Articular diseases are listed among the most common and important disorders in equine athletes. The disturbance of articular homeostasis and inefficacy of healing processes are crucial factors for the outcome of cartilaginous lesions. Homeostasis ultimately could be defined as a balance between anabolic (synthesis and constitution) and catabolic (degradation) responses to stresses imposed on articular cartilage.
Cytokines are essential components of intercellular communication and responsible for orchestrating molecular events involved in the anabolic and catabolic processes of the extracellular matrix of articular cartilage. Thus, characterizing the structure, functions, and interactions of cytokines is necessary for developing treatment strategies. These treatments would be able to modulate articular metabolism and reestablish tissue homeostasis.
Because the intent is to modulate catabolic and anabolic processes, the discovery of natural receptor antagonists or synthetic analogs with the capacity to block mediators of degenerative processes is of great clinical relevance. There is evidence indicating that IL-1β and TNF-α are the most important proinflammatory cytokines in joint diseases.1 Interleukin-1β and TNF-α can stimulate their own production as well as induce chondrocytes to produce other cytokines, PGE2, and matrix metalloproteases-1 and −3, which leads to degradation of the extracellular matrix of articular cartilage.2
One of the methods for inhibition of IL-1 is through a natural receptor antagonist of IL-1ra that competes for occupation of surface IL-1 receptors and acts in a dose-dependent manner.3 A commercial autologous conditioned serum product has been developed.4 In another study,5 investigators compared equine blood processed by the use of the commercial autologous conditioned serum and an IRAP device with blood processed by use of heparinized and non-heparinized tubes and found an increase in the concentration of anti-inflammatory cytokines (including IL-1ra and IL-10) in serum and plasma without the use of the commercial IRAP device. Investigators of another study6 processed plasma using heparinized glass tubes to prepare a blood-derived product and found that the plasma obtained after blood incubation and centrifugation elicited an antioxidant effect equivalent to that of the commercial autologous conditioned serum product for stimulated equine synovial fluid cells treated with both blood-derived products. This blood-derived product was named APP.7
Other treatment modalities (eg, platelet-rich products) have gained popularity for their high concentrations of growth factors. In addition, the use of PRP in the treatment of joint diseases has been advocated because it contains growth factors and also has anti-inflammatory effects.8
Hyaluronic acid is responsible for most of the viscoelastic properties of synovial fluid.9 Hyaluronic acid binds with high affinity to the cell membrane receptor CD44, which is expressed by many cell types including leukocytes and synoviocytes. Therefore, hyaluronic acid may modulate the inflammatory response by various mechanisms, such as downregulation of PGE2 synthesis10 and reduction of ROS by activation of protein kinase B in chondrocytes.11
The use of natural receptor antagonists or synthetic analogs that prevent progression of joint degeneration and act to restore joint homeostasis has been increasingly considered for horses as well as humans and other domestic animals. In a recent study12 of horses, joints with osteochondritis dissecans with no clinical signs of the disease had evidence of homeostatic imbalance as detected by an increase in the concentration of inflammatory cells and chondroitin sulfate in synovial fluid. In joints with clinical signs of osteochondritis, this fact became more evident because an increase in the concentration of low-molecular-weight hyaluronic acid was detected concomitantly.12 Without appropriate treatment, these joints are subject to develop secondary osteoarthritis characterized by the appearance of irreversible lesions in the articular cartilage.
Treatment of osteochondritis dissecans consists of arthroscopic removal of the osteochondral fragment. However, this procedure causes an increase in WBC counts and concentrations of total protein, PGE2, and chondroitin sulfate in synovial fluid as well as an oxidative burst during the immediate postoperative period.13,14 Thus, the use of adjuvant treatments immediately after arthroscopy would be beneficial in the recovery process to restore the loss of homeostasis caused by the primary disease and to minimize the inflammatory process attributable to the surgical procedure.
To the authors' knowledge, there have been no studies conducted to compare the anti-inflammatory and antioxidant effects of various treatment modalities, including blood products or commercial receptor antagonist products, for the reestablishment of articular homeostasis in joints after arthroscopic removal of osteochondrotic fragments. Therefore, the purpose of the study reported here was to evaluate the proinflammatory, anti-inflammatory, and antioxidant effects of PRP, IRAP, APP, and sodium hyaluronate on synovial fluid cells challenged in vitro and on osteochondrotic joints after arthroscopy.
Materials and Methods
Sample
For the in vitro experiment, 8 healthy equine tibiotarsal joints were used to obtain synovial fluid cells. All horses had no evidence of tibiotarsal joint disease as determined on the basis of results of lameness and radiographic evaluations. For the in vivo experiment, 40 tibiotarsal joints with osteochondritis dissecans in the intermediate ridge of the distal portion of the tibia in 25 client-owned horses of various breeds were used. The horses comprised 19 males and 6 females, and mean ± SD age was 3.2 ± 2.0 years. Informed consent was obtained for use of the client-owned horses. The study protocol was approved by an institutional animal care and use committee.
Drugs and blood-derived products
Homologous blood-derived products were used for the in vitro experiment, and autologous blood-derived products were used for the in vivo experiment. As reported in another study,15 for each PRP preparation, 20 mL of blood was collected from healthy horses and placed in tubes containing sodium citrate. Tubes were then centrifuged (150 × g for 5 minutes at 24°C). Supernatant was harvested and placed in tubes without anticoagulant; these tubes were centrifuged again (800 × g for 5 minutes at 24°C). Then, three-fourths of the supernatant plasma was removed, and the remaining fraction (PRP) was homogenized for 1 hour. Preparation of PRP in this manner concentrates platelets in amounts sufficient to elicit therapeutic responses without a concomitant increase in leukocytes.15
The IRAPa preparation was created in accordance with the manufacturer's instructions. Briefly, 50 mL of blood was collected from a jugular vein of a horse into a 60-mL syringe. The blood was then injected into the IRAP device at an angle of approximately 60°, homogenized, and incubated at 37°C for 24 hours. After incubation was completed, the blood was centrifuged (3,200 × g for 20 minutes). Serum was harvested and transferred to a 20-mL polypropylene syringe and filtered through a 0.22-μm filter.
For the APP preparation, a blood sample (20 mL/horse) was collected and placed in two 10-mL plastic sodium heparin tubes. Tubes were homogenized and incubated in a vertical position at 37°C for 24 hours. Tubes then were centrifuged (300 × g for 10 minutes at 24°C). Supernatant plasma was harvested and transferred to a 15-mL sterile conical centrifuge tube. Plasma was centrifuged again (900 × g for 10 minutes at 24°C) and then filtered through a 0.22-μm filter.7
A commercial sodium hyaluronate productb that contained 20 mg of sodium hyaluronate/2 mL was obtained. Molecular weight of the sodium hyaluronate was 3,000 kDa.
Culture of synovial fluid cells
Samples of synovial fluid were aseptically collected from a tibiotarsal joint of each of 8 horses and processed as described elsewhere.16 Briefly, 2 mL of each synovial fluid sample was suspended in 3 mL of DMEM supplemented with 10% (vol/vol) fetal bovine serum, 1% penicillin-streptomycin, 1% glutamine (200mM), 1% pyruvic acid, and 0.25% amphotericin B; samples were then plated in a 25-cm2 flask. Synovial cell samples were allowed to attach during incubation at 37°C in a humidified atmosphere containing 5% CO2. On the fourth day of incubation, the medium was aspirated to remove nonadherent cells and replaced with fresh medium. Cell cultures were maintained for sufficient time to allow investigators to monitor cell growth via inverted microscopy, and fresh medium was provided every 48 hours until cells reached > 80% confluence. At that point, the DMEM was removed from the flasks, and cells were washed with 2.0 mL of PBS solution. Then, 1 mL of 0.25% trypsin was added to each flask, and samples were incubated at 37°C for 5 minutes. Subsequently, 2.0 mL of culture medium supplemented with fetal bovine serum was added to inactivate the trypsin. The cell suspension was aspirated and transferred into a 15-mL conical tube and centrifuged (287 × g for 5 minutes) to remove the trypsin. For each sample, the cell pellet was resuspended in 1.0 mL of supplemented DMEM, and an aliquot (10 μL) was used for cell counting in a Neubauer chamber. The remaining cells were transferred into a 75-cm2 flask to which 9 mL of medium was added, and cells were incubated as previously described until they reached > 80% confluence. Trypsin was then used on the cell cultures as previously described. Cultured synovial fluid cells were used in in vitro assays.
In vitro inflammatory assay
Cells from each of the 8 horses were distributed in eight 24-well plates (final concentration, 1 × 105 cells/well). Then, 1.0 mL of DMEM-supplemented culture medium was added to each well, and the plates were incubated by use of standard conditions (37°C, approx 100% relative humidity, and 5% CO2) for 24 hours.
Subsequently, cells were microscopically examined to assess cell attachment to the plates. Cells were challenge exposed by replacing the medium with 1.0 mL of DMEM supplemented with 20 ng of LPS (Escherichia coli O55:B5). Plates were incubated by use of standard conditions for 3 hours. All wells of each plate were washed with 1.0 mL of PBS solution and randomly assigned to receive 1.0 mL of DMEM (control treatment), DMEM supplemented with phenylbutazone (10 μg/mL; assay positive control sample), PRP, IRAP, APP, or sodium hyaluronate (final concentration, 20% [vol/vol]). Each set of 4 treatments and 2 controls was randomly allocated in 6 wells of each of the 8 plates. Samples were evaluated in duplicate in the same corresponding sequence by use of 6 other wells in the same plate.
After samples were incubated for 24 hours, medium supernatant was collected, divided into aliquots, and frozen at −80°C until analyzed. Concentrations of PGE2,c IL-1β,d TNF-α,e IL-10,f and IL-1rag were measured with commercial kits.
In vitro oxidative burst
The oxidative burst from cultured synovial fluid cells was evaluated as described for cells cultured from whole blood.6,17,18 After cultured synovial fluid cells from the 8 equine tibiotarsal joints reached > 80% confluence, they were treated with trypsin, harvested, and centrifuged (395 × g for 5 minutes). Cells were resuspended in 1.0 mL of PBS solution, and an aliquot (10 μL) was used for cell counting in a Neubauer chamber. Briefly, 1 × 105 synovial cells/50 μL were mixed with 200 μL of 0.3mM dichlorofluorescein diacetate and 950 μL of sterile PBS solution (without stimulants) to induce basal ROS production. To evaluate the stimulated oxidative burst, identical incubation mixtures were prepared that contained the same number of cells, and incubation with dichlorofluorescein diacetate was followed by incubation with 100 μL of PMA (1 ng/mL) or LPS (1 mg/mL) as a stimulus. Additionally, respective tubes received 50 μL (final concentration, 5%) of sodium hyaluronate, PRP, IRAP, APP, or phenylbutazone (4.0 mg/mL; assay positive control sample). The same treatments were added to cells incubated with dichlorofluorescein diacetate followed by PMA or LPS and an amount of PBS solution sufficient to achieve a final volume of 1.1 mL. All cells were incubated by use of the same conditions.
Samples were incubated in a water bath at 37°C for 20 minutes. Samples were then centrifuged (454 × g for 5 minutes at 4°C), and the cell pellet was re-suspended in 150 μL of PBS solution and analyzed with flow cytometry.h Data from 10,000 events were collected by use of commercially available software.i Samples were evaluated in duplicate.
Effects of each treatment on oxidative burst from synovial cells were evaluated by direct measurements of mean fluorescence of green channels; the fluorescence was directly proportional to the amount of ROS generated. Fluorescence data were recorded on a logarithmic scale. Green fluorescence of dichlorofluorescein diacetate was measured at a mean ± SD of 530 ± 30 nm. Quantification of oxidative burst was estimated as the mean fluorescence intensity per cell.
In vivo inflammatory response
Diagnosis of tibiotarsal joints with osteochondritis dissecans in the intermediate ridge of the distal portion of the tibia for the 40 samples was made on the basis of results of radiographic and ultrasonographic examinations of 25 horses admitted for arthroscopy at the University of São Paulo School of Veterinary Medicine and Animal Science. Joints were randomly assigned to 5 treatment groups (8 joints/treatment). Opaque envelopes containing the treatment that each horse would receive were shuffled, sequentially numbered, then closed by an individual not involved in the study. Envelopes were opened in a sequential manner after each horse was evaluated arthroscopically. Joints received no treatment (control treatment), sodium hyaluronate, APP, PRP, or IRAP. A final volume of 2.0 mL of the selected treatment was administered into each joint at the end of the arthroscopic procedure.
Horses were anesthetized and positioned in dorsal recumbency for arthroscopy and treatment administration. A sample of synovial fluid (6 to 10 mL) was collected aseptically by arthrocentesis, and the joint was then distended with lactated Ringer solution to perform the arthroscopic procedure. Osteochondral fragments were identified, separated with an elevator, and grasped and removed with a rongeur. Osteochondritis dissecans fragment beds were then curetted until subchondral bone that had good ossification and vascularization was exposed. Joints were then copiously flushed to remove debrided fragments. Beginning during the preoperative period, amikacin (15 mg/kg, IV) and phenylbutazone (2.2 mg/kg, IV) were administered every 24 hours for 3 days.
A second sample of synovial fluid was collected 48 hours after surgery. Horses were restrained in a standing position. A sample of synovial fluid was collected aseptically by arthrocentesis from each of the treated joints.
Immediately after samples of synovial fluid were collected, they were centrifuged (2,000 × g for 15 minutes at 4°C). Supernatant was harvested and stored at −80°C until analyzed. Concentrations of PGE2, IL-1ra, IL-10, TNF-α, IL-1β, chondroitin sulfate, and hyaluronic acid were measured in synovial fluid samples. Concentrations of PGE2,c IL-1β,d TNF-α,e IL-10,f and IL-1rag were measured by use of ELISA kits.19–21 Assays were performed in accordance with the manufacturers’ recommendations. Concentrations of chondroitin sulfate and hyaluronic acid were determined, as described elsewhere.12,22,23 In brief, synovial fluid samples (100 μL) were subjected to proteolysis, and debris was removed by centrifugation (3,000 × g for 15 minutes at 24°C). Supernatant was harvested, freeze-dried, and resuspended in water (50 μL). Aliquots (5 μL) were subjected to agarose gel electrophoresis. Gels were stained with toluidine blue in 1% acetic acid and then in sodium acetate buffer (pH, 5.0). Band density was quantified with densitometry.j
Molecular weight of hyaluronic acid was determined by electrophoresis in 1% agarose gels in 0.04M tris-acetate-EDTA buffer (0.02M acetate and 0.01M EDTA; pH, 8.0), as described elsewhere.24,k Gels were calibrated with hyaluronic acids of known molecular weights; 2 hyaluronic acid standardsl (rooster comb hyaluronic acid, 2 mg/mL [molecular weight, approx 800 kDa]; and bovine trachea hyaluronic acid, 1 mg/mL [molecular weight, approx 20 kDa]) were included on each gel. Bromophenol blue was used as an indicator of the migrated distance. After electrophoresis was completed, gels were stained by immersion in 0.1% toluidine blue in 0.025M sodium acetate (pH, 5.0) for 15 minutes. Excess dye was removed by washing with 0.025M sodium acetate. Migration patterns and distances were obtained with densitometry.j Migration distance was inversely proportional to the logarithm of the hyaluronic acid molecular weight.
Statistical analysis
The Shapiro-Wilk test was used to evaluate distribution of the data. For the in vitro experiment, all variables were not normally distributed; therefore, nonparametric tests were used. The Friedman test was used to evaluate the difference among treatments for each variable, followed by the Wilcoxon test when there were significant differences (cell cultures were treated as dependent sample units). Comparisons among horses were made by use of the Kruskal-Wallis test. For the in vivo experiment, only 2 variables (TNF-α and IL-10 concentration) were normally distributed. To compare treatments and test for a treatment-by-time interaction, a 2-way ANOVA with treatment and time as fixed effects was used. The Kruskal-Wallis test was used, followed by the Mann-Whitney U test as a post hoc test, to evaluate differences among treatments. Time was considered a dependent factor and evaluated by use of the Wilcoxon test. All analyses were conducted with commercial statistical software.m For all analyses, differences were considered significant at P < 0.05.
Results
In vitro inflammatory response
To confirm validity of the assays, PGE2 and cytokine concentrations were also measured in culture medium added to treatments without the presence of cells. Some samples did not contain a sufficient volume to enable all analyses to be performed.
Concentrations of PGE2, IL −1β, TNF-α, IL-10, and IL-1ra were measured in culture medium after LPS challenge exposure and addition of the various treatments (Table 1). Only phenylbutazone resulted in a PGE2 concentration significantly less than the concentration for the control treatment. On the other hand, PRP resulted in a significantly higher PGE2 concentration, compared with the concentration for the control treatment. Treatment with APP and IRAP resulted in higher, but not significantly different, IL-1β concentrations, compared with concentrations for the other treatments. Treatment with PRP, IRAP, and APP resulted in significantly higher IL-1ra concentrations than for phenylbutazone, sodium hyaluronate, and the control treatment. Additionally, APP resulted in a significantly higher IL-1ra concentration than for IRAP and PRP. No difference was detected in TNF-α and IL-10 concentrations among the treatments.
Median (interquartile [25th to 75th percentile] range) concentrations of PGE2, IL-1β, TNF-α, IL-10, and IL-1ra in supernatant of equine synovial cell cultures (n = 8 samples) after challenge exposure with LPS for 3 hours and incubation with various treatments for 24 hours.
| Treatment | PGE2 (pg/mL) | IL-1β (pg/mL) | TNF-α (pg/mL) | IL-10 (pg/mL) | IL-1ra (pg/mL) |
|---|---|---|---|---|---|
| Control | 4,332.5a (3,683.0–5,805.4) | 0 (0–0) | 0 (0–0) | 0 (0–0.5) | 0a (0–7.3) |
| Phenylbutazone | 153.1b (90.2–917.3) | 0 (0–0) | 0 (0–0) | 0 (0–28.2) | 0a (0–26.2) |
| PRP | 7,481.1c (6,390.5–9,249.2) | 0 (0–0) | 0 (0–0) | 0 (0–2.3) | 1,141.2b (584.7–1,397.4) |
| IRAP | 6,499.5a,c,d (0–8,803.6) | 135.2 (34.4–168.5) | 0 (0–0) | 19.2 (0–48.1) | 9,603.1c (6,364.7–10,163.4) |
| APP | 4,476.5a,d (0–7,100.9) | 180.3 (31.4–239.1) | 0 (0–0) | 18.9 (7.7–52.9) | 20,996.1d (19,333.0–25,816.0) |
| Sodium hyaluronate | 5,050.3a,c,d (4,777.0–7,147.3) | 0 (0–0) | 0 (0–0) | 0 (0–12.4) | 0a (0–18.8) |
The control treatment consisted of untreated cells.
Within a column, values with different superscript letters differ significantly (P < 0.05; Friedman test followed by Wilcoxon test).
In vitro oxidative burst
Two distinct cell peaks were identified, which indicated the presence of 2 cell populations (Figure 1). Mean fluorescence intensity for the cell populations after the various treatments and challenge exposure with PMA and LPS was measured (Table 2). Cellular responses were similar after stimulation with both PMA and LPS. All treatments decreased the mean fluorescence intensity, compared with results for the untreated control cells, for stimulation with both PMA and LPS. Cell oxidative responses to phenylbutazone, APP, PRP, and IRAP were plotted (Figure 2).
Cytograms (A and C) and histograms (B and D) of synovial cells after challenge exposure with PMA (A and B) and LPS (C and D). Notice the separation between the populations of interest and the differentiated response to stimuli (R5 vs R6 and M2 vs M1). Green fluorescence of dichlorofluorescein diacetate was measured as arbitrary units, and cells with values > 101 for fluorescence intensity were considered to be producing ROS. SSC = Side scatter.
Citation: American Journal of Veterinary Research 80, 7; 10.2460/ajvr.80.7.646
Cytograms (A and C) and histograms (B and D) of synovial cells after challenge exposure with PMA (A and B) and LPS (C and D). Notice the separation between the populations of interest and the differentiated response to stimuli (R5 vs R6 and M2 vs M1). Green fluorescence of dichlorofluorescein diacetate was measured as arbitrary units, and cells with values > 101 for fluorescence intensity were considered to be producing ROS. SSC = Side scatter.
Citation: American Journal of Veterinary Research 80, 7; 10.2460/ajvr.80.7.646
Cytograms (A and C) and histograms (B and D) of synovial cells after challenge exposure with PMA (A and B) and LPS (C and D). Notice the separation between the populations of interest and the differentiated response to stimuli (R5 vs R6 and M2 vs M1). Green fluorescence of dichlorofluorescein diacetate was measured as arbitrary units, and cells with values > 101 for fluorescence intensity were considered to be producing ROS. SSC = Side scatter.
Citation: American Journal of Veterinary Research 80, 7; 10.2460/ajvr.80.7.646
Cytograms (A, C, E, G, and I) and histograms (B, D, F, H, and J) of synovial cells after challenge exposure with LPS and treatment with phenylbutazone (A and B), APP (C and D), IRAP (E and F), PRP (G and H), or sodium hyaluronate (I and J). Notice the separation between the populations of interest and the differentiated response to stimuli (R5 vs R6 and M2 vs M1). Green fluorescence of dichlorofluorescein diacetate was measured as arbitrary units, and cells with values > 101 for fluorescence intensity were considered to be producing ROS. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 80, 7; 10.2460/ajvr.80.7.646
Cytograms (A, C, E, G, and I) and histograms (B, D, F, H, and J) of synovial cells after challenge exposure with LPS and treatment with phenylbutazone (A and B), APP (C and D), IRAP (E and F), PRP (G and H), or sodium hyaluronate (I and J). Notice the separation between the populations of interest and the differentiated response to stimuli (R5 vs R6 and M2 vs M1). Green fluorescence of dichlorofluorescein diacetate was measured as arbitrary units, and cells with values > 101 for fluorescence intensity were considered to be producing ROS. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 80, 7; 10.2460/ajvr.80.7.646
Cytograms (A, C, E, G, and I) and histograms (B, D, F, H, and J) of synovial cells after challenge exposure with LPS and treatment with phenylbutazone (A and B), APP (C and D), IRAP (E and F), PRP (G and H), or sodium hyaluronate (I and J). Notice the separation between the populations of interest and the differentiated response to stimuli (R5 vs R6 and M2 vs M1). Green fluorescence of dichlorofluorescein diacetate was measured as arbitrary units, and cells with values > 101 for fluorescence intensity were considered to be producing ROS. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 80, 7; 10.2460/ajvr.80.7.646
Median (interquartile [25th to 75th percentile] range) fluorescence intensity for equine synovial cells (n = 8 samples) challenge exposed with PMA and LPS and incubated with various treatments.
| Treatment | PMA | LPS |
|---|---|---|
| Control | 826.5a (708.4–967.5) | 850.5a (663.0–990.7) |
| Phenylbutazone | 61.2b (27.4–240.5) | 177.5b,c,d (72.5–241.5) |
| PRP | 129.5b (102.7–308.2) | 103.5b,d (98.7–153.5) |
| IRAP | 142.8b (91.0–200.5) | 93.5b,c (78.2–184.2) |
| APP | 93.5b (72.7–252.7) | 104.5d (89.1–193.5) |
| Sodium hyaluronate | 156b (35.7–290.7) | 303.9c,d (114.5–564.8) |
Fluorescence intensity was reported as arbitrary units.
See Table 1 for remainder of key.
In vivo inflammatory response
Concentrations of PGE2, IL-1β, TNF-α, IL-10, and IL-1ra in synovial fluid were measured before and 48 hours after arthroscopy and treatment (Table 3). The PGE2 concentration decreased significantly in the postoperative period, compared with the concentration before surgery, for only the control and IRAP treatments. On the other hand, the PGE2 concentration following PRP treatment was significantly increased 48 hours after surgery. An increase in IL-1ra concentration was detected 48 hours after surgery for the control, IRAP, APP, and sodium hyaluronate treatments. Synovial fluid concentrations of TNF-α and IL-10 did not differ significantly before and after the arthroscopic procedure for all treatments.
Median (interquartile [25th to 75th percentile] range) concentrations of PGE2, IL-1β, and IL-1ra and least squares mean (95% confidence interval) concentrations of TNF-α and IL-10 in synovial fluid of osteochondrotic tibiotarsal joints of horses (8 joints/treatment) before (time 0) and 48 hours after arthroscopy and injection of various treatments.
| PGE2 (pg/mL) | IL-1β (pg/mL) | IL-1ra (pg/mL) | TNF-α (pg/mL) | IL-10 (pg/mL) | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Treatment | 0 hours | 48 hours | 0 hours | 48 hours | 0 hours | 48 hours | 0 hours | 48 hours | 0 hours | 48 hours |
| Control | 33.9a (23.9 to 52.1) | 19.8* (15.5 to 29.9) | 2.0 (1.5 to 2.2) | 2.1 (1.6 to 2.5) | 0 (0 to 0) | 1,721* (536 to 3,266) | 1.14 (0.34 to 1.94) | 1.52 (0.33 to 2.70) | 23.9 (−52.7 to 100.0) | 33.2 (−54.4 to 120.0) |
| PRP | 15.8a (11.1 to 32.7) | 30.3* (19.8 to 33.1) | 3.1 (1.7 to 3.6) | 2.1 (1.8 to 3.6) | 0 (0 to 0) | 3,730* (802 to 5,965) | 1.59 (1.3 to 1.9) | 1.67 (0.84 to 2.5) | 47.7 (8.3 to 87.2) | 52.4 (−53.4 to 158.0) |
| IRAP | 70.9b (39.8 to 77.5) | 29.1* (21.3 to 37.7) | 2.5 (1.9 to 2.5) | 2.9 (2.6 to 3.2) | 0 (0 to 0) | 3,229* (1,779 to 5,126) | 1.83 (1.13 to 2.53) | 1.53 (0.84 to 2.48) | 33.1 (−29.3 to 95.6) | 30.6 (−17.4 to 78.3) |
| APP | 13.8a (12.5 to 19.0) | 18.4 (16.4 to 43.1) | 2.7 (2.2 to 4.1) | 2.6 (2.4 to 3.5) | 0 (0 to 0) | 1,827* (292 to 4,487) | 1.74 (0.44 to 3.04) | 1.89 (1.46 to 2.33) | 23.1 (−12.3 to 58.6) | 77.1 (−119.0 to 273.0) |
| Sodium hyaluronate | 32.4a,b (21.2 to 37.7) | 27.5 (16.4 to 38.6) | 3.2 (2.5 to 4.1) | 3.6 (1.7 to 5.9) | 0 (0 to 0) | 9,520* (247 to 12,627) | 1.55 (0.78 to 2.31) | 2.5 (−0.23 to 5.22 | 17.4) (−23.5 to 58.4) | 29.2 (−39.4 to 97.9) |
The control treatment consisted of joints that were examined arthroscopically but that received no additional treatment. Concentrations of TNF-α and IL-10 were analyzed by use of a 2-way ANOVA with treatment and time as fixed effects.
Within a treatment, value differs significantly (P < 0.05; Wilcoxon test) from the value at 0 hours.
Within a column, values with different superscript letters differ significantly (P < 0.05; Kruskal-Wallis followed by Mann-Whitney U test).
Glycosaminoglycan analysis of synovial fluid
Synovial fluid concentrations of chondroitin sulfate and hyaluronic acid and the percentage of high-molecular-weight hyaluronic acid in samples obtained before and 48 hours after arthroscopy were determined for each group (Table 4). Chondroitin sulfate concentrations were significantly higher at 48 hours, compared with concentrations before surgery, for all treatments (including the control treatment). Mean chondroitin sulfate concentrations of approximately 200 μg/mL were detected. Only treatment with APP resulted in a significant change (a decrease) in the hyaluronic acid concentrations at 48 hours, compared with the concentrations before surgery. All treatments, except for IRAP, caused a significant decrease in the percentage of high-molecular-weight hyaluronic acid in synovial fluid at 48 hours.
Median (interquartile [25th to 75th percentile] range) concentrations of chondroitin sulfate and hyaluronic acid and percentage of high-molecular-weight (HMW) hyaluronic acid in synovial fluid of osteochondrotic tibiotarsal joints of horses (8 joints/treatment) before and 48 hours after arthroscopy and injection of various treatments.
| Chondroitin sulfate (μg/mL) | Hyaluronic acid (μg/mL) | HMW hyaluronic acid (%) | ||||
|---|---|---|---|---|---|---|
| Treatment | 0 hours | 48 hours | 0 hours | 48 hours | 0 hours | 48 hours |
| Control | 75.4 (40.6–136.7) | 195.8* (88.6–356.4) | 676.0 (419.0–744.9) | 802.7a,b (326.9–877.3) | 74.0 (62.2–87.0) | 45.5* (14.7–61.2) |
| PRP | 38.0 (23.9–58.2) | 68.9* (61.2–164.4) | 377.6 (267.6–630.7) | 390.4a (241.8–583.9) | 80.0 (75.0–89.0) | 54.0* (35.0–82.0) |
| IRAP | 40.5 (34.1–66.8) | 109.4* (74.0–165.1) | 288.2 (193.0–426.2) | 206.5c (174.5–247.9) | 77.0 (63.0–81.0) | 57.7 (39.5–63.0) |
| APP | 65.5 (50.7–103.2) | 181.0* (127.4–382.6) | 541.6 (309.2–609.4) | 406.2*a,b (239.9–605.4) | 79.0 (38.5–90.3) | 43* (29.3–60.3) |
| Sodium hyaluronate | 47.3 (19.6–53.1) | 159.9* (135.0–180.7) | 492.2 (476.4–577.5) | 584.1b (526.7–664.5) | 83.5 (62.7–91.5) | 51.5* (24.7–71.5) |
Within a column, values with different superscript letters differ significantly (P < 0.05; Kruskal-Wallis test followed by Mann-Whitney U test).
See Table 1 for remainder of key.
Discussion
For the in vitro oxidative burst assay, all treatments reduced the production of ROS by the cells challenged with PMA and LPS. Unexpectedly, only treatment with the NSAID phenylbutazone resulted in a significant reduction in PGE2 concentrations when compared with results for the control treatment in the in vitro inflammatory assay. On the other hand, phenylbutazone did not cause changes in IL-1β, TNF-α, IL-10, and IL-1ra concentrations when compared with results for the control treatment.
For decades, phenylbutazone has been the NSAID most commonly administered to horses. Surprisingly, given its prominent place in the pharmacopoeia for horses, there are few studies on the effects of phenylbutazone on equine joint metabolism. Phenylbutazone inhibits the enzymatic system directly responsible for the synthesis of prostanoids25 (eg, PGE2), and the use of phenylbutazone reduces PGE2 concentrations in the synovial fluid of horses with experimentally induced osteoarthritis.26,27 In addition, PGE2 is inhibited when chondrocytes in cell culture are stimulated with inflammatory mediators and then treated with phenylbutazone.28
Several anti-inflammatory drugs have been tested as potential inhibitors of IL-1–mediated stimulation of proteinase and PGE2 synthesis. Most drugs, including phenylbutazone, are ineffective for inhibiting the IL-1–mediated increase of proteinase synthesis and sulfated glycosaminoglycan release.29 In the present study, there also was no effect of phenylbutazone on IL-1β, TNF-α, IL-10, and IL-1ra concentrations.
On the basis of results of the in vivo experiment, we assumed that the use of phenylbutazone in the postsurgical period affected the synovial fluid concentration of PGE2 but not the concentrations of the cytokines IL-1β, TNF-α, IL-10, and Il-1ra. The use of NSAIDs, particularly phenylbutazone, in the postoperative period is in accordance with the typical recommendation for postoperative pain control in horses.30
The PGE2 concentration is increased in the joint fluid of horses after arthroscopy.13,31 However, in the present study, the PGE2 concentration 48 hours after arthroscopy was lower, even for the control treatment. Despite the possibility that the systemic administration of phenylbutazone affected PGE2 concentrations, the inclusion of inflammatory markers was intended to detect differences between treatment and control groups because both received this anti-inflammatory drug.
It is important to remember that NSAIDs, including phenylbutazone, are fundamentally inhibitors of cyclooxygenase, but they may also counteract the generation and stimulation of ROS.32–34 Although several in vivo experiments have not confirmed the interference of NSAIDs in the generation of oxidative stress,35 phenylbutazone caused a reduction in the production of ROS by cells challenged with PMA and LPS, compared with results for the control treatment, in the study reported here. This indicated the need for additional studies to confirm the in vivo role of these drugs in oxidative stress.
The addition of PRP to cells challenge exposed with LPS or PMA caused increased concentrations of PGE2 and IL-1ra. An increase in the PGE2 concentration was also detected in the synovial fluid of joints with osteochondritis dissecans at 48 hours after PRP injection. This result suggested that caution should be used for the injection of PRP into joints with osteochondritis dissecans during the immediate postoperative period. In 1 study,15 the inflammatory response associated with PRP injection was detected as an increase in the PGE2 concentration in the synovial fluid of PRP-treated joints within 6 hours after administration and by an increase in the total protein concentration and WBC counts in the synovial fluid of PRP-treated joints until 48 hours after injection. In another study,36 effects of PRP were evaluated in vivo and in vitro, which revealed a mild and transient inflammatory process in the in vivo experiment on the basis of increases of IL-6 and TNF-α concentrations after intra-articular administration of PRP activated by thrombin. However, in the in vitro experiment, there was no increase in proinflammatory cytokine concentrations. Considering that there is diversity in commercial and laboratory methods for obtaining PRP, data reported here pertain only to the method described for the present study.
All horses received phenylbutazone after surgery, and a robust inflammatory response was not detected for any of the treatments, including the control treatment, as determined on the basis of a lack of increase in IL-1 and TNF-α concentrations. However, the IRAP treatment and the control treatment were the only ones that resulted in a significant decrease in the synovial fluid concentration of PGE2 at 48 hours after arthroscopy. Because phenylbutazone has a pronounced anti-inflammatory effect through inhibition of the formation of proinflammatory PGs, it was difficult to evaluate the contribution of IRAP alone to the reduction of PGE2 concentrations for the study reported here.
The concentration of IL-1ra in culture medium without the presence of cells was considerably lower than the concentration in the medium after cell culture in PRP, IRAP, and APP for 24 hours. These results confirmed the potential of blood-derived products to stimulate the release of IL-1ra by synovial cells in vitro. Another relevant result was the higher concentration of IL-1ra by cells treated with APP (a low-cost, noncommercial product), compared with concentrations for cells treated with IRAP (a commercial product).
An increase in IL-1ra concentrations 48 hours after arthroscopy was detected in the synovial fluid of all treatments, including the control treatment. Sodium hyaluronate increased the concentration of IL-1ra more than did the other treatments; however, this increase did not differ significantly. Increased IL-1ra concentrations in the synovial fluid after intra-articular application of PRP in healthy joints were also evident in another study.15 Additionally, increased IL-1ra concentrations were detected in experiments involving IRAP, APP, and PRP preparations.5,7,15 Existence of a natural antagonist for the IL-1 receptor indicates that the body mounts its own response against inflammation. Synthesis of IL-1ra is part of this response, which is intended to resolve an underlying process.37 Endogenously produced IL-1ra may be important for preventing or reducing local damage resulting from overproduction of IL −1.38 However, the balance between IL-1 and IL-1ra concentrations in tissues and synovial fluid determines the physiologic or pathophysiologic effects of IL −1.39 The production of small amounts of IL-1 and large amounts of IL-1ra in tissues would appear to be a typical response in clinical situations. Arthroscopy probably induced an increase in IL-1ra concentrations for all treatments, which would be expected to be the body's typical response to insults.
The anti-inflammatory properties of IL-10 include a reduction in the production of IL-1β, TNF-α, IL-6, IL-8, PGE2, and matrix metalloproteinases and stimulation of the production of IL-1ra and tissue inhibitor of metalloproteinases.40,41 Treatment with IRAP and APP increased IL-10 concentrations in relation to results for the control treatment in the in vitro experiment of the present study, but this increase was not significantly different from that of the control treatment and was not evident in the synovial fluid 48 hours after application of the treatments in the in vivo experiment. We also cannot infer that the increase of IL-1ra concentrations for the IRAP, APP, and PRP treatments was attributable to an increase in IL-10 concentration because for the PRP treatment, there also was an increase in the IL-1ra concentration, compared with the concentration for the control treatment, without a concomitant increase in the IL-10 concentration.
Significant increases in the concentration of ROS (ie, oxidative stress) contribute to the imbalance between anabolic and catabolic events in cartilage and result in oxidative damage to cartilage components.42,43 In the present study, PRP, IRAP, APP, and sodium hyaluronate caused a reduction in the production of ROS by cells challenge exposed with PMA and LPS, compared with results for the control treatment. It has been reported that hyaluronic acid reduces ROS production in mechanical stress–loaded bovine cartilage44 and that a crucial function of hyaluronic acid is the control of oxidative damage.45
In contrast to results of another study11 in which chondrocyte culture was used to determine that the mechanism of action whereby hyaluronic acid reduces ROS production involves the regulation of nuclear factor-erythroid-2–related factor 2 by the activation of protein kinase B, the present study involved the culture of synovial cells. Because nuclear factor-erythroid-2–related factor 2 is constitutively expressed in all tissues of vertebrates and plays an important role in cell survival and maintaining cellular integrity by enhancing the protective capacity against oxidative stress, we assumed that the same mechanism of action was responsible for the effects of sodium hyaluronate on ROS production by synovial cells in the study reported here.
All blood-derived products reduced ROS production, compared with results for the control treatment. The ROS can be synthesized by various cytoplasmic enzymes, but most ROS are derived from NADPH oxidase.46 Several NADPH oxidase inhibitors have been described, including biological molecules (eg, nitric oxide,47 IL-10,48 and IL-449). Both IRAP and APP increased IL-10 concentrations in cell culture, but PRP did not. Therefore, it appeared that the reduction of ROS induced by the blood-derived products was attributable to the increased concentration of IL-10 and, consequently, inhibition of NADPH oxidase.
Concentrations of the glycosaminoglycans chondroitin sulfate and hyaluronic acid were measured in synovial fluid to characterize events related to extra-cellular matrix metabolism and joint homeostasis. The significant increase in chondroitin sulfate concentrations 48 hours after arthroscopy indicated the effects of the surgical procedure on matrix synthesis and catabolism events in horses affected by osteochondritis dissecans. Evidence of degradation of the extracellular matrix in synovial fluid has been reported in animals with osteochondritis dissecans, regardless of the course and severity of joint disease.12,19,50–52 Thus, the insult inherent to the surgical procedure adds to the effects on the extracellular matrix in animals with osteochondritis dissecans.13,31
It would be beneficial to use treatments that reduce the breakdown of the extracellular matrix that occurs with osteochondritis dissecans. In the present study, none of the treatments prevented increases in the chondroitin sulfate concentration in the synovial fluid 48 hours after arthroscopy; however, it is possible that a longer treatment period with an increased number of administrations would have yielded a better result. Treatment with IRAP was the only one that did not result in a significant decrease in the percentage of high-molecular-weight hyaluronic acid in synovial fluid samples obtained 48 hours after arthroscopy. On the basis of these results, it could be inferred that IRAP was most effective for preventing the reduction of the percentage of high-molecular-weight hyaluronic acid. This would be relevant because the influence of the molecular weight of hyaluronic acid on its biological properties has been reported, and it was found that high- and intermediate-molecular-weight hyaluronic acid increases the healing process, whereas very-low-molecular-weight hyaluronic acid causes inflammation.53 Other studies54,55 have also indicated proinflammatory activity for low-molecular-weight hyaluronic acid in pathological processes.
Expression of hyaluronidase is increased in inflammatory reactions.56 Therefore, it may be plausible that part of the prevention of the partial depolymerization of the hyaluronic acid molecule in IRAP-treated joints could have been related to its anti-inflammatory action, which was evident as a decreased concentration of PGE2 at 48 hours after arthroscopy.
Analysis of results of the study reported here suggested caution in the use of PRP in animals with osteochondritis dissecans during the immediate postoperative period. Antioxidant effects were detected for all treatments, and it can be inferred that sodium hyaluronate, APP, and IRAP contributed to the control of joint inflammation.
Acknowledgments
Supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; Nos. 11/19096-2 and 11/12929-9) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES; finance code 001).
Funding sources had no involvement in the study design, analysis and interpretation of data, or writing and publication of the manuscript.
The authors declare there were no conflicts of interest.
ABBREVIATIONS
| APP | Autologous processed plasma |
| DMEM | Dulbecco modified Eagle medium |
| IL | Interleukin |
| IL-1ra | Interleukin-1 receptor antagonist |
| IRAP | Interleukin-1 receptor antagonist protein |
| LPS | Lipopolysaccharide |
| NADPH | Reduced form of nicotinamide adenine dinucleotide phosphate |
| PG | Prostaglandin |
| PMA | Phorbol 12-myristate 13-acetate |
| PRP | Platelet-rich plasma |
| ROS | Reactive oxygen species |
| TNF | Tumor necrosis factor |
Footnotes
IRAP II system, Arthrex Vet Systems, Fort Myers, Fla.
Hylartil Vet, Zoetis Inc, Parsippany, NJ.
PG E2 EIA kit–monoclonal, Cayman Chemical, Ann Arbor, Mich.
Equine IL-1β DuoSet ELISA (DY3340), R&D Systems Inc, Minneapolis, Minn.
Equine TNF-α DuoSet ELISA (DY1814), R&D Systems Inc, Minneapolis, Minn.
Equine IL-10 DuoSet ELISA (DY1605), R&D Systems Inc, Minneapolis, Minn.
Equine IL-1ra/IL-1F3 DuoSet (DY2466), R&D Systems Inc, Minneapolis, Minn.
FACSCalibur, Becton Dickinson Immunocytometry Systems, San Jose, Calif.
Cell Quest Pro, Becton Dickinson Immunocytometry Systems, San Jose, Calif.
VisionWorks LS, Upland, Calif.
Neuenschwander HM. Comparação dos efeitos da aplica-ção intra-articular de ácido hialurônico de diferentes pesos moleculares em modelo de sinovite aguda induzida por LPS em equinos. Master's dissertation, Faculty of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil, 2016.
Select-HA, Sigma-Aldrich Corp, St Louis, Mo.
SPSS, IBM Corp, Armonk, NY.
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