , Wayne McIlwraith , Wimmer MA , and Kathryn Seabaugh Authors Info & Affiliations https://doi.org/10.22541/au.177258447.71381217/v1 190 views 79 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background: Osteoarthritis (OA) is a leading cause of lameness in horses. Injectable polyacrylamide hydrogels (iPAAGs) have emerged as an intra-articular (IA) treatment option though the mechanism of action is not fully understood. Objectives: To evaluate the symptom- and disease-modifying effects of 2.5% iPAAG in an experimentally-induced model of OA in horses. Study design: In vivo experimental study. Methods: Sixteen horses had an osteochondral fragment created (d0) in one randomly selected middle carpal joint (MCJ); opposite MCJs were sham-operated. OA joints were treated (d14) with 2 mL saline (n=8) or 2.5% iPAAG (n=8). High-speed treadmill exercise commenced on d16 to induce OA. Clinical parameters (lameness, joint effusion, range of motion, flexion response) and synovial fluid were collected weekly to biweekly, respectively. Radiographs were obtained before, d14, and d70 post-surgery. Post-mortem evaluation (d70) included gross, arthroscopic and histopathologic evaluation of the MCJs and measurement of joint capsule stiffness. Results: There were no significant differences in clinical parameters nor endpoint arthroscopic, macroscopic and synovial membrane histopathology scores between treated and control groups. 2.5% iPAAG-treated OA joints had significantly lower (better) overall articular cartilage histology scores and better proteoglycan staining of the intermediate layer of the 3 rd carpal bone articular cartilage. Saline-treated OA joints had significantly increased joint capsule stiffness whereas 2.5% iPAAG-treated joints had stiffness values similar to sham-operated MCJs. Main limitations: Experimentally-induced OA does not fully mimic naturally occurring OA. Horses were followed 56 days post 2.5% iPAAG treatment, which may not have been long enough to fully elucidate treatment effects. Conclusions: Treatment with 2.5% iPAAG showed disease-modifying effects including some ability to mitigate the negative effects of OA on articular cartilage. Treatment also resulted in retention of normal joint capsule elasticity during experimental progression of OA, which likely contributes to the symptom-modifying effects that have been shown in several clinical trials. [1]¿p#1 Effects of intra-articular 2.5% polyacrylamide hydrogel in an experimental model of equine osteoarthritis Authors names and institutional affiliations xxx [1]¿p#1 ABSTRACT Background: Osteoarthritis (OA) is a leading cause of lameness in horses. Injectable polyacrylamide hydrogels (iPAAGs) have emerged as an intra-articular (IA) treatment option though the mechanism of action is not fully understood. Objectives: To evaluate the symptom- and disease-modifying effects of 2.5% iPAAG in an experimentally-induced model of OA in horses. Study design: In vivo experimental study. Methods: Sixteen horses had an osteochondral fragment created (d0) in one randomly selected middle carpal joint (MCJ); opposite MCJs were sham-operated. OA joints were treated (d14) with 2 mL saline (n=8) or 2.5% iPAAG (n=8). High-speed treadmill exercise commenced on d16 to induce OA. Clinical parameters (lameness, joint effusion, range of motion, flexion response) and synovial fluid were collected weekly to biweekly, respectively. Radiographs were obtained before, d14, and d70 post-surgery. Post-mortem evaluation (d70) included gross, arthroscopic and histopathologic evaluation of the MCJs and measurement of joint capsule stiffness. Results: There were no significant differences in clinical parameters nor endpoint arthroscopic, macroscopic and synovial membrane histopathology scores between treated and control groups. 2.5% iPAAG-treated OA joints had significantly lower (better) overall a rticular cartilage histology scores and better proteoglycan staining of the intermediate layer of the 3 rd carpal bone articular cartilage. Saline-treated OA joints had significantly increased joint capsule stiffness whereas 2.5% iPAAG-treated joints had stiffness values similar to sham-operated MCJs. Main limitations: Experimentally-induced OA does not fully mimic naturally occurring OA. Horses were followed 56 days post 2.5% iPAAG treatment, which may not have been long enough to fully elucidate treatment effects. Conclusions: Treatment with 2.5% iPAAG showed disease-modifying effects including some ability to mitigate the negative effects of OA on articular cartilage. Treatment also resulted in retention of normal joint capsule elasticity during experimental progression of OA, which likely contributes to the symptom-modifying effects that have been shown in several clinical trials. MANUSCRIPT Introduction Osteoarthritis (OA) remains a leading cause of lameness in horses 1 affecting nearly 3 million horses annually in the United States (US) alone. 2,3 There are many strategies 4 , from systemic to targeted therapies, to manage this debilitating and economically impactful disease but intra-articular (IA) therapies remain a cornerstone. In the last fifteen years, injectable polyacrylamide hydrogels (iPAAGs) have been used with increasing frequency to treat OA in horses. There are currently two iPAAG products of differing characteristics and concentrations (2.5% and 4%) available commercially in the US veterinary market. In humans, 2.5% iPAAG has been used for over two decades, initially for urethral bulking and as a cosmetic filler 5,6 and more recently, for IA treatment of knee OA. 7-10 In horses, 2.5% iPAAG has shown clinical efficacy for treatment of OA in multiple clinical studies. 11-17 In a study of 43 horses with single joint OA, 59% were sound one month following 2.5% iPAAG treatment; response improved over time with 69%, 79%, 81% and 82.5% of horses being sound at 3, 6, 12 and 24 months post treatment, respectively. 12 Other studies have also shown increasing response over time with 43% of 49 Thoroughbred racehorses and 55% of 40 Sport horses showing improvement at 1 month compared to 63% and 75%, respectively, showing improvement 6 months post-treatment. 13,14 Additionally, studies have shown better efficacy of 2.5% iPAAG compared to IA triamcinolone and sodium hyaluronate. 14,15 In two smaller studies of 12 and 18 horses with chronic distal or proximal interphalangeal OA that failed other previous treatments, two-thirds of horses were sound or fully functional 6 to 12 months following treatment with 2.5% iPAAG. 16,17 Despite the clinical use, the mechanism of action of 2.5% iPAAG is not yet fully understood. It is well documented that 2.5% iPAAG, which is inert and non-degradable, physically integrates into the synovial lining in multiple species 5,18,19 and does so via the normal synovial macrophage-guided response. 20 Investigation in goats has demonstrated that 2.5% iPAAG increases joint capsule elasticity (i.e. decreases stiffness) leading to a proposed mechanism of action via a biomechanical effect. 18,19 To date, there have been no controlled experimental studies in horses, and there is a need to further investigate the mechanism of action for 2.5% iPAAG. The primary objective of this study was therefore to test the efficacy of 2.5% iPAAG in a well-established and validated model of OA in the MCJ of skeletally mature horses and additionally, to gain more insight on the mechanism of action of 2.5% iPAAG. We hypothesized that treatment with 2.5% iPAAG in OA limbs would result not only in symptom- but also disease-modifying effects when compared to non-treated OA control limbs. math_shortcuts Materials and Methods Horses, OA induction and treatment: With IACUC approval, 16 skeletally mature horses free of musculoskeletal disease were utilized. Prior to surgery, horses underwent subjective lameness assessment (described below) to determine baseline soundness. At d0, arthroscopic-guided osteochondral fragmentation was performed in one randomly selected MCJ using a well-established model. 21 Briefly, an 8 mm osteochondral fragment was created on the distal aspect of the radial carpal bone and the fragment gap widened on the parent bone to 15 mm using a motorized burr. The fragment and bone debris were not removed from the joint. Diagnostic arthroscopy was performed in the contralateral MCJ to ensure normality of the joint. Following suture removal (d10), horses underwent subjective lameness evaluation and were ranked by lameness score to assign them, in alternating (balanced) fashion, to one of two groups: a control or a treatment group. Control horses (horses that had an osteochondral fragment in one MCJ but no treatment; n=8) had both MCJs aseptically injected with 2 mL phosphate-buffered saline (PBS). Horses in the treatment group (n=8) had 2 mL 2.5% iPAAG (Arthramid, {masked for review}, Franklin, TN, USA) aseptically injected into the fragmented MCJ while the opposite sham MCJ received an equal volume of PBS. For blinding purposes, all treatments were performed by a clinician not performing the lameness examinations. Horses were treated on d14, stall rested until d16, and began high-speed treadmill exercise (6 min, 5 days/week) for the remainder of the 70-day study. Clinical and dynamic evaluation: On a weekly basis, beginning 1 week prior to surgery (week -1), subjective lameness evaluations were performed by trotting the horses with both a markerless AI motion sensor system (Sleip AI, Stockholm, Sweden) and a horse-mounted inertial sensor system (Lameness Locator, Equinosis Q, St. Louis, MO, USA). For both objective measurement systems, the fore signed vector sum was utilized for analysis; for the markerless AI system this was calculated from the head impact mean and head pushoff mean values. The fore signed vector sum indicates the direction and magnitude of lameness with a positive value indicating a right forelimb lameness and a negative value indicating a left forelimb lameness. Additional clinical parameters that were evaluated weekly included response to carpal flexion and MCJ effusion [both graded 0 (none) to 4 (severe)], carpal range of motion [graded 0 (normal) to 3 (severely restricted)] and objective goniometric ROM of the carpal joint [measured in triplicate (in degrees) and averaged]. Bilateral carpal radiographs (lateromedial, dorsopalmar, dorso30°medial-palmarolateral oblique, dorso45°lateral-palmaromedial oblique and flexed lateromedial projections) were obtained at week -1, d14 and d70. Radiographs were scored by a board-certified radiologist unaware of the treatment administered and analyzed for enthesopathy, subchondral lysis and subchondral sclerosis of the radial carpal bone, subchondral sclerosis of the third carpal bone, and osteophyte formation. Each parameter was graded 0 (no abnormality) to 4 (severe) and a total radiographic score was calculated for each limb based on a summation of the scores (0-20). Kinetic and kinematic analysis was performed as previously described 22 prior to d0, and on d14, d42 and d63. Kinetic analysis entailed recording Ground Reaction Forces (GRFs) for all 4 limbs as horses were jogged in-hand over 2 sequential in-ground force platforms. A trial was considered successful when ipsilateral thoracic and pelvic limb pairs contacted the center of a single force platform; five valid trials were collected during each time point. The vertical and craniocaudal GRFs were analyzed and the following parameters were calculated from each trial: stance duration, peak vertical, braking and propulsive forces and impulses and vertical loading rates. Kinetic variables were averaged across the five trials and normalized to subject body mass and expressed as N/kg or Ns/kg. For kinematic analysis retro-reflective markers were adhered to the skin overlying anatomic landmarks on both fore limbs. Fore limb joint angles were calculated based on the anatomic flexor surface of each joint. Raw coordinate data were filtered with a low-pass fourth-order recursive Butterworth filter at 12 Hz. Kinematic variables calculated for each trial included maximum and minimum joint angles during the stance and swing phases, time to peak joint angles during each stride, stride length, stance phase duration and swing phase duration. Synovial fluid and serum evaluation: Synovial fluid (SF) was collected from both MCJs every two weeks (Day 0, 14, 28, 42, 56 and 70), placed into EDTA and serum (red top tube; RTT) blood tubes, refrigerated and analyzed for cellularity and total protein (TP) within 12 hours. After centrifugation of the RTT, the cellular portion was utilized for next generation bulk RNA gene sequencing (data presented separately) and the supernatant stored at -80°C until it was analyzed for concentrations of prostaglandin E 2 (PGE 2 ) and glycosaminoglycan (GAG) as previously described. 23,24 PGE 2 was measured using a commercially available assay (Enzo Life Sciences, Farmingdale, NY, USA). GAG concentrations were analyzed with a dimethylmethylene blue (DMMB) assay. The synovial fluid was also analyzed for the concentrations of 23 cytokines [Fibroblast Growth Factor 2 (FGF2), Eotaxin (CCL11), Granulocyte Colony-Stimulating Factor (G-CSF), Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Growth-Regulated Oncogene (GRO), Fractalkine (CX3CL1), Interferon Gamma (IFN-γ), IL-1α, IL-1β, IL-2, IL-4 IL-5, IL-6, IL-8 (CXCL8), IL-10, IL-12 (p70), IL-13, IL17A, IL-18, Interferon Gamma-Induced Protein 10 (IP-10; CXCL10), Monocyte Chemoattractant Protein 1 (MCP-1), RANTES (CCL5; regulated on activation, normal T cell expressed and secreted), and TNFα] using a commercially available magnetic bead panel (Immunology Multiplex Assay Millipore Sigma, Darmstadt, Germany). Each synovial fluid sample (192 samples) was analyzed in duplicate in the magnetic bead panel for cytokines (384 tested samples). Only cytokines in which ≥10% of tested samples had detectable levels were further evaluated. For statistical analysis, cytokine and PGE 2 values that were below the lower limit of detection (BDL) for the individual assays were recorded as 0.5 times the value of the minimal limit of detection. Blood was collected on Days 0, 14, 28, 42 and 70 and the resulting serum was stored at −80°C until analysis. Serum GAG concentrations were analyzed using a DMMB assay; values that were BDL were recorded as 0.5 times the minimal limit of detection for statistical analysis. Post-mortem evaluation: Horses were humanely euthanized on Day 70 (+/- 2 days) with an overdose of pentobarbital. The forelimbs were harvested, and the following were performed aseptically in this order: synovial fluid collection, endpoint arthroscopy, collection of joint capsule for rheological assessment, cartilage sample collection from the intermediate carpal bone for radiolabeled 35 S GAG synthesis analysis and synovial membrane harvesting for RNA extraction (data presented separately). Joint capsule collection entailed harvesting a 2.5 cm x 10 cm horizontal strip of joint capsule, including the synovial membrane, from the dorsal aspect of the joint; samples were stapled to a tongue depressor, labeled dorsally and laterally for orientation, placed into a conical tube with PBS and frozen prior to shipping on dry ice to co-authors for rheologic testing. Then, macroscopic grading was performed, and additional cartilage and synovial membrane samples were collected for histology (Figure 1). Via a dorsal approach, the MCJ was evaluated arthroscopically. Each individual bone was graded for severity (score 0-3) and extent (0-4) of cartilage erosion and for cartilage fibrillation (0-3). These individual scores were summed from each bone to calculate a total arthroscopic cartilage score per joint. Fragment healing, synovial hyperplasia and synovial hyperemia (0-3) were also graded. The disarticulated joint was similarly evaluated macroscopically, with the exception of cartilage fibrillation which was not assessed macroscopically (Table 1). The arthroscopic and macroscopic grading scales were adopted from Anderson et al. (2022). 25 Articular cartilage biopsies from the radial carpal bone and intermediate carpal bone were collected for GAG quantification and normalized to DNA content. Additionally, cartilage samples were aseptically collected from the intermediate carpal bone and assessed for GAG synthesis. GAG quantification was carried out with the DMMB assay and sample DNA was quantified using a Hoechst assay (Invitrogen, Carlsbad, CA, USA). The concentrations for GAG were normalized to the concentrations of DNA, producing an endpoint value of µg of GAG per µg of DNA. GAG synthesis was measured with the use of a 35 S radioisotope normalized to GAG concentrations. Articular cartilage and synovial membrane samples were collected, processed, embedded in paraffin and sectioned for histologic analysis. Synovial membrane samples, collected from the lateral, central and medial aspects of each joint, were stained with Hematoxylin and Eosin (H&E) while cartilage samples were collected from the radial, intermediate, ulnar, second, third and fourth carpal bones and stained with H&E and Safranin O and Fast Green (SOFG). Samples were consensus graded by two investigators according to the OARSI histopathology initiative. 26 Synovial membrane samples were evaluated at 20x objective for cellular infiltration (0-4), vascularity (0-4), edema (0-4), fibrosis (0-4) and synovial hyperplasia (0-4) (Table 2). The scores of all five parameters were summed to calculate a total histology score for each sample (0-20). These scores were then averaged across the three sample locations to determine an average synovial membrane score per joint. Articular cartilage samples stained with H&E were graded (0-4) for presence of surface fissuring and/or fibrillation, complex chondrone (cluster) formation, surface chondrocyte necrosis and focal cell loss. The scores from these four variables were summed to calculate a total H&E score per carpal bone (0-16). Further, these total H&E scores were averaged across all locations within the joint to calculate average joint H&E score. SOFG-stained cartilage samples were graded based on the amount of stain in each the tangential, intermediate, radiate territorial and radiate interterritorial zones (0-4; Table 2). The SOFG scores from each zone were summed to calculate a total SOFG score per sample (0-16); these total SOFG scores were averaged across all locations within the joint to calculate an average joint SOFG score. Finally, the average H&E and SOFG scores were summed to determine a total cartilage score per joint. Additional synovial membrane samples were collected from both carpi of each horse at termination and were stained routinely with H&E and also for immunohistochemistry (IHC) with S100, Glial Fibrillary Acidic Protein (GFAP), and anti-neurofilament. These slides were then assessed for the presence of nerves by a boarded veterinary pathologist. Nerve counts were performed for the H&E and the three IHC stained samples and then averaged per joint. For rheological assessment of joint capsule stiffness, samples were thawed at room temperature in Tris-buffered saline (10 mM Tris, 0.14 M NaCl, pH 7.5) for 4–5 hours. Each sample was punched using an 8 mm OATS tool to create circular plugs that were tested in rotational shear; two plugs per sample were obtained for amplitude sweeps, i.e. oscillatory tests with gradually increasing rotation. Amplitude sweeps were conducted using a rheometer (Anton Paar MCR 302e rheometer , Graz, Austria) with an 8 mm parallel-plate system and a water-cooled Peltier system for ambient temperature control. To obtain sufficient friction between tissue and plates, silicon-carbide waterproof abrasive paper (320 grit size, Struers ApS, Ballerup, Denmark) as fixed onto both the oscillation piston and the stationary lower plate. Tissue plugs were centered on the lower plate and compressed by 5% of their thickness, a value established during pilot testing to minimize disturbance of tissue structure while preventing slippage. Tris-buffered saline was added around the specimen to maintain hydration throughout testing. Shear strain was then incrementally increased from 0.1% to 100% at a constant frequency of 1 Hz. The linear viscoelastic region (LVE) was identified from the amplitude sweep. For stiffness, the storage modulus (G′; primary outcome), which represents the tissue’s elastic spring characteristics, and loss modulus (G″; secondary outcome), which represents the damping characteristics, were calculated as the mean of G′ and G″ across all data points within the LVE plateau. Each plug was tested once and averaged with values from the replicate plugs for each joint. Data analysis: Data are reported as mean ± standard deviation. The four limb groups were defined as control horse OA limb, control horse sham limb, treatment horse OA limb and treatment horse sham limb. Serially acquired outcomes (clinical, lameness, imaging and synovial fluid data) were compared between groups and over time using a repeated measures mixed model ANOVA. Random effects included horse and horse-by-joint interaction to account for repeated measures. Fixed effects included treatment groups, study day and their interaction. Endpoint outcomes (histology and nerve analysis data) were compared between groups and sample location within the joint (where applicable) using a mixed model ANOVA. Random effects included horse and horse-by-joint interaction. Fixed effects included treatment group, sample location and their interaction. The type 3 tests of fixed effects of each variable were adjusted using a Bonferroni correction to address multiple testing bias and reduce type I error. Variables that passed this threshold had pairwise comparisons examined using a Tukey-Kramer adjustment. Model assumptions were assessed visually using residual versus quantile diagnostic plots. Ordinal response variables showing a significant difference in pairwise comparisons between treatment groups using mixed model ANOVA and Bonferroni correction were further assessed using ordinal mixed logistic regression with multinomial distribution and cumulative logit link function. Treatment groups were compared to the control horses OA limb (reference category) in the ordinal mixed regression model. Data analysis of storage moduli of OA and contralateral sham samples within each treatment group (saline or 2.5% iPAAG) were compared using paired t-tests. Statistical significance for all tests was defined at p<0.05. Statistical analysis was performed using SAS (SAS On Demand version 9.4, Cary, NC, USA). Statistical significance was defined at P<0.05. Results There were 9 geldings and 7 mares included with a mean age of 2.8 ± 0.7 years and mean weight of 404 ± 31 kg. Clinical and radiographic outcomes This model of OA produced a mild but statistically significant increase in lameness evidenced by significantly higher mean subjective lameness grades in the OA limbs compared to sham-operated limbs at all time points following fragment formation (d14: 1.25 ± 0.86 vs 0.06 ± 0.25; p<0.0001; d70: 1.00 ± 0.73 vs 0.19 ± 0.40; p<0.001). Prior to treatment on d14, the mean subjective lameness was significantly higher in the OA limbs of the treatment group (1.75 ± 0.89) compared to the control group (0.75 ± 0.46; p=0.017); there were no differences in mean subjective lameness scores between groups at any other timepoints . Within the treatment OA limb group, horses showed a decrease in mean lameness from 1.75 ± 0.89 at d14 to 1.25 ± 0.71 at the end of the study, though not statistically significant. A significant improvement was observed within the treated OA group at d49 however, where the mean lameness decreased to 0.88 ± 0.84 compared to pre-treatment at d14 (p=0.024) (Table 3). Overall, the inertial sensor and AI objective lameness measurement systems did not reveal any significant effects of limb, study day or limb by study day interaction. On d14, prior to treatment, the mean calculated vector sum of the AI system was higher in treatment OA limbs (0.91 ± 0.96) versus control OA limbs (0.31 ± 0.23) but this was not statistically significant. At this same time point, kinetic and kinematic analysis also showed significantly lower normalized peak vertical ground reaction force measurements—indicating more lameness—in the treatment group compared to the control group (9.56 ± 0.71 vs. 10.29 ± 0.65 N/kg; p=0.001). There were otherwise no significant differences in OA limbs between the treatment and control groups. Induction of OA worsened mean passive ROM, effusion, average goniometry, and response to flexion scores but there were no significant differences between treatment groups at any time points (Table 3). Mean effusion scores decreased in the treatment group between d14 (2.0 ± 0.76) and d70 ( 1.5 ± 1.07) whereas they remained unchanged in the control group (d14: 1.88 ± 0.64; d70: 1.88 ± 0.84) but the difference was not significant. In both treatment and control groups, mean total radiographic scores of the OA limbs were significantly increased from d0 (Treated: 0.25 ± 0.71, Control: 0.125 ± 0.35) to d14 (Treated: 2.25 ± 2.05, p=0.009; Control: 2.13 ± 1.13, p=0.009) and further from d14 to d70 (Treated: 5.63 ± 3.20, p<0.001; Control: 6.88 ± 3.00, p<0.0001). At d70, the mean total radiographic score was lower (5.63 ± 3.2) in treated OA limbs compared to control OA limbs (6.88 ± 3.0), but after correction this did not reach statistical significance. There was a statistically significant effect of study day by limb interaction on each individual radiographic variable (joint capsule enthesopathy, osteophytosis, radial carpal bone lysis, radial carpal bone sclerosis, and third carpal bone sclerosis) due to the increased mean radiographic scores of the OA-induced limbs at d70 but no significant differences between treatment groups. There was, however, a strong trend for treated OA limbs to have less mean radial carpal bone sclerosis compared to control OA limbs (1.5 ± 1.2 vs 2.3 ± 0.89; p=0.07) at d70 (Figure 3). Synovial fluid and serum GAG: Across groups, mean synovial fluid TP peaked at d14 and steadily declined to near baseline levels by d70. There was a significant effect of study day by limb interaction but no significant differences between the OA limb treatment groups. At d14 the OA limbs of both treatment and control horses had significantly higher mean synovial fluid TP concentrations compared to contralateral sham limbs (Treated: 3.08 ± 0.39 vs 2.15 ± 0.51 g/dL, p<0.0001; Control: 3.45 ± 0.41 vs 2.45 ± 0.49 g/dL, p<0.0001); this remained at d28 (Treated: 2.50 ± 0.34 vs 1.78 ± 0.43, p=0.0002; Control: 2.78 ± 0.26 vs 1.85 ± 0.35 g/dL, p<0.0001). Additionally, at d56 TP was significantly higher in the OA limbs (1.88 ± 0.47 g/dL) compared to the sham limbs (1.40 ± 0.47 g/dL) in the treatment group (p=0.032). Synovial fluid mean white blood cell (WBC) concentrations were variable with highest levels in the OA limbs at d14 in the control group (760 ± 930 cells/µL) and at d70 in the treatment group (750 ± 330 cells/µL). There was a significant effect of study day*limb for total WBC, neutrophil and lymphocyte concentration but no significant differences between the treatment and control groups in the OA limbs. For the cytokine analysis, most samples were below detectable limits and only six cytokines (TNFα, IP-10, IL-6, IL-8, IL-10 and FGF-2) met the evaluation criteria. Following treatment at d14, there were no significant differences between treatment groups for TNFα, IP-10, IL-6, IL-10 or FGF-2. Synovial fluid concentrations of IL-8 were significantly lower in treated versus control OA limbs at d28 (29.63 ± 0.24 vs. 341.03 ± 294.17 pg/mL; p<0.001) and d56 (29.5 ± 0 vs. 259.27 ± 250.84 pg/mL; p=0.007) but this difference stemmed from pre-treatment differences at d14 (treated OA limbs: 49.82 ± 45.44 vs. control OA limbs 414.84 ± 292.85 pg/mL; p=0.0004) thus was not considered a treatment effect. The mean synovial fluid PGE 2 concentration was lower in treated OA limbs compared to control OA limbs at d28 (71.72 ± 26.39 vs. 135.71 ± 70.11 pg/mL) but this did not reach statistical significance. Synovial fluid GAG was lowest across groups at d14 which contributed to the significant study day*limb interaction (p=0.036) but was then similar to baseline levels at all subsequent timepoints for all groups. Serum GAG concentrations were largely BDL (90/95) and there was no difference across time points nor between treatment groups. Endpoint macroscopic and arthroscopic evaluation: Total macroscopic cartilage scores were higher in OA vs. sham-operated limbs in both groups (Treated: 12.25 ± 6.11 vs. 6.25 ± 4.68; Control: 11.13 ± 5.64 vs. 4.75 ± 3.24) but the differences did not reach statistical significance. There were no significant differences between OA groups for any of the macroscopic outcome variables. The total arthroscopic score of treated OA limbs (12.38 ± 8.30) was not significantly different than either the control OA limbs (13.75 ± 3.92) nor the contralateral sham limbs (6.75 ± 5.26). Endpoint arthroscopic grading showed an effect of limb group for the extent and severity of cartilage defects of the radial carpal bone as well as synovial hyperemia but no significant differences between the control and treated OA limbs. Biochemistry and histology: There were no significant differences for any of the cartilage viability biomarkers including decorin, proteoglycan, biglycan, GAG and DNA, as evaluated with Hoechst stain. Similarly, there was no significant difference in GAG synthesis. The 2.5% iPAAG was observed within the synovium in all 8 of the treated limbs (Figure 2). By location, 2.5% iPAAG was visible in the medial, central and lateral synovial membrane samples in 2 limbs, in the medial and central synovial membrane samples in 4 limbs, and in the medial and lateral synovial membrane samples in 1 limb; in one limb the 2.5% iPAAG was only visualized in the central sample. There were no significant differences in any of the synovial membrane histology outcome parameters by limb group (Figure 4). Further, there were no differences in the number of nerves present in the synovial membrane based on H&E and IHC-stained samples between groups. In the 3 rd carpal bone, treated OA limbs had significantly lower (better) mean total cartilage histology scores (6.38 ± 4.10) compared to control OA limbs (11.25 ± 4.20, p=0.023). Treated OA limbs also had significantly lower (better) SOFG staining of the 3 rd carpal bone intermediate zone compared to control OA limbs (0.38 ± 0.74 vs. 1.88 ± 1.25; p=0.022) (Figure 5). Unlike the synovial membrane, there were no cartilage samples in which 2.5% iPAAG was observed histologically. Mean average joint H&E and SOFG histology scores were lower (better) in treated OA limbs compared to control OA limbs but these differences were not statistically significant. Ordinal mixed logistic regression analysis further revealed that 2.5% iPAAG-treated OA limbs had significantly lower SOFG scores in the intermediate zone of the 3 rd carpal bone compared to control OA limbs (p=0.037). [1]¿p#1 Joint Capsule Stiffness Joint capsule stiffness, measured as storage modulus (G′), was significantly increased in OA limbs of the control (saline) group compared to their contralateral sham-operated limbs (1824 ± 1105 Pa vs 1002 ± 495 Pa; p=0.043). In contrast, OA limbs in the 2.5% iPAAG treated group had storage moduli similar to their matched sham limbs (1099 ± 647 Pa vs 1184 ± 504 Pa; p=0.718; Figure 6). Relative to the contralateral sham limb for each horse, joint capsule stiffness increased approximately 2.7-fold in saline-treated OA limbs but showed no detectable change (1.0-fold) in 2.5% iPAAG-treated OA limbs. The loss modulus (G″), addressing the damping characteristics of the tissue, showed a similar pattern, although differences did not reach statistical significance. OA-saline limbs tended to have higher G″ values than their sham limbs (400 ± 251 Pa vs 205 ± 91 Pa; p=0.102), whereas OA limbs in the 2.5% iPAAG group had loss moduli comparable to their matched sham limbs (250 ± 168 Pa vs 276 ± 140 Pa; p=0.724). Discussion As expected, induction of OA in this model was successful as evidenced by increased (worsened) scores for multiple outcome parameters in OA compared to sham including lameness scores, joint effusion, response to carpal flexion, radiographic scores, synovial fluid total protein, macroscopic and arthroscopic scores. The hypothesis was partially supported in that treatment with 2.5% iPAAG resulted in disease-modifying effects including reduced joint capsule stiffness and better 3 rd carpal bone articular cartilage histopathology scores. The only symptom-modifying effect was a decrease in subjective lameness in the 2.5% iPAAG treated OA limbs at d49 compared to d14. One of the major findings of this study was the multiple outcomes that demonstrated an improvement in the articular cartilage of the 3 rd carpal bone of treated joints, indicating that treatment with 2.5% iPAAG had some ability to mitigate the negative effects of OA. The radial facet of the 3 rd carpal bone—which is most prone to injury in this model as it articulates with the osteochondral fragment created on the distal aspect of the radial carpal bone—revealed significantly less articular damage in treated horses compared to controls. This was most evident in SOFG stained samples, in which minimal leaching of counterstain into the articular cartilage was seen, indicating a functionally more proteoglycan-rich cartilage matrix. As the third carpal bone is a good barometer for the treatment effect using this model, these results support 2.5% iPAAG as a disease-modifying OA product and provide support for its use for the treatment of OA in horses. This study also confirmed, through rheological assessment, that OA was associated with a substantial increase in joint capsule stiffness, whereas treatment with 2.5% iPAAG preserved a stiffness value comparable to that of sham-operated joints. Specifically, joint capsule storage moduli were significantly higher in saline-treated OA joints than in their contralateral sham controls, consistent with fibrotic remodeling and loss of normal tissue compliance. In contrast, OA joints treated with 2.5% iPAAG had storage moduli that were indistinguishable from their corresponding sham joints, indicating that treatment mitigated the OA-associated stiffening of the joint capsule. These findings support a biomechanical, disease-modifying mechanism of action for 2.5% iPAAG at the level of the joint capsule. The normalization of joint capsule stiffness observed in the treated joints parallels previous work in a caprine model, where 2.5% iPAAG increased joint capsule elasticity and counteracted OA-related fibrotic changes. 18,19 In the present equine model, the combination of preserved joint capsule viscoelastic properties and improved 3rd carpal bone cartilage histopathology suggests that maintaining near-normal joint capsule mechanics may contribute to the mitigation of cartilage degeneration under cyclic loading. It is well established that 2.5% iPAAG integrates into the synovial lining. 5,18,19 Specifically, 2.5% iPAAG integrates into the subintima and initiates a normal synovial foreign body-type reaction that is primarily macrophage-driven in that location. 20 The rheological results of this study align with the histologic and immunohistochemical findings, which showed integration of 2.5% iPAAG into the synovial membrane without an associated increase in inflammatory cell infiltrates. In fact, even at their peak, synovial fluid WBC concentrations were well within the normal reference range. Together, these data indicate that 2.5% iPAAG can modify synovial biomechanics in the direction of normal joint homeostasis without inducing a deleterious cellular response. The normal joint capsule elastic modulus observed in 2.5% iPAAG treated OA joints likely contributes to the reduction in pain and improvement in function that has been documented in clinical studies of 2.5% iPAAG in both horses 11–15 and humans. 7–10 Synovial fluid PGE 2 , a key marker of inflammation in diseased joints, was almost half the concentration in treated OA limbs compared to control OA limbs (71.72 ± 26.39 pg/mL vs. 135.71 ± 70.11 pg/mL) at the first sampling timepoint post 2.5% iPAAG treatment (d28). Although this difference failed to reach statistical significance, this coincides with the results of the transcriptomic analysis (presented separately) in which the maximal difference in gene expression—in an anti-inflammatory and normal joint homeostasis direction—were also noted at d28. Collectively, these findings may indicate that the derived cellular effects of 2.5% iPAAG treatment occur earlier than previously realized and may warrant its earlier clinical use. The model produced a very mild degree of lameness (averaging 1.06 in OA limbs across all time points), making it difficult to detect a treatment effect, especially with the small sample size of only 8 horses per group. This could be one reason that the treatment effect on lameness outcomes did not reach statistical significance. Additionally, multiple clinical trials have shown that the proportion of horses with improved lameness following 2.5% iPAAG treatment increases over time, up to at least six months. 11-13 Thus the 70-day timeline of this study may have been too short to fully elucidate the clinical improvements that have been demonstrated in other clinical studies. The mean lameness was significantly higher for the treated OA limbs (Grade 1.75) compared to control OA limbs (Grade 0.75) but this was prior to treatment administration. This was unexpected as the horses were graded for lameness on Day 10, ranked, and assigned to treatment groups in an alternating fashion. The reason for the statistical difference was likely two-fold. Horses were ranked and assigned to treatment groups on d10 but not treated until d14; as lameness and clinical signs associated with lameness are dynamic, some horses’ lameness grades changed between days 10 and 14. Additionally, one horse was excluded from the study at d14 due to having a fatal complication—the horse developed a slab fracture of the radial carpal bone prior to treatment and was humanely euthanized—requiring a replacement horse that could not be included in the rank. Regardless, in the statistical model each horse was its own control and each lameness score was compared to the ‘baseline’ d14 value. Therefore, ultimately the difference in lameness between the groups at Day 14 did not influence the ability to detect a treatment effect. This study utilized one subjective and three objective systems of assessing lameness and gait asymmetry. It is noteworthy that all four methods, and particularly the objective systems, correlated well with one another. For example, the continuous data from the markerless AI and body-mounted inertial sensor systems had a very strong correlation (r=0.95). When comparing to the subjective lameness scores, by categorizing the objective data into ordinal data, the correlation was still strong with r-values of 0.82 (AI markerless system) and 0.74 (body-mounted inertial sensor system). Therefore, it would be reasonable for future investigations to utilize only one of the four systems to evaluate lameness as the results did not vary appreciably between the systems. There has been anecdotal concern over potential neurotoxicity of 2.5% iPAAG due to it being polymerized from acrylamide, a well-known neurotoxin. A recent in vitro study investigated the potential cytotoxic and neurotoxic effects of 2.5% iPAAG using human cortical neurons. 27 Exposure to concentrations of up to 20% by volume of 2.5% iPAAG did not significantly affect cell viability, including cell survival, apoptotic and non-apoptotic cell death, unlike positive controls. The results of the current study did not demonstrate a difference in the synovial membrane nerve counts between treatment groups. Similarly, a study in goats also reported no difference in synovial membrane nerve counts with 2.5% iPAAG treatment. 19 Taken collectively, there is no evidence to suggest that 2.5% iPAAG is neurotoxic and the reduction of pain that has been demonstrated in other studies is unlikely to be from a direct effect on nerves. In this study, the administration of 2.5% iPAAG was via a standard dorsolateral approach into the MCJ. Despite the dorsolateral deposition of a very viscous substance, histology revealed that the 2.5% iPAAG was distributed widely throughout the joint. This demonstrates that the hydrogel diffuses throughout the joint prior to incorporating into the synovial membrane and does not necessarily remain focused at the site of injection. This is in contrast to one study that demonstrated that 4% polyacrylamide gel tended to stay local to the site of injection. 28 There is often misunderstanding about the various concentrations and properties of polyacrylamide hydrogels. It is important to note that 2.5% and 4% polyacrylamide hydrogels may differ significantly in their structure, characteristics and mechanisms of action. Specifically, 4% polyacrylamide hydrogel in vitro has been proposed to adhere to the cartilage surface and, compared to saline, decreased the friction coefficient of cartilage 2 days post administration. 28,29 Additionally, 4% iPAAG is proposed to eventually be phagocytosed by the synovial membrane 30 whereas 2.5% iPAAG is non-degradable and incorporates, for up to 2 years or more, into the synovial membrane. 5 These differences are likely critical in understanding the mechanism of action of different iPAAGs. LIMITATIONS This study was limited by the relatively short 70-day timeline and small sample size. Although this model of OA is well-established and validated, it does not perfectly mimic naturally occurring OA, and clinical improvement in horses often develops over a longer time course than the 56-day follow-up used here. These factors may help explain discrepancies between our findings and those reported in clinical case series of horses with naturally occurring OA. [1]¿p#1 CONCLUSIONS The model produced symptoms and pathologic changes consistent with OA and represents changes seen in both acute and chronic equine OA. Treatment with 2.5% iPAAG demonstrated some ability to mitigate the negative effects of OA via positive effects on articular cartilage. Additionally, treatment with 2.5% iPAAG resulted in a joint capsule elastic modulus that was similar to non-OA joints; the retention of normal joint capsule elasticity may be a major factor, and a likely mechanism of action, to account for the decrease in pain that has been shown in humans and horses following treatment. Despite the incorporation of 2.5% iPAAG into the synovial membrane, no significant cellular infiltration was appreciated, which supports the theory that 2.5% iPAAG exerts its effect via biomechanical mechanisms. Further study into these mechanisms of action is warranted. ACKNOWLEDGEMENTS Acknowledgements: The authors would like to acknowledge the support staff at xxx for their help with all aspects of study design, data collection and sample analysis. Additionally, we acknowledge xxx for her consultation on statistical analysis and xxx for evaluating the nerve-stained synovial membrane histopathology slides. Declaration of Ethics: The authors have adhered to the Principles of Veterinary Medical Ethics of the AVMA. The project was approved by the institutional animal care and use committee. Conflicts of Interest: One of the authors (xxx) serves on the Scientific Advisory Council for {masked for review}. Funding Sources: Funding for this study was provided by {masked for review}. REFERENCES 1. McIlwraith, C.W. (2016) Traumatic arthritis and posttraumatic osteoarthritis in the horse. In: Joint Disease in the Horse , 2nd edn., Elsevier, St. Louis. pp 33-48. 2. USDA (2017) Equine 2015 Report 3: Equine Management and Select Equine Health Conditions, 2015 , USDA-APHIS-VS-CEAH-NAHMS, Fort Collins. 3. Oke, S.L. and McIlwraith, C.W. (2010) Review of the economic impact of osteoarthritis and oral joint-health supplements in horses. Proc. Am. Ass. equine Practnrs. 56, 12-18. 4. Caron, J.P. and Genovese, R.L. (2003) Principles and practices of joint disease treatment. In: Diagnosis and Management of Lameness in the Horse , Elsevier, St. Louis. pp 746-764. 5. Christensen, L., Camitz, L., Illigen, K.E., Hansen, M., Sarvaa, R. and Conaghan, P.G. (2016) Synovial incorporation of polyacrylamide hydrogel after injection into normal and osteoarthritic animal joints. Osteoarthritis Cartilage 24, 1999-2002. 6. Lose, G., Mouritsen, L. and Nielsen, J.B. (2006) A new bulking agent (polyacrylamide hydrogel) for treating stress urinary incontinence in women. BJU Int. 98, 100–104. 7. Bliddal, H., Overgaard, A., Hartkopp, A., Beier, J., Conaghan, P.G. and Henriksen, M. (2021) Polyacrylamide hydrogel injection for knee osteoarthritis: A 6 months prospective study. J. Orthop. Res. Ther. 6, 1188. 8. Bliddal, H., Beier, J., Hartkopp, A., Conaghan, P.G. and Henriksen, M. (2024) Effectiveness and safety of polyacrylamide hydrogel injection for knee osteoarthritis: results from a 12-month follow up of an open-label study. J. Orthop. Surg. Res. 19, 274. 9. Henriksen, M., Overgaard, A., Hartkopp, A. and Bliddal, H. (2018) Intra-articular 2.5% polyacrylamide hydrogel for the treatment of knee osteoarthritis: an observational proof-of-concept cohort study. Clin. Exp. Rheumatol. 36, 1082–1085. 10. Overgaard, A., Bliddal, H. and Henriksen, M. (2019) Safety of intra-articular polyacrylamide hydrogel for the treatment of knee osteoarthritis symptoms: a retrospective case series. Clin. Ortho. Adv. Res. J. 2019, 1–6. 11. Tnibar, A., Schougaard, H., Camitz, L., Rasmussen, J., Koene, M., Jahn, W. and Markussen, B. (2012) Efficacy of a polyacrylamide hydrogel in horses with symptomatic osteoarthritis: an international multi-centre prospective study. Equine vet. J. 44, 16. 12. Tnibar, A., Schougaard, H., Camitz, L., Rasmussen, J., Koene, M., Jahn, W. and Markussen, B. (2015) An international multi-centre prospective study on the efficacy of an intraarticular polyacrylamide hydrogel in horses with osteoarthritis: a 24 months follow-up. Acta vet. scand. 57, 1-8. 13. deClifford, L.T., Lowe, J.N., McKellar, C.D., Bolwell, C. and David, F. (2019) Use of a 2.5% cross-linked polyacrylamide hydrogel in the management of joint lameness in a population of flat racing Thoroughbreds: a pilot study. J. equine vet. Sci. 77, 57-62. 14. Tnibar, A., Schougaard, H., Koene, M. and Markussen, B. (2014) A controlled clinical trial on the efficacy of an intra-articular polyacrylamide hydrogel in horses with osteoarthritis. Vet. Surg. 43, E138. 15. deClifford, L.T., Lowe, J.N., McKellar, C.D., McGowan, C. and David, F. (2021) A double-blinded positive control study comparing the relative efficacy of 2.5% polyacrylamide hydrogel against triamcinolone acetonide and sodium hyaluronate in the management of middle carpal joint lameness in racing Thoroughbreds. J. equine vet. Sci. 107, 103780. 16. Janssen, I., Koene, M. and Lischer, L. (2012) Intraartikuläre applikation von polyacrylamid hydrogel zur behandlung von osteoarthritis des hufgelenkes: fallserie von 12 pferden. Pferdeheilkunde 28, 650–656. 17. Bathe, A.P., Read, R. and Briggs, C. (2016) Intra-articular polyacrylamide hydrogel for the treatment of 20 horses with non-responsive osteoarthritis of the interphalangeal joints: a prospective study. Proc. Vet. Orthop. Soc. 43, 4-5. 18. Tnibar, A., Persson, A.B., Nielsen, H., Svalastoga, E. and Westrup, U. (2014) Evaluation of a polyacrylamide hydrogel in the treatment of induced osteoarthritis in a goat model: a randomized controlled pilot study. Osteoarthritis Cartilage 22, S477. 19. Tnibar, A., Persson, A.B. and Jensen, H.E. (2017) Mechanisms of action of an intraarticular 2.5% polyacrylamide hydrogel (Arthramid Vet) in a goat model of osteoarthritis: Preliminary Observations. SM J. biomed. Eng. 3, 1022. 20. Lowe, J., Clifford, L., Julian, A. and Koene, M. (2024) Histologic and cytologic changes in normal equine joints after injection with 2.5% injectable polyacrylamide hydrogel reveal low-level macrophage-driven foreign body response. J. Am. vet. med. Ass. 262, 649-677. 21. Frisbie, D.D., Ghivizzani, S.C., Robbins, P.D., Evans, C.H. and McIlwraith, C.W. (2002) Treatment of experimental equine osteoarthritis by in vivo delivery of the equine interleukin-1 receptor antagonist gene. Gene Ther. 9, 12-20. 22. King, M.R., Haussler, K.K., Kawcak, C.E., McIlwraith, C.W., Reiser, R.F. 2nd, Frisbie, D.D. and Werpy, N.M. (2017) Biomechanical and histologic evaluation of the effects of underwater treadmill exercise on horses with experimentally induced osteoarthritis of the middle carpal joint. Am. J. vet. Res. 78, 558-569. 23. Frisbie, D.D., Kawcak, C.E., Werpy, N.M., Park, R.D. and McIlwraith, C.W. (2007) Clinical, biochemical, and histologic effects of intra-articular administration of autologous conditioned serum in horses with experimentally induced osteoarthritis. Am. J. vet. Res. 68, 290-296. 24. Carroll, G.J. (1987) Spectrophotometric measurement of proteoglycans in osteoarthritic synovial fluid. Ann. Rheum. Dis. 46, 375-379. 25. Andersen, C., Jacobsen, S., Walters, M. and Lindegaard, C. (2022) A detailed macroscopic scoring system for experimental post-traumatic osteoarthritis in the equine middle carpal joint. BMC Res. Notes 15, 226. 26. McIlwraith, C.W., Frisbie, D.D., Kawcak, C.E., Fuller, C.J., Hurtig, M. and Cruz, A. (2010) The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the horse. Osteoarthritis Cartilage 18, S93-S105. 27. Walmod, P.S., Kusk, P., Jøhnk, N., Ankorina-Stark, I. and Essex, A. (2025) An injectable 2.5% cross-linked polyacrylamide hydrogel (2.5 iPAAG) demonstrates no neurotoxicity in human induced pluripotent stem cells-derived iCell® GlutaNeurons. Front. Toxicol. 7, 1585430. 28. Vishwanath, K., McClure, S. and Bonassar, L. (2024) Heterogenous distribution of viscosupplements in vivo is correlated to ex vivo frictional properties of equine cartilage. J. biomed. Mater. Res. 112, 2149-2159. 29. Vishwanath, K., McClure, S. and Bonassar, L. (2023) Polyacrylamide hydrogel lubricates cartilage after biochemical degradation and mechanical injury. J. Orthop. Res. 41, 63-71. 30. McClure, S.R., Peitzmeier, M.D., Jackman, B.R., Evans, R.B., Ziegler, C.L. and Ganta, C.K. (2024) Serial injections of 4% polyacrylamide hydrogel have no detrimental effects in equine joints following clinical, histologic, and synovial biomarker evaluation. Am. J. vet. Res. 85(6). math_shortcuts TABLES AND FIGURES Table 1. Grading rubric utilized for macroscopic and arthroscopic scoring of the middle carpal joints (MCJs). math_shortcuts Grade Description Grade Description 0 None 0 Absent 1 Partial thickness erosions, each 5mm in diameter 2 Moderate 3 Full thickness erosion 3 Severe Carpal Cartilage Extent of Erosion Score assigned to each individual bone of MCJ Osteochondral Fragment Healing & Erosions Score assigned to OA joint only (N/A for sham joints) Grade Description Grade Description 0 None 0 Full healing with integration with surrounding cartilage 1 1-25% of the cartilage surface 1 Incomplete healing, demarcated border, healthy cartilage around fragment 2 26-50% of the cartilage surface 2 Fragment attached to parent bone, mild erosions around fragment 3 51-75% of the cartilage surface 3 Fragment attached to parent bone but with severe erosions around fragment 4 76-100% of the cartilage surface 4 Fragment not attached and severe erosions around defect Cartilage Fibrillation – Arthroscopic Evaluation Only Score assigned to each individual bone of MCJ Grade Description 0 Absent 1 Mild 2 Moderate 3 Severe math_shortcuts Table 2. Synovial membrane and articular cartilage histology scoring rubric in accordance with the OARSI histopathology initiative. [1]¿p#1 Outcome parameter Score Description Outcome parameter Score Description Cellular Infiltration 0 No mononuclear cells in the section Chondrocyte necrosis 0 Normal section without necrosis 1 Occasional small areas of mononuclear cells throughout the section 1 ≤ 1necrotic cell located near the articular surface per 20x objective 2 Mild presence of mononuclear cells in 25% of the section 2 1-2 necrotic cells located near the articular surface per 20x objective 3 Moderate presence of mononuclear cells in 25-50% of the section 3 2-3 necrotic cells located near the articular surface per 20x objective 4 Marked presence of mononuclear cells in >50% of the section 4 3-4 necrotic cells located near the articular surface per 20x objective Intimal Hyperplasia 0 None Cluster formation 0 No cluster formation throughout section 1 Villi w/ 2-4 rows of intimal cells w/in the section 1 2 chondrocytes within same lacunae along superficial aspect of the articular cartilage section 2 Villi w/ 4-5 rows of intimal cells over 25-50% the section 2 2-3 chondrocytes within same lacunae along superficial aspect of articular cartilage section 3 Villi w/ 4-5 rows of intimal cells over 50% the section 3 3-4 chondrocytes within same lacunae along superficial aspect of the articular cartilage section 4 Villi w/ 5 or greater rows of intimal cells over 50% of the section 4 > 4 chondrocytes within same lacunae along superficial aspect of the articular cartilage section Subintimal edema 0 No edema Fibrillation/ fissuring 0 No fibrillation/ fissuring of the articular cartilage surface 1 Slight edema detected within section 1 Fibrillation/ fissuring of the articular cartilage restricted to surface and superficial zone 2 Mild edema within 25% of the section 2 Fissuring that extends into the middle zone 3 Moderate edema within 25-50% of the section 3 Fissuring that extends to the level of the deep zone 4 Marked edema in greater than 50% of the section 4 Fissuring that extends into the deep zone Subintimal fibrosis 0 Normal Focal cell loss 0 Normal cell population throughout the section 1 Slight increase in fibrosis w/in section 1 10-20% area of acellularity per 20x field 2 Mild increase in fibrosis in 25% of section 2 20-30% area of acellularity per 20x field 3 Moderate increase in fibrosis in 25-50% of the section 3 40-50% area of acellularity per 20x field† 4 Marked increase in fibrosis in >50% of the section 4 >50% area of acellularity per 20x field Vascularity 0 Normal SOFG stain uptake 0 Normal staining 1 Slight increase in vessels in focal locations throughout the section 1 Less than 25% loss of staining characteristics 2 Mild increase in number & dilation of vessels focally throughout section 2 25-50% loss of staining characteristics 3 Moderate increase in number & dilation of vessels in up to 50% of the section 3 50-75% loss of staining characteristics 4 Marked increase in number & dilation of vessels in up to 50% of the section 4 >75% loss of staining characteristics † Samples with 30-40% area of acellularity per 20x field were scored as a 3. Table 3. Mean values of clinical variables in horses the underwent osteochondral fragmentation of the middle carpal joint to induce osteoarthritis; horses were treated intra-articularly at d14 with either saline (control; n=8) or 2.5% iPAAG (n=8). A mixed model ANOVA was performed and variables that reached significance after Bonferroni correction (subjective lameness) underwent pairwise comparison; significant differences (p<0.05) from pairwise comparisons are noted with different letters. Figure 1. Study timeline (A) indicating time points in which treatments were administered and data and clinical samples were collected. Flags above the timeline indicate time points procedures were performed and flags below the timeline indicate time points at which samples were collected. At study end (d70), various samples were collected from the middle carpal joint (B). math_shortcuts Figure 2. Hematoxylin and Eosin (H&E) stained synovial membrane of the middle carpal joint of horses with experimentally-induced osteoarthritis. Horses were treated with intra-articular saline (A) or 2.5% iPAAG (B) on d14 and samples were collected at d70. The amorphous to somewhat globular purple stained material (white arrows) within the subintima is the 2.5% iPAAG (20x magnification). math_shortcuts Figure 3. Radiographic evaluation 70 days after osteochondral fragmentation of the middle carpal joint. A dorsolateral-palmaromedial oblique radiograph of the left front limb in a control horse (A) and a horse treated with 2.5% iPAAG (B). Blue arrows indicate radial carpal bone sclerosis, green arrows indicate the defect (A) and fragment (B), and yellow arrows indicate the lack of sclerosis with good corticomedullary distinction. Radiographs were graded for joint capsule enthesopathy (Jt Caps Enthes), radial carpal bone (RCB) lysis and sclerosis, 3 rd carpal bone (3CB) sclerosis and osteophytosis (C). There was a trend (p=0.07) for treated horses to have less radial carpal bone sclerosis compared to controls Figure 4. Mean histologic grades of synovial membrane harvested from the medial, middle and lateral aspects of the middle carpal joint. The individual parameters were each graded 0-4 and then summed to create a total synovial membrane histologic score. There were no significant differences between limb groups for any of the outcome parameters. Figure 5. Safranin O and Fast Green (SOFG) stained articular cartilage from the third carpal bones of horses with induced osteoarthritis of the middle carpal joint. The intermediate zone of the cartilage was graded from 0 (no fast green counter stain present) to 4 (complete loss of safranin O stain retention); grades 1 (A), 2 (B) and 3 (C) are shown above. Cumulative histology grades of H&E and SOFG-stained articular cartilage samples of the 3 rd carpal bone are shown graphically (D); different letters signify a statistical difference of p<0.05. math_shortcuts Figure 6. Storage modulus (G′) of synovial membrane from osteoarthritis (OA) limbs and contralateral sham limbs in horses treated with intra-articular saline or 2.5% iPAAG. For each treatment, storage moduli of OA limbs are compared with their contralateral sham control limbs (n=8 horses per treatment group). Data are displayed as boxplots; in each box, the thick horizontal bar marks the median of the dataset. Supplementary Material File (arthramid evj tables figs final 2.27.26.docx) Download 15.83 MB File (image12.emf) Download 736.84 KB File (image7.emf) Download 881.10 KB File (image8.emf) Download 1.21 MB Information & Authors Information Version history V1 Version 1 04 March 2026 Copyright This work is licensed under a Non Exclusive No Reuse License. Authors Affiliations Erin Contino 0000-0002-1756-510X [email protected] Colorado State University Department of Clinical Sciences View all articles by this author AbuAlia M Rush University Department of Orthopedic Surgery View all articles by this author Brad Nelson 0000-0002-0205-418X Colorado State University Department of Clinical Sciences View all articles by this author Laurie Goodrich Colorado State University Department of Clinical Sciences View all articles by this author Myra Barrett 0000-0001-5060-6009 Colorado State University Department of Clinical Sciences View all articles by this author Melissa King 0000-0002-1203-3059 Colorado State University Department of Clinical Sciences View all articles by this author Lynn Pezzanite 0000-0003-4990-5006 Colorado State University Department of Clinical Sciences View all articles by this author Wayne McIlwraith Colorado State University Department of Clinical Sciences View all articles by this author Wimmer MA Rush University Department of Orthopedic Surgery View all articles by this author Kathryn Seabaugh Colorado State University Department of Clinical Sciences View all articles by this author Metrics & Citations Metrics Article Usage 190 views 79 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Erin Contino, AbuAlia M, Brad Nelson, et al. Effects of intra-articular 2.5% polyacrylamide hydrogel in an experimental model of equine osteoarthritis. Authorea . 04 March 2026. DOI: https://doi.org/10.22541/au.177258447.71381217/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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