• View in gallery

    Illustrative representation of relative quantities of cellular and protein contents in equine venous blood (left), intermediate cell solution (middle), and APS (right). Notice that APS contains higher amounts or numbers of anti-inflammatory proteins, growth factors, WBCs, and platelets and fewer RBCs than venous blood.

  • View in gallery

    Mean ± SEM subjective lameness grades (A) and AI-VFP (B) in horses treated with saline (0.9% NaCl) solution (n = 20 [control]; black bars) or APS (20; gray bars) at days 0, 7, and 14 after intra-articular injection. *Within a time point, value is significantly (P < 0.05) different between control and treatment groups. †Within a group, value is significantly (P < 0.05) different, compared with day 0.

  • View in gallery

    Mean ± SEM range of joint motion (A), pain flexion score (B), joint circumference (C; percentage change from day −1), and joint swelling score (D) in horses treated with saline solution (n = 20 [control]; black bars) or APS (20; gray bars). †Within a group, value is significantly (P < 0.05) different, compared with day −1. See Figure 2 for remainder of key.

  • View in gallery

    Mean ± SEM total protein concentration (A), WBC count (B), percentage of neutrophils (C), IL-1β concentration (D), TNFα concentration (E), and IL-1 receptor antagonist concentration (F) in joint fluid of horses treated with saline solution (n = 20; black bars) or APS (20; gray bars) at days 0 and 14. See Figure 2 for remainder of key.

  • View in gallery

    Mean ± SEM subjective scores assigned by clients before (white bars) and 3 months (gray bars) and 1 year after (black bars) intra-articular APS injection in horses. *Significant (P < 0.05) decrease in scores at 3 months and 1 year, compared with baseline (before APS injection). †Significant (P < 0.05) increase in scores at 1 year, compared with 3 months.

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Evaluation of a single intra-articular injection of autologous protein solution for treatment of osteoarthritis in horses

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  • 1 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.
  • | 2 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.
  • | 3 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.
  • | 4 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.
  • | 5 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.
  • | 6 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.
  • | 7 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.
  • | 8 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.
  • | 9 South Towns Equine, 925 Quaker Rd, East Aurora, NY 14052.
  • | 10 Cleveland Equine Clinic, 3340 Webb Rd, Ravenna, OH 44266.

Abstract

Objective—To evaluate intra-articular autologous protein solution (APS) for the treatment of osteoarthritis in horses.

Animals—40 client-owned horses with naturally occuring osteoarthritis.

Procedures—APS was generated from a dual-device system that concentrated plasma and WBC proteins and enriched platelet growth factors. Horses were randomly assigned to receive an intra-articular injection of 5 mL of saline (0.9% NaCl) solution (n = 20) or APS (20), exercised on a treadmill, and evaluated on the basis of lameness grades, kinetic gait analysis, joint circumference, and range of motion for 14 days. Horses that received saline solution were administered APS at termination of the study, and clients scored horses for lameness and discomfort before, 12 weeks after, and 52 weeks after the APS injection.

Results—The APS group had significant improvements in lameness grade, asymmetry indices of vertical peak force, and range of joint motion by 14 days, compared with baseline or control group values. No adverse effects associated with APS treatment were evident. Clients assessed lameness and comfort as improved at 12 and 52 weeks. The APS had greater likelihood (OR, 4.3 to 30.0) of a therapeutic response in horses with a lameness score < 4, < 10% vertical force asymmetry, or absence of marked osteophyte formation, subchondral sclerosis, or joint space narrowing. Concentration of interleukin-1 receptor antagonist in APS was 5.8 times that in blood.

Conclusions and Clinical Relevance—Intra-articular administration of APS can be considered an effective treatment option for equine osteoarthritis, with the potential for disease-modifying effects.

Abstract

Objective—To evaluate intra-articular autologous protein solution (APS) for the treatment of osteoarthritis in horses.

Animals—40 client-owned horses with naturally occuring osteoarthritis.

Procedures—APS was generated from a dual-device system that concentrated plasma and WBC proteins and enriched platelet growth factors. Horses were randomly assigned to receive an intra-articular injection of 5 mL of saline (0.9% NaCl) solution (n = 20) or APS (20), exercised on a treadmill, and evaluated on the basis of lameness grades, kinetic gait analysis, joint circumference, and range of motion for 14 days. Horses that received saline solution were administered APS at termination of the study, and clients scored horses for lameness and discomfort before, 12 weeks after, and 52 weeks after the APS injection.

Results—The APS group had significant improvements in lameness grade, asymmetry indices of vertical peak force, and range of joint motion by 14 days, compared with baseline or control group values. No adverse effects associated with APS treatment were evident. Clients assessed lameness and comfort as improved at 12 and 52 weeks. The APS had greater likelihood (OR, 4.3 to 30.0) of a therapeutic response in horses with a lameness score < 4, < 10% vertical force asymmetry, or absence of marked osteophyte formation, subchondral sclerosis, or joint space narrowing. Concentration of interleukin-1 receptor antagonist in APS was 5.8 times that in blood.

Conclusions and Clinical Relevance—Intra-articular administration of APS can be considered an effective treatment option for equine osteoarthritis, with the potential for disease-modifying effects.

Osteoarthritis is one of the most important joint diseases in horses because of a high prevalence and major economic impact on the equine industry.1,2 Even though various systemically or locally administered medications such as NSAIDs (either orally administered paste3 or topically administered ointment4), corticosteroids,5,6 polysulfated glycosaminoglycan,7,8 and hyaluronan8,9 have been used, the treatment and management of equine osteoarthritis remain challenging.10,11

Regenerative treatment with autologous products, including PRP, has been broadly applied for various musculoskeletal disorders in horses, including superficial digital flexor tendinitis,12,13 suspensory desmitis,14 and wound healing.15 Autologous protein solution is a new modified blood product in this category. Platelets contain high concentrations of growth factors released from alpha granules, such as TGF-β, epidermal growth factor, and platelet-derived growth factor. Conversely, ACS is a separate form of blood product containing high concentrations of growth factors and anti-inflammatory cytokines, such as IL-1 receptor antagonist, IL-10, IGF-1, and TGF-β.16 In horses, ACS has been reported to improve clinical signs of osteoarthritis in horses with experimentally induced chip fractures,17 purportedly through increased concentration of IL-1 receptor antagonist, a protein recognized to influence equine osteoarthritis.18 Autologous conditioned serum has been prepared by incubating blood overnight in a container filled with glass beads. In humans, recombinant human IL-1 receptor antagonista has been approved for treatment of rheumatoid arthritis; however, a recent study19 failed to reveal significant improvement in clinical measurements of knee osteoarthritis. Treatment of osteoarthritis may require the targeting of multiple inflammatory and degradative pathways. A recent report20 has been published of in vitro research that supported this assessment in that single recombinant anti-inflammatory cytokines were not as effective at inhibiting the production of matrix metallopeptidase-13 from chondrocytes as multiple recombinant anti-inflammatory cytokines (targeting IL-1 and TNFα). Specifically, the simultaneous analysis of a cytokine and its binding receptor, such as IL-1 with IL-1 receptor antagonist and TNFα with TNF receptor 1, may provide more insight into relative functional amounts of cytokine active locally.20

Intra-articular use of autologous products like PRP is a relatively novel but promising therapeutic strategy.21 In vitro, PRP induces chondrogenesis by stimulating proliferation of chondrocytes and extracellular matrix synthesis of proteoglycans and type II collagen.22 Platelet-rich plasma could also induce chondroprotection by increasing the production and secretion of hyaluronic acid from synoviocytes23 and inhibiting inflammatory processes in chondrocytes.24 In vivo, PRP has been administered intra-articularly to treat osteoarthritis in humans,25–28 dogs,21 and horsesb; however, its efficacy and safety have not been comprehensively investigated in a prospective, randomized, masked, placebo-controlled clinical trial in horses. In addition, prognostic factors enabling clinicians to predict responses to intra-articular administration of PRP have not been investigated for equine osteoarthritis. It would be advantageous to develop an autologous treatment that further refined and combined the high concentrations of anti-inflammatory cytokines and growth factors as well as the point-of-care treatment options of PRP.

Autologous protein solution is a therapeutic blood product designed to achieve these goals and is created in a 2-step process. Blood is first processed in an APS separator,c which sequesters WBCs and platelets in a small fraction of plasma. This separated blood component is then transferred to an APS concentrator,c which desiccates the product by filtration through polyacrylamide beads. Autologous protein solution contains not only concentrated WBCs and platelets, but also plasma proteins. For this reason, APS from humans has significantly greater concentrations of platelet- and plasma-derived growth factors and cytokines (eg, TGF-β, epidermal growth factor, platelet-derived growth factor, IL-10, or IGF-1), compared with PRP products.20,29 In addition, neither the humand nor the equine APS producte requires the 24-hour incubation period.16,29 In a previous study,20 APS inhibited matrix metallopeptidase-13 production of stimulated human articular chondrocytes by multiple pathways, including IL-1β and TNFα. The purpose of the study reported here was to evaluate the efficacy, safety, and prognostic factors associated with use of intra-articular APS injection treatment of naturally occurring osteoarthritis in horses.

Materials and Methods

Study design—The study was designed as a prospective, randomized, masked, placebo-controlled clinical trial. Forty horses (age range, 2 to 16 years) with osteoarthritis in various high-motion joints were assigned, by use of an online random number generator,f into either a treatment group (n = 20) or control group (20) in the order they entered the study. At day 0, the horses in treatment and control groups received an intra-articular injection of APS or saline (0.9% NaCl) solution,g respectively. All horses were evaluated by use of subjective lameness grades from 0 to 5 on the American Association of Equine Practitioners lameness scale (days 0, 7, and 14), kinetic gait analysis (days −1, 7, and 14), joint signs of pain and swelling assessments (days 0, 4, 7, 10, and 14), joint fluid and blood analysis (days 0 and 14), and radiography (days −1 and 14). Horses were exercised twice weekly on a treadmill (at days −1, 4, 7, 10, and 13). At day 14, owners of horses in the control group were offered the option for their horses to receive an intra-articular injection of APS following the same treatment protocol as the APS treatment group horses. The horses were evaluated by the clients via a questionnaire for lameness, comfort, and adverse events before and 12 and 52 weeks after the APS treatment.

Inclusion criteria at admission included clinical evidence of osteoarthritis (enlarged joint with reduced range of joint motion accompanied by signs of pain) predominantly in 1 joint of 1 limb, observable lameness in the osteoarthritic limb at the trot (lameness grade of 2 to 4), a minimum 4-week history of lameness at the trot, radiographic evidence of osteoarthritis (osteophytes, subchondral bone changes, joint space narrowing, or irregular joint space) obtained within 30 days, no intra-articular injections or articular surgeries within 60 days, and no medications or orally administered supplements within 7 days. Exclusion criteria included presence of a fracture, active infection, or history of chronic infection associated with the joint.

All procedures were approved by the Institutional Animal Care and Use Committee at The Ohio State University. Informed consent was obtained from owners of horses before enrollment in the study.

Validation and characterization of APS in horses—Blood from 5 healthy adult horses (age, 6 years) was processed separately with 5 APS kits.h Each kit consisted of a separation devicec and a concentration device (Figure 1).c After processing through the first device, the blood product is an intermediate cell solution, specifically a cell and platelet solution in plasma. The intermediate cell solution is further processed through the second device to produce concentrated APS. The intermediate cell solution and final APS product were analyzed by CBC and for concentration of IL-1 receptor antagonist (equine), soluble TNF receptor 1 (human), and IL-10 (equine) measured with commercially available ELISA kits.g

Figure 1—
Figure 1—

Illustrative representation of relative quantities of cellular and protein contents in equine venous blood (left), intermediate cell solution (middle), and APS (right). Notice that APS contains higher amounts or numbers of anti-inflammatory proteins, growth factors, WBCs, and platelets and fewer RBCs than venous blood.

Citation: American Journal of Veterinary Research 75, 2; 10.2460/ajvr.75.2.141

Horses—Forty client-owned horses, with diagnosed osteoarthritis in the metacarpophalangeal or metatarsophalangeal (fetlock) joint, carpus, stifle joint, or tarsocrural or proximal intertarsal joints, were enrolled in the study between April and December 2011. Diagnosis of osteoarthritis was determined within 30 days prior to arrival for enrollment by use of lameness and radiographic examination and, when indicated, diagnostic anesthesia to confirm the location of the lameness. In past medical records, lameness had been located to the joint by use of regional or intra-articular anesthesia in all horses.

Treatment—For horses in the treatment group, APS was produced at day 0 by centrifugation and processing 110 mL of venous blood on the basis of the manufacturer's protocol at 30 to 60 minutes prior to the injection with 2 APS kitsf/horse (Figure 1). Briefly, 55 mL of jugular venous blood was aspirated into each of 2 syringes containing 5 mL of acid citrate dextrose.h After thorough mixing, the blood was transferred to 2 separation devices and centrifuged for 15 minutes. Then, approximately 5 to 6 mL of intermediate cell solution was transferred to each of 2 APS concentration devices, where the cell solutions were mixed with polyacrylamide beads and centrifuged for 2 minutes. This process resulted in a final volume of 5.0 to 6.0 mL of APS. For the control group, 5.0 mL of saline solution was injected in each control joint.

At day 0, joint fluid aspiration followed by intra-articular injection of APS (treatment group) or saline solution (control group) was performed by 1 investigator (ALB), who was kept unaware of treatment assignments by disguising and capping the syringe contents with aluminum foil. The blood draws and device processing were performed by staff personnel who were not unaware of treatment groups. Aspiration of and injection into the joints were performed by passing a needle percutaneously into the joint. Joint fluid aspiration confirmed needle placement in the joint, and up to 5.0 mL was aspirated prior to the injection. The amount of fluid withdrawn was recorded, and the sample was frozen at −80°C. No bandages were applied after the joint aspiration and injection of APS or saline solution. For the control group, joint fluid aspiration followed by intra-articular injection of APS was performed at day 14 following the same protocol as for the APS group. For the APS group, joint fluid aspiration was performed at day 14.

Horses were exercised twice per week (at days −1, 4, 7, 10, and 13) on a high-speed equine treadmilli with the following regimen: walking (7.2 km/h) for 10 minutes, followed by trotting (14.4 km/h) for 10 minutes and walking (7.2 km/h) for 10 minutes. If horses were or became lame at the walk on the treadmill, they were not trotted and were exercised at the walk for only 20 minutes. Throughout the experiment, the horses were housed individually in stalls in a temperature-controlled environment, fed a commercial grain mixture twice daily, and provided access to hay and water ad libitum. No medications or supplements were given during the 2-week period.

Lameness examination—Evaluation of lameness was performed on the basis of the American Association of Equine Practitioners lameness scale at days 0, 7, and 14 by 1 investigator (ALB) unaware of group assignments. In a 30-m-long lameness examination area with a hard paved surface, lameness was graded with the horse being walked in 1 trip (back and forth), trotted in 2 trips, and trotted in 1 trip after joint flexion, with 30 seconds for flexion tests of the distal portion of the limb (osteoarthritis in fetlock joint) and 60 seconds for flexion tests of the proximal portion of the limb (osteoarthritis in carpus, tarsus, or stifle joint). Lameness grades were as follows: 0 = not lame at the walk and trot, 1 = intermittently lame at the trot, 2 = mildly lame at the trot, 3 = obviously lame at the trot, 4 = lame at the walk and trot, 5 = partially or fully non–weight bearing at the walk.

Kinetic gait analysis—An examination aisle (3 × 20 m) with an in-ground stationary force platej and computer analysis systemk was used for kinetic gait analysis of each horse at days −1, 7, and 14.30,31 The central force plate and the aisle were covered by a mat to prevent horses from slipping and to avoid recognition of the plate, and the mat was cut around the force plate to prevent distracting forces. Data sampling rate was 500 Hz, and data were filtered by a stop-band frequency of 80 Hz. Five valid repetitions were recorded for both the lame limb and contralateral limb while the horse was trotting. A valid measurement was defined as a passage by the horse over the force plate during which the hoof of the limb of interest fully contacted the surface of the plate and the gait velocity was within the range of 2.5 to 3.5 m/s. For each repetition, VFP was calculated by the computer analysis system on the basis of the vertical force-time curve, where VFP values were normalized by horses' weights. The mean for data of 5 repetitions was calculated for both the lame and contralateral limbs (VFPLame and VFPContralateral). To adequately compare the data among different joints in forelimbs and hind limbs, AI-VFP between lame and contralateral limbs was calculated within horse as follows30:

article image

Evaluation of signs of joint pain during flexion and joint swelling—To measure the range of joint motion without signs of pain, the treated joint was flexed until each horse raised its head or moved the limb in resistance, and a handheld goniometer was positioned so that the center of the rotation was over the center of rotation of the joint.30 The maximal angle of flexion was obtained for 3 consecutive evaluations, and the mean was calculated and expressed as percentage change from day 0 values. Signs of pain during flexion were graded on a scale from 0 to 3 (0 = minimal resistance during flexion, 1 = mild resistance, 2 = moderate resistance, and 3 = marked resistance). Joint circumference (mm) was measured with a tape at the following locations: at the height of the midpoint of the proximal sesamoid bones for the fetlock joint, at the height of antebrachiocarpal and midcarpal joints for the carpus, at the height of the femorotibial joint or distal point of the patella (femoropatellar joint) for the stifle joint, and at the height of the middle of the medial trochlear ridge of the tarsus (tarsocrural joint) for the tarsus. Joint circumference was obtained for 3 consecutive evaluations, and the mean was calculated and expressed as percentage change from day 0 values. Joint swelling was graded by means of a scale ranging from 0 to 4 (0 = no swelling, 1 = minimal swelling localized to the injection site, 2 = mild swelling localized to the level of the joint near the injection site, 3 = moderate swelling extending < 10 cm proximally and distally from the injection site, and 4 = marked swelling extending > 10 cm proximally and distally from the injection site).

Synovial fluid and blood analysis—Synovial fluid was collected from the joints at days 0 and 14 and placed in an glass tubek containing EDTA and in a plastic cryopreservation vial.l Samples in the EDTA-containing tubes were immediately evaluated microscopically for total WBC count (cells/L), WBC differential (percentage of polymorphonuclear and mononuclear cells), and total protein concentration, which was measured with a refractometer.m Samples in the cryopreservation tubes were frozen at −80°C and thawed once for ELISA. By use of commercially available kits,g,n concentrations of IL-1β (equine), TNFα (equine), IL-1 receptor antagonist (equine), and soluble TNF receptor 1 (human) were measured for each sample. Complete blood counts and serum biochemical analysis of jugular venous blood samples were performed at days 0 and 14.

Radiographic examination—Routine radiographic images of the affected joint were obtained at days −1 and 14 and evaluated by an investigator (LSZ) unaware of group assignments. Each image was assessed semiquantitatively (0 = none, 1 = minimum, 2 = mild, 3 = moderate, or 4 = marked) for osteophyte formation, joint narrowing, and sclerosis and irregularity of subchondral bone as well as the presence of subchondral bone cysts.

Evaluation by clients—All horses (n = 40) were subjectively evaluated by the clients, by use of a mailed questionnaire, before and 12 and 52 weeks after the APS treatment for lameness, comfort levels, and adverse events. Seven categories were graded on a scale ranging from 0 to 10: degree of lameness (0 = sound; 10 = won't bear weight), comfort at rest in stall (0 = comfort; 10 = discomfort), comfort at turnout (0 = comfort; 10 = discomfort), general attitude (0 = bright, alert, and interactive; 10 = dull, signs of depression, and noninteractive), appetite (0 = eats all food and looks for more; 10 = will not eat), body condition (0 = excellent; 10 = poor), and hair condition (0 = excellent; 10 = poor).

Statistical analysis—All statistical analyses were performed by use of commercially available statistical software programs.o,p The effects of injections on lameness scores, kinetic gait measurements, signs of joint pain and swelling, and synovial fluid values were analyzed by use of repeated-measures ANOVA. Explanatory variables included horse, limb (forelimb or hind limb on the right or left), joint (forelimb fetlock joint, hind limb fetlock joint, carpus, stifle joint, or tarsus), time points, and treatment (saline solution or APS). Each horse was treated as a random variable, and repeated measures (time points) were considered to be nested within the horse.

For the lameness grade data at days 7 and 14, the changes in subjective lameness grades were categorized as improved (lameness grade was decreased from day 0), unchanged (lameness grade was equal to day 0), worsened (lameness grade was increased from day 0), or became sound (lameness grade became 0). For the kinetic gait analysis data at days 7 and 14, the changes in AI-VFP were categorized as improved (AI-VFP was decreased > 50% from day 0), unchanged (AI-VFP was decreased or increased < 50% from day 0), or worsened (AI-VFP was increased > 50% from day 0). The categories of responses in lameness grade (days 7 and 14) and AI-VFP (days 7 and 14), such as improved, unchanged, worse, or became sound were compared between the explanatory variables for frequencies by the χ2 test.

The potential prestudy prognostic variables of lameness and radiographic severity were correlated with 2-week, 12-week, and 52-week outcomes (good vs poor outcomes) by crude ORs and the Fisher exact test. The 2-week outcomes were defined as good (decreased lameness grade between weeks 0 and 2) or poor (increased or unchanged lameness grade between weeks 0 and 2). The 12-week and 52-week outcomes were defined as good (more than half reduction of total client questionnaire scores, compared with before APS injection) or poor (increased, unchanged, or less than half reduction of total client questionnaire scores, compared with before APS injection). If a horse was euthanized because of a reason associated with the treated osteoarthritic joint, it was also considered as poor outcome. Values of P < 0.05 were considered significant for all analyses.

Results

Characterization of APS in horses—Autologous protein solution contained significantly greater concentrations of IL-1 receptor antagonist (5.8-fold increase; P = 0.002), soluble TNF receptor 1 (3.6-fold increase; P = 0.013), and IL-10 (3.4-fold increase; P = 0.031), compared with concentrations in blood (Table 1). Also, APS contained significantly (P < 0.001) increased WBC (12.1-fold increase) and significantly (P < 0.001) decreased RBC count (5-fold decrease) but no significant (P = 0.204) difference in platelet counts (1.6-fold increase), compared with concentrations in blood.

Table 1—

Mean ± SEM values of selected variables in blood and APS obtained from 5 horses.

VariableBloodAPSAPS:blood ratio     
Cellular components     
 WBC count (× 106/mL)6.2 ± 0.375.0 ± 4.8*12.1     
 Platelet count (× 106/mL)151 ± 13243 ± 53*1.6     
 RBC count (× 106/mL)6.6 ± 0.71.3 ± 0.4*0.2     
Anti-inflammatory proteins        
 IL-1 receptor antagonist (pg/mL)303 ± 1751,757 ± 100*5.8     
 Soluble TNF receptor 1 (pg/mL)4.6 ± 0.116.9 ± 2.9*3.6     
 IL-10 (ng/mL)970 ± 4793,271 ± 807*3.4     

Significant (P < 0.05) difference from blood value.

Horses—There were no significant differences in affected limbs, affected joints, or breed distribution between the treatment and control groups (Table 2). No significant differences were detected in age, body weight, or height between the treatment and control groups.

Table 2—

Comparison of signalment variables of 40 client-owned horses with naturally occurring osteoarthritis in various joints; 20 horses were treated with intra-articular injection of saline (0.9% NaCl) solution (control), and 20 horses were treated with intra-articular injection of APS.

SignalmentControlAPSP value
Age (y)  0.634
 Mean ± SD9.25 ± 4.638.50 ± 5.22 
 Median (range)8.5 (4–24)7.0 (2–21) 
Height (m)  0.961
 Mean ± SD1.59 ± 0.071.59 ± 0.06 
 Median (range)1.55 (1.52–1.72)1.63 (1.44–1.66) 
Weight (kg)  0.479
 Mean ± SD535 ± 50521.36 ± 68.64 
 Median (range)542.73 (432.27–608.18)505.91 (404.55–630.45) 
Sex (No. of horses)   
 Sexually intact male10 
 Castrated male1514 
 Sexually intact female46 
Breed (No. of horses)   
 Thoroughbred24 
 Standardbred14 
 Quarter Horse138 
 Warmblood32 
 Other12 
Forelimb vs hind limb (No. of horses)  0.057
 Forelimb126 
 Hind limb814 
Limb  0.292
 Left forelimb63 
 Right forelimb63 
 Left hind limb46 
 Right hind limb48 
Joint (No. of horses)  0.377
 Metacarpophalangeal joint84 
 Metatarsophalangeal joint12 
 Carpus42 
 Stifle joint711 
 Tarsus01 

Treatment—No complications occurred in the preparation or injections of APS. The volumes of joint fluid aspiration and APS injection did not have a significant effect on any outcome variables. A mean ± SEM of 1.37 ± 0.12 mL of joint fluid was obtained before injection, and 14 days later, 1.69 ± 0.20 mL was obtained in the APS group and 1.66 ± 0.16 mL was obtained in the control group. Joint fluid was collected in all aspiration attempts from stifle joints, but was of insufficient volume to analyze in 11 of 41 aspirates.

Lameness examination—The lameness grades in APS-treated limbs were significantly (P < 0.001) improved at days 7 and 14, compared with baseline and compared with the corresponding time points in the control group (Figure 2). The lameness grades in the control group were not significantly changed between days 0, 7, and 14. The APS group had a significantly greater number of horses with sound gait (lameness grade = 0; P < 0.001) or improved gait (lameness grade decreased from day 0; P < 0.001) at days 7 and 14, compared with the control group (Table 3).

Figure 2—
Figure 2—

Mean ± SEM subjective lameness grades (A) and AI-VFP (B) in horses treated with saline (0.9% NaCl) solution (n = 20 [control]; black bars) or APS (20; gray bars) at days 0, 7, and 14 after intra-articular injection. *Within a time point, value is significantly (P < 0.05) different between control and treatment groups. †Within a group, value is significantly (P < 0.05) different, compared with day 0.

Citation: American Journal of Veterinary Research 75, 2; 10.2460/ajvr.75.2.141

Table 3—

Frequency (No. of horses) of change in lameness grade in the same horses as in Table 2 at days 7 and 14, compared with day 0.

 Day 7Day 14    
VariableControlAPSP valueControlAPSP value
Degree of change in lameness grade  < 0.001  < 0.001
 Worsened40 60 
 Unchanged156 107 
 Improved110 44 
 Became nonlame04 09 
Degree of change in AI-VFP  0.024  < 0.001
 Worsened72 72 
 Unchanged119 126 
 Improved29 112 

Kinetic gait analysis—The AI-VFP values in the APS group were significantly improved at day 14, compared with those at day −1 (P = 0.046) and compared with the corresponding time points in the control group (P = 0.021 and 0.002, respectively; Figure 2). The AI-VFP values in the control group were significantly (P = 0.029) worse at day 14, compared with those at day −1. Also, the APS group had a significantly greater number of horses with improved gait symmetry (AI-VFP became < 5% or the baseline value decreased > 50%) at days 7 (P = 0.024) and 14 (P < 0.001), compared with the control group (Table 3).

Joint pain on flexion and joint swelling—The range of joint motion without signs of pain and the signs-of-pain score on flexion in the APS treatment group were significantly improved at days 4 to 14, compared with those values on day 0 (P = 0.003) and compared with the control group at days 7 to 14 (P < 0.05; Figure 3). The range of joint motion and the signs-of-pain score on flexion in the control group were significantly worse at days 4 to 14, compared with those values day 0 data. Joint circumference and swelling scores were not significantly different between the groups at any time points, although there was a significant increase from baseline in both groups at day 4.

Figure 3—
Figure 3—

Mean ± SEM range of joint motion (A), pain flexion score (B), joint circumference (C; percentage change from day −1), and joint swelling score (D) in horses treated with saline solution (n = 20 [control]; black bars) or APS (20; gray bars). †Within a group, value is significantly (P < 0.05) different, compared with day −1. See Figure 2 for remainder of key.

Citation: American Journal of Veterinary Research 75, 2; 10.2460/ajvr.75.2.141

Synovial fluid and blood analysis—The joint fluid total protein concentration in the control group was significantly (P = 0.005) greater at day 14, compared with that on day 0 (Figure 4), whereas the joint fluid total WBC count and percentage of neutrophils were not significantly different among time points or between groups. In addition, the joint fluid concentrations of IL-1β, TNFα, and IL-1 receptor antagonist were not significantly different among time points or between groups. Also, the ratios of IL-1β and IL-1 receptor antagonist were not significantly different among time points or between groups. All blood variables in all horses at all time points were within reference range, and no blood analysis variables were significantly different between treatment and control groups or compared with day 0 values within the treatment group.

Figure 4—
Figure 4—

Mean ± SEM total protein concentration (A), WBC count (B), percentage of neutrophils (C), IL-1β concentration (D), TNFα concentration (E), and IL-1 receptor antagonist concentration (F) in joint fluid of horses treated with saline solution (n = 20; black bars) or APS (20; gray bars) at days 0 and 14. See Figure 2 for remainder of key.

Citation: American Journal of Veterinary Research 75, 2; 10.2460/ajvr.75.2.141

Radiographic examination—The radiographic signs of osteoarthritis were not significantly different between APS and control groups at day 0 and were not significantly changed between day −1 and day 14 in either group (Table 4). There were no radiographic signs of adverse effects associated with APS injection.

Table 4—

Results (median [range] or proportion) of radiographic assessment of osteoarthritic joints of the same horses as in Table 2.

VariableControlAPSP value     
Periarticular osteophyte score     
 Day 02.5 (0–4)2 (1–4)0.880     
 Day 142.5 (0–4)2 (1–4)0.852     
Subchondral sclerosis score        
 Day 01.5 (0–4)1 (0–4)0.590     
 Day 141.5 (0–4)1 (0–4)0.847     
Articular irregularity score        
 Day 01.5 (0–3)1 (0–4)0.189     
 Day 141.5 (0–3)1 (0–4)0.149     
Joint space narrowing score        
 Day 00 (0–2)0 (0–4)0.129     
 Day 140 (0–2)0 (0–4)0.153     
Total radiographic score        
 Day 05.5 (1–11)5 (1–12)0.867     
 Day 145.5 (1–11)5 (1–12)0.932     
 Subchondral bone cyst        
 Day 06/202/200.235     
 Day 146/202/190.235     
Progression of osteoarthritis between day 0 and day 14        
 Improved0/200/191.000     
 Unchanged20/2019/19      
 Worsened0/200/19      

Evaluation by clients—All 40 horses completed the 12-week evaluation. Two horses were lost to follow-up at the 52-week evaluation time point (1 in the APS group and 1 in the control group). Three horses were euthanized at 2 weeks, 7 weeks, and 8 months after discharge because of the progression of osteoarthritis. Of these, 2 horses euthanized at 2 and 7 weeks had severe lameness (lameness grade = 4) at baseline.

Client-evaluated subjective grades of lameness (P < 0.001 and P < 0.001, respectively), comfort at rest in the stall (P < 0.001 and P = 0.029, respectively), and comfort at turnout (P < 0.001 and P = 0.004, respectively) were significantly improved at the 12-week and 52-week follow-up evaluations on the questionnaire (Figure 5). The subjective grades of general attitude, appetite, body condition, and coat were not significantly changed between baseline and 12-week and 52-week evaluations by clients. No adverse events associated with APS injection were reported.

Figure 5—
Figure 5—

Mean ± SEM subjective scores assigned by clients before (white bars) and 3 months (gray bars) and 1 year after (black bars) intra-articular APS injection in horses. *Significant (P < 0.05) decrease in scores at 3 months and 1 year, compared with baseline (before APS injection). †Significant (P < 0.05) increase in scores at 1 year, compared with 3 months.

Citation: American Journal of Veterinary Research 75, 2; 10.2460/ajvr.75.2.141

Prognostication—Prestudy lameness grade and gait analysis variables (eg, AI-VFP) significantly predicted outcome at 14 days, radiographic scores for periarticular osteophytes and subchondral sclerosis significantly predicted outcomes at 12-week, and joint space narrowing significantly predicted outcomes at 12 and 52 weeks (Table 5). A horse with no lameness observable at the walk (grade < 4), < 10% gait asymmetry, or no radiographic signs of moderate to severe osteophytes, subchondral sclerosis, or joint space narrowing was 4 to 30 times as likely (OR, 4.3 to 30.0) to have a therapeutic improvement after APS treatment. Of the 13 horses with good outcomes at 2 weeks, 9 horses maintained the good outcomes at 12 weeks, and 6 horses maintained the good outcomes at 52 weeks. Of the 7 horses with poor outcomes at 2 weeks, 4 continued to have poor outcomes at both 12 and 52 weeks (the other 3 horses were improved to have good outcomes). Interestingly, all 4 of these horses had the prestudy radiographic sign of joint space narrowing (score ≥ 1).

Table 5—

Prestudy prognostic variables and crude ORs for good outcomes at 14 days, 12 weeks, and 52 weeks in horses with osteoarthritis treated with an APS solution.

 14 days (n = 20)12 weeks (n = 40)52 weeks (n = 38)         
VariableGood (n = 13)Poor (n = 7)ORP valueGood (n = 25)Poor (n = 15)ORP valueGood (n = 17)Poor (n = 21)ORP value
Gait (lameness grade)
 ≥ 414Referent0.03156ReferentNS47ReferentNS
 < 412316.0* 2092.7 13141.6 
AI-VFP            
 ≥ 10%15Referent0.007107ReferentNS87ReferentNS
 < 10%12230.0* 1581.3 9140.6 
Radiographic periarticular osteophyte score            
 ≥ 344ReferentNS810Referent0.050612ReferentNS
< 3933.0 1754.3* 1192.4 
Radiographic subchondral sclerosis score            
 ≥ 236Referent0.017109ReferentNS811ReferentNS
 < 210120.0* 1562.3 9101.2 
Radiographic joint space narrowing score            
 ≥ 134ReferentNS38Referent0.00919Referent0.023
 01034.4 2278.4* 161212.0* 

The day 14 outcomes were defined as good (decreased lameness grade between days 0 and 14) or poor (increased or unchanged lameness grade between days 0 and 14). The 12- and 52-week outcomes were defined as good (more than half reduction of total client questionnaire score, compared with score before APS injection) or poor (increased, unchanged, or less than half reduction of total client questionnaire score, compared with before APS injection). If a horse was euthanized for a reason associated with the treated osteoarthritic joint, it was also considered to be a poor outcome. Radiographic scores: 0 = none, 1 = minimum, 2 = mild, 3 = moderate, and 4 = marked.

Significant (P < 0.05) prestudy prognostic variable and OR.

NS = No significant (P > 0.05) difference between time periods.

Two horses were lost to follow-up at 52 weeks. Of the 38 remaining horses in the 52-week group, 3 were euthanized because of progression of osteoarthritis, and outcome was categorized as poor.

Discussion

This study revealed that intra-articular APS injection can induce clinical sign– modifying effects on osteoarthritis with significant improvement in lameness, weight-bearing symmetry, and range of joint motion without signs of pain. The results also indicated that intra-articular APS injection was associated with normal synovial fluid in osteoarthritic joints of exercised horses, gait improvement with exercise, and sustained client assessment of improvement in the 40 horses for up to 1 year. Saline solution–treated horses worsened with treadmill exercise, and their synovial fluid had characteristics of a transudate with the exercise protocol. Therapeutic responses were also indicated by clients' evaluations at 12 and 52 weeks after treatment for lameness and comfort levels at rest in the stall or at turnout. Because of the lack of masking and an untreated control comparison, this finding is of low evidenciary value but reflects positive client perception of the treatment. No adverse effects or worsening of clinical or radiographic signs associated with the APS injection was evident. Fibrosis of the joint capsule from increased concentrations of TGF-β after hyaluronan injection in mice has been noted,32 and increased TGF-β concentration has been measured in PRP from horses.33 Increased TGF-β concentration has not been reported with APS injection but could occur; however, fibrosis was not detected in the present study. Therefore, intra-articular APS injection can be considered as a promising sign-modifying option for osteoarthritis. Because of the short time required for APS preparation (< 20 minutes) and portable centrifugation equipment, intra-articular APS injection can be applicable as a quick, point-of-care, nonlaboratory, technically undemanding, ambulatory-based practice.

Intra-articular PRP injection has been studied in human and equine osteoarthritis25–28,b; however, this is the first report of a prospective, randomized, masked, placebo-controlled clinical trial to study efficacy and clinical responses to intra-articular APS injection for osteoarthritis. No safety issues were observed in this study. The regimented study design controlled the environment of the horses such that gait could be reliably and accurately measured and compared with a control group. The prospective and random assignment design minimized biased outcome assessments or inequitable case selection. In the literature, only the ACS treatment with intra-articular application has been studied in a prospective, randomized, masked, placebo-controlled clinical trial of human knee osteoarthritis.34,35 The present study revealed APS to be effective in treating the natural, chronic disease state of osteoarthritis.

There are several potential mechanisms of action for intra-articular application of APS. The previous in vitro work in human blood revealed that because APS contained a high concentration of autologous IL-1 receptor antagonist, it could reduce the effect of IL-1β and limit the production of IL-8 and TNFα.36 In addition, another study20 revealed that APS could inhibit production of matrix metallopeptidase-13 by IL-1- and TNFα-stimulated articular chondrocytes. Platelet-rich protein releasate reduces multiple inflammatory IL-1β–mediated effects on osteoarthritic chondrocytes, including inhibition of activation of nuclear factor κ-light-chain-enhancer of activated B cells,24 and other in vitro studies revealed that PRP can induce not only chondrogenesis by stimulating chondrocyte proliferation and extracellular matrix synthesis22 but also chondroprotection by increasing hyaluronic acid production by synoviocytes.23 In addition to concentrating platelet growth factors, the APS in the present study concentrated anti-inflammatory proteins produced by WBCs (IL-1 receptor antagonist) and soluble anti-inflammatory and anabolic plasma proteins known to be important in cartilage health, including IGF-1.37 The present study revealed that intra-articular APS injection had a sign-modifying effect of improving lameness and decreasing signs of joint pain on flexion. However, it remains to be determined whether the intra-articular APS injection has chondrogenic or chondroprotective effects; therefore, future investigation is warranted to determine the biological improvement in cartilage health or acceleration of cartilage healing, possibly with MRI or histologic evaluation.

To the authors' knowledge, this study is the first to correlate various clinical variables of osteoarthritis to clinical response following intra-articular APS or other biotherapy. Results indicated that therapeutic response to APS injection was significant if the horse did not have severe lameness (lameness grade ≥ 4), marked vertical force asymmetry (AI-VFP > 10%), or radiographic signs of moderate to severe periarticular osteophytes, subchondral sclerosis, or joint space narrowing. On the basis of this retrospective analysis, with the absence of these pretreatment conditions, the odds of achieving good clinical outcomes could be 4- to 30-fold greater (Table 5). Our study also had a greater, although not significantly greater, number of horses with hind limb lameness enrolled to receive APS, compared with forelimb lameness, so future studies could assess whether this is associated with response. A prospective study could be designed to validate these associations and determine whether the clinical criteria could be prognostic for a response to treatment. It is therefore probable that comprehensive patient evaluation prior to APS injection, including lameness examination, kinetic gait analysis, and radiography, can provide useful and reliable prognostic variables for screening of clinical cases in which lameness may be largely improved by intra-articular APS injection. This is in agreement with a previous equine studyb of intra-articular PRP injection that found a negative effect of the chronicity and radiographic change factors on athletic prognosis. Together, results of the present study indicated that APS injection is safe and effective for treatment of equine osteoarthritis.

ABBREVIATIONS

ACS

Autologous conditioned serum

AI-VFP

Asymmetry indices of vertical peak force

APS

Autologous protein solution

IGF

Insulin-like growth factor

IL

Interleukin

PRP

Platelet-rich plasma

TGF

Transforming growth factor

TNF

Tumor necrosis factor

VFP

Vertical force peak

a.

Kineret, Amgen Manufacturing Ltd, Thousand Oaks, Calif.

b.

Abellanet I, Prades M. Intraarticular platelet rich plasma (PRP) therapy: evaluation in 42 sport horses with OA (abstr), in Proceedings. 11th Int Cong World Equine Vet Assoc 2009. Available at: www.ivis.org/proceedings/weva/2009/72.pdf?LA=1. Accessed Sep 30, 2013.

c.

Biomet Biologics, Warsaw, Ind.

d.

Orthokine, Arthrex Inc, Naples, Fla.

e.

IRAP, Arthrex Inc, Naples, Fla.

f.

Random number generator, Stat Trek. Available at: stattrek.com/statistics/random-number-generator.aspx. Accessed Jun 3, 2013.

g.

R&D Systems Inc, Minneapolis, Minn.

h.

NStride Arthritis Treatment, Biomet Biologics, Warsaw, Ind.

i.

Sato I, Uppsala, Sweden.

j.

Kistler Instrument Corp, Amherst, NY.

k.

Vacutainer, Tyco Healthcare, Mansfield, Mass.

l.

Nalgene Nunc Corp, Rochester, NY.

m.

10482 ABBE Mark II Refractometer, Reichert Scientific Instruments, Buffalo, NY.

n.

Bethyl Laboratories Inc, Montgomery, Tex.

o.

SAS, version 9.2, SAS Institute Inc, Cary, NC.

p.

STATA, version 11, Stata Corp LP, College Station, Tex.

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Contributor Notes

Supported by Biomet Biologics, the 2011 summer research scholarship at The Ohio State University College of Veterinary Medicine, and the animal science internship program at The Ohio State University.

Presented in abstract form in the proceedings of the American College of Veterinary Surgeons Veterinary Symposium, National Harbor, Md, November 2012. Presented in abstract form at the 3rd North American Veterinary Regenerative Medicine Association Annual Meeting, Savannah, Ga, November 2012.

The authors thank Krista O'Shaughnessey and Jacy Hoeppner for product technical support; Megan Cline, Stephanie Vijan, Jill Stephens, Melissa Roemer, Lauren Eisemann, Rachel Wermertm, and Rebekah Sanchez-Hodge for technical assistance; and Drs. Harold Kemp, Jane Kennedy, Jim Chase, Patricia Balzer, Peter Meuse, Brett Berthold, Chris Beinlich, Don Palmer, Hugh Worsham, John Stanek, Keith Brown, Robert Schwartz, Thomas Beckman, Thomas Walrond, William Gesel, and William Wise for patient referral.

Address correspondence to Dr. Bertone (bertone.1@osu.edu).