Osteoarthritis is a progressive deterioration of articular cartilage combined with changes in the subchondral bone and soft tissues.1–4 In horses, the prevalence of osteoarthritis is high, and osteoarthritis is a frequent cause of morbidity as a result of pain that is typically involved with this disease.5
To gain insight into the pathogenesis of osteoarthritis and to evaluate the effect of new treatments in various animal species, experimental manipulations of research animals to simulate the events observed in naturally occurring osteoarthritis are customarily used.6 In horses, the carpal osteochondral fragment procedure is most frequently used to induce osteoarthritis.7,8 However, naturally occurring osteoarthritis is more frequently observed in the MCP joint than in the carpus.8 For the MCP joint, different techniques to induce osteoarthritis have been described and include intra-articular injection of chemicals,8–10 transection of ligaments to induce joint instability,11 osteochondral fragment creation in combination with exercise,8,12 an arthroscopic grooving procedure with controlled exercise,13 and impact injury of the palmar aspect of the metacarpus.14 However, most of these procedures did not result in appropriate degenerative changes in the entire joint or had confounding joint instability or severe inflammation. For example, chemically induced osteoarthritis is typically associated with severe inflammation; therefore, this procedure is suited for investigation of the analgesic effect of a new treatment and less useful for assessment of the cartilage repair capacity of a new treatment.9 Transection of the lateral collateral ligament of the MCP joint and the lateral collateral sesamoidean ligament induces permanent joint instability, which makes this procedure less suitable for assessing the effect of a treatment, given that the trigger of the osteoarthritis cannot be removed.11 On the other hand, in 1 study12 of horses, osteochondral fragment creation in the MCP joint resulted in very mild lameness (ie, no visually evident lameness and only changes in symmetry indices in an early stage, compared with presurgical findings, but a positive flexion test response at 4 postsurgical time points) and onset of osteoarthritis was slow.
Therefore, the aim of the study reported here was to develop a procedure for use in horses to experimentally induce MCP joint osteoarthritis without permanent joint instability or severe inflammation but with a relative rapid onset of osteoarthritis (within 11 weeks) and observable lameness. We hypothesized that creation of a standardized osteochondral fragment at the dorsoproximal aspect of the PP that remained attached to the dorsal joint capsule and an opposing groove in the dorsal aspect of the metacarpal condyle would induce moderate osteoarthritis of the MCP joint after an intensive exercise regimen in horses.
Materials and Methods
Animals
The present study included the placebo group of a larger experiment to evaluate effectiveness of stem cell–based therapy on experimentally induced MCP joint osteoarthritis. That larger study also contained a second group of horses (n = 6) that was treated locally in their experimentally injured MCP joint with a stem cell–based therapy (chondrogenic-induced mesenchymal stem cells). Those horses, however, were not involved in the present study in any way. Because the horses of the present study comprised the placebo group of the larger study, they received a 2-mL injection of saline (0.9% NaCl) solution in their injured MCP joint at day 35. At the same time point in the larger study, the other group of horses was treated with the stem cell–based product. Because the horses in the present study comprised the placebo group, data from the evaluations in these horses were compared with those obtained from the horses treated with the stem cell–based therapy in the larger study. However, those analyses were separate and did not interfere with the analyses performed for the present study. Sample size calculations for the present study were based on the set-up of the larger study. With the exception of the saline solution injection, the study design would not have differed if the horses of the present study had not been used as the placebo group of the larger study.
The group of horses in the present study was composed of 6 warmbloods (5 mares and 1 gelding) with a mean ± SD age of 9 ± 4 years. Prior to study participation, the horses had no visible forelimb lameness or any radiographic abnormalities of the MCP joints. The independent Ethics Committee of Global Stem Cell Technology (permit No. LA1700607) approved the protocol (approval No. EC_2015_002).
Surgery and exercise program
Each horse was anesthetized for arthroscopic surgery. The horse was placed in dorsal recumbency, and both MCP joints were prepared and draped for aseptic surgery. The dorsal aspects of both MCP joints were arthroscopically assessed for the presence of preexisting cartilage lesions through a standard dorsolateral portal. The osteoarthritis induction procedure was applied in the right MCP joint for practical reasons. The correct localization of the instrument portal was determined by insertion of a 19-gauge needle medially in the dorsal joint pouch approximately 8 to 10 mm proximal to the dorsomedial edge of the PP. With a No. 11 scalpel, an incision was made through the joint capsule. A standard straight 8-mm osteotomea was positioned on the dorsoproximal border of the PP, approximately 15 mm abaxial to the sagittal ridge of the third metacarpal bone, and was held at an angle of 55° with respect to the longitudinal axis of the metacarpus. This was verified by use of a custom-made template (Figure 1). Next, the osteotome was tapped with a mallet until a dorsomedial PP osteochondral fragment was created that remained attached to the dorsal joint capsule (Figure 2). Creation of the osteochondral fragment was combined with debridement of the fragment bed with an arthroburr.b Debridement was performed toward the center of the bone, medially and laterally, to decrease apposition between the fragment and the fracture bed. In addition, the arthroburr was used to create a transverse full-thickness cartilage lesion on the dorsal aspect of the medial condyle of the third metacarpal bone at the level of the fragment (a horizontal groove). Whereas fragment creation was performed during joint distention with saline solution, the procedures with the arthroburr were performed during CO2 distention to keep the bone and cartilage debris inside the joint. The osteoarthritis induction procedure had been first optimized in a pilot study on cadavers and 2 experimental horses (data not shown). During optimization of the procedure, no lameness attributable to the presence of the osteochondral fragment alone in either experimental horse was noted. Therefore, the fragment bed was enlarged with a motorized burr to delay reattachment and increase the potential to develop osteoarthritis. The opposing groove was added to initiate cartilage damage as a trigger of osteoarthritis. It was also determined during optimization of the procedure to retain the debris from groove creation and fragment bed drilling in the joint to increase irritation.
Photograph of the surgical set-up for performing an osteochondral fragment–groove procedure as a means of experimental induction of osteoarthritis in an MCP joint of a horse, illustrated in a cadaveric limb. An arthroscope is inserted into the right MCP joint through a dorsolateral portal made lateral to the common digital extensor tendon. To increase standardization of the creation of the osteochondral fragment, the osteotome is held at an angle of 55° with respect to the longitudinal axis of the metacarpus, which is verified by a custom-made template, before tapping the osteotome with a mallet.
Citation: American Journal of Veterinary Research 80, 3; 10.2460/ajvr.80.3.246
Representative arthroscopic views obtained during application of the osteochondral fragment–groove procedure in the right MCP joint of 1 of 6 study horses. A—Through an instrument portal, an osteotome is tapped with a hammer until an osteochondral fragment is created at the dorsomedial edge of the PP that remains attached to the dorsal joint capsule. B—The fragment bed is debrided medially and laterally toward the center of the bone with an arthroburr to decrease apposition between the fragment and the fracture bed. C—The arthroburr is used to create a transverse full-thickness cartilage lesion on the dorsal aspect of the medial condyle of the third metacarpal bone adjacent to the fragment (appearing as a horizontal groove [arrow]).
Citation: American Journal of Veterinary Research 80, 3; 10.2460/ajvr.80.3.246
Skin incisions were sutured with monofilament resorbable suture material.c The right MCP region was protected with a sterile bandage. After arthroscopic inspection, the contralateral MCP joint underwent a sham operation with saline solution joint distension and, subsequently, CO2 joint distention; skin incisions were then closed and a bandage was applied. The entire procedure was documented with high-resolution digital video and photographs.
During surgery, each horse received a single dose of morphine hydrochloride (0.1 mg/kg, IV) and an antimicrobial drug (107 U of benzylpenicillin sodium, IV).d No postoperative medication was given. Bandages were changed after 24 hours and on day 7 and were removed in combination with suture removal on day 14. After the procedure, each horse was stall rested for 1 week. Beginning on day 7, the horses underwent a treadmill exercise program daily (7 d/wk) during the subsequent 10 weeks as follows: 2 minutes of walking at 7 km/h, 5 minutes of trotting at 17 km/h, 1 minute of walking at 7 km/h, 5 minutes of trotting at 17 km/h, and 2 minutes of walking at 7 km/h. On days 35 to 38, the horses were rested because they had received a 2-mL saline solution injection in the right MCP joint as part of the aforementioned concurrent study of a stem cell–based treatment.
Radiographic analysis
Lateromedial, dorsopalmar, 45° dorsolateral-palmaromedial oblique, and 45° dorsomedial-palmarolateral oblique radiographic views of both MCP joints of each horse were obtained on days 0 (before surgery), 1, 35, and 77. Radiographic abnormalities at each time point were recorded.
Lameness evaluation
All horses underwent visual and objective lameness examinations before surgery on day 0 and at 7-day intervals up to and including day 77. Lameness was evaluated when the horses were moving on the treadmill, circling on a soft surface, and trotting on a straight line on a hard surface before and after distal forelimb joint flexion. A lameness scale (generated by the American Association of Equine Practitioners15) was used for visual assessment as follows: 0 = lameness is not detectable under any circumstances; 1 = lameness is difficult to observe and is not consistently apparent, regardless of circumstances; 2 = lameness is difficult to observe at a walk or when trotting in a straight line but is consistently apparent under certain circumstances; 3 = lameness is consistently observable at a trot under all circumstances; 4 = lameness is obvious at a walk; and 5 = lameness produces minimal weight bearing in motion or at rest or a complete inability to move. Objective lameness examinations were performed with an inertial sensor-based system.e The system generated a vector sum to represent forelimb lameness.16,17 Visual and objective lameness evaluations were performed by a qualified veterinarian (who was trained in good clinical practice and had > 5 years of experience in lameness evaluations and performing clinical studies). The observer was unaware of which forelimb had undergone the osteochondral fragment-groove procedure. On days 0 (before surgery), 35, and 77, pressure-plate analysis was performed for each horse with a 2-m pressure platef (as previously described18) and a force plateg that provided dynamic calibration of the pressure plate.19 Symmetry indices were determined and expressed as percentage symmetry ([left forelimb index/right forelimb index] × 100%).
Synovial fluid sample collection and analysis
Synovial fluid samples were collected in tubes containing EDTA during surgery (day 0) and on days 35, 36, 49, 63, and 77 by means of a standard MCP joint arthrocentesis procedure.20 Samples were centrifuged at 1,500 × g for 10 minutes at 4°C. The supernatants were stored at −20°C pending biomarker analyses. Commercial ELISA kits were used according to the manufacturers’ instructions to assay concentrations of the following biomarkers: IL-10,h IL-1 receptor antagonist protein,i PGE2,j matrix metalloproteinase 13,i IL-6,k serum amyloid A,h TNF-αi, interferon-γ, HA,l GAGs,m and transforming growth factor-β3.k Cytologic evaluations were performed with a hematology analyzer.
Postmortem examination
On day 77 after the procedure, all horses were euthanized (after sedation with romifidine hydrochloride and induction of anesthesia with a combination of ketamine and midazolam) with an IV injection containing 1 g of embutramide, 2.5 g of mebezonium, and 250 mg of tetracaine hydrochloride.n
Macroscopic and histologic examination of MCP joints
For each horse, both MCP joints were opened dorsally, exposing the joint surfaces. The entire joint (PP joint surface, third metacarpal bone joint surface, joint surface of sesamoid bones, and synovium) was scored according to the semiquantitative macroscopic guidelines of the Osteoarthritis Research Society International21 (Appendix). Macroscopic consensus scores were derived after reaching an agreement between the 2 scoring individuals (an ECVP diplomate and a qualified veterinarian who was trained in good clinical practice with > 5 years of experience in performing clinical studies).
Cartilage samples were collected from the area adjacent to the created fragment and at the level of the groove lesion. In the sham-operated joints, cartilage samples were taken from areas corresponding with these locations. In addition, samples of synovial membrane including the joint capsule of the dorsal joint were obtained. All cartilage and synovial membrane samples were fixed with neutral-buffered 4% formalin, embedded in paraffin, sectioned at a thickness of 4 μm, and stained with H&E stain. Cartilage samples were also stained with Alcian blue stain (for GAGs). Cartilage damage was evaluated by use of modified Osteoarthritis Research Society International histologic guidelines21 (Appendix). For each section, 3 areas were randomly selected and photographed (at a magnification of 200×), and the percentage of the total area that was stained (Alcian blue uptake representing GAG content) was calculated with software.22,o Histologic scoring was performed by an ECVP diplomate who was unaware of whether the sections came from an injured or uninjured MCP joint.
Immunohistochemical analysis
Immunohistochemical analyses for COMP and collagen type II were performed on cartilage samples obtained from the area adjacent to the osteochondral fragment and at the level of the created groove. In the sham-operated joints, cartilage samples were collected from areas corresponding with those locations. Tissue sections were stained with rabbit polyclonal anti-COMPp (1:50) or anti-collagen type IIq(1:50) antibody. Immunolabeling was achieved with a high-detection–sensitivity horseradish peroxidase–conjugated goat anti-rabbit antibody diaminobenzidine kit, with blocking of endogenous peroxidaser in an autoimmunostainer. A commercially available antibody diluent with background-reducing components was used to block hydrophobic interactions. Positive staining was confirmed microscopically. For each section, 3 areas were randomly selected and photographed (at a magnification of 200×), and the percentage of the total area that was stained (representing COMP or collagen type II content) was calculated with software.22,o
Statistical analysis
Data from the objective lameness examinations and synovial fluid sample analyses were analyzed by use of a mixed model, with horse as a random effect and time point as a fixed effect. The affected area percentages for Alcian blue, COMP, and collagen type II staining were analyzed by a fixed-effects model, with side as a fixed effect and separated for the 2 locations (osteochondral fragment and groove). All model assumptions were tested and met. A value of P < 0.05 was considered significant. All statistical analyses were performed with statistical analysis software.s
For this study, osteoarthritis was considered to be successfully induced when lameness of a horse was detected and histologic signs of right MCP joint inflammation and cartilage degeneration with or without (subchondral) bone changes could be observed.8
The sample size of the present study was determined on the basis of the set-up of the aforementioned larger study. For the larger study, a treatment success of 83% was assumed in the stem cell–based therapy group. In the placebo group, a treatment success of 17% was assumed. On the basis of power of 80% and an α value of 0.05, this resulted in a sample size of 6 animals/group. Moreover, 6 animals/group has been previously used to generate statistically relevant data.23,24
Results
Radiographic examination
On radiographic views of both MCP joints of each horse obtained on day 0 (before surgery), there were no degenerative changes (eg, sclerosis or osteophytes). The day after surgery, the presence of a dorsomedially located osteochondral fragment was confirmed radiographically in all right MCP joints (Figure 3). On day 77, no radiographic signs other than the induced osteochondral fragment were evident.
Forty-five–degree dorsolateral-palmaromedial oblique digital radiographic views of the right MCP of 1 of 6 study horses that underwent the osteochondral fragment–groove procedure. The day of surgery was designated as day 0. Findings for this horse were representative of findings for the treated MCP joint in all 6 horses. A—On day 1, a dorsomedial osteochondral fragment (arrow) is evident. B—On day 77, the induced osteochondral fragment is still visible (arrow), but no other radiographic signs are present.
Citation: American Journal of Veterinary Research 80, 3; 10.2460/ajvr.80.3.246
Lameness evaluation
All horses were clinically nonlame on day 0 prior to surgery, which resulted in an overall lameness score of 0/5. Corresponding mean vector sums on day 0 were within physiologic variation (ie, < 8.5 mm),25 except for the data obtained from horses during circling to the left (mean vector sum, 13.5 mm; Figure 4). The evolution of the lameness scores during the study period were summarized (Table 1).
Mean ± SEM vector sum values (representing forelimb lameness) determined with an inertial sensor-based system for 6 horses in which the right MCP joint underwent the osteochondral fragment–groove procedure. Values presented here were determined during performance of 4 maneuvers before surgery on day 0 and at various time points after surgery. Evaluations were performed when horses were moving on a treadmill (A), during circling to the left on a soft surface (B), and trotting at a straight line on a hard surface before (C) and after (D) right distal forelimb joint flexion. In each panel, the black horizontal bar indicates the clinically relevant threshold for lameness detection (ie, 8.5 mm). *Value is significantly (P < 0.05) different from that determined on day 0.
Citation: American Journal of Veterinary Research 80, 3; 10.2460/ajvr.80.3.246
Results of lameness evaluations over an 11-week period for 6 horses in which the right MCP joint underwent an osteochondral fragment–groove procedure and the left MCP joint underwent a sham operation.
| Lameness score (No. of horses) | ||||||
|---|---|---|---|---|---|---|
| Day | 0 | 1 | 2 | 3 | 4 | 5 |
| 0 | 6 | 0 | 0 | 0 | 0 | 0 |
| 7 | 0 | 3 | 2 | 1 | 0 | 0 |
| 14 | 0 | 3 | 2 | 1 | 0 | 0 |
| 21 | 0 | 2 | 3 | 1 | 0 | 0 |
| 28 | 0 | 1 | 4 | 1 | 0 | 0 |
| 35 | 0 | 0 | 4 | 2 | 0 | 0 |
| 42 | 0 | 0 | 4 | 2 | 0 | 0 |
| 49 | 0 | 0 | 5 | 1 | 0 | 0 |
| 56 | 0 | 0 | 5 | 1 | 0 | 0 |
| 63 | 0 | 0 | 5 | 1 | 0 | 0 |
| 70 | 0 | 0 | 5 | 1 | 0 | 0 |
| 77 | 0 | 1 | 4 | 1 | 0 | 0 |
Surgery was performed on day 0; lameness evaluations were performed before surgery (on day 0) and at 7-day intervals until day 77. Lameness was evaluated when horses were moving on a treadmill, circling left on a soft surface, and trotting in a straight line on a hard surface before and after right distal forelimb joint flexion. A lameness scale (generated by the American Association of Equine Practitioners15) was used for visual assessment as follows: 0 = lameness is not detectable under any circumstances; 1 = lameness is difficult to observe and is not consistently apparent, regardless of circumstances; 2 = lameness is difficult to observe at a walk or when trotting in a straight line but is consistently apparent under certain circumstances; 3 = lameness is consistently observable at a trot under all circumstances; 4 = lameness is obvious at a walk; and 5 = lameness produces minimal weight bearing in motion or at rest or a complete inability to move.
On day 7, lameness (assessed with the inertial sensor-based system) was observed when horses moved on a straight line before right distal forelimb joint flexion had significantly increased, compared with the mean vector sum on day 0 (P = 0.01), and was still visible on days 14, 21, 35, and 42 (P = 0.04, P = 0.006, P = 0.020, and P = 0.030, respectively; Figure 4). On day 14, lameness (as evidenced by a significantly [P < 0.001] greater mean vector sum) was observed when horses moved on a straight line after right distal forelimb joint flexion. On day 21, the mean vector sum was significantly (P = 0.049) greater than that on day 0 when horses circled to the left. After a decrease of the mean vector sum on days 21 and 28, lameness when horses moved on a straight line after right distal forelimb joint flexion increased and mean vector sums on days 35, 42, and 49 were significantly (P = 0.004, P = 0.047, and P = 0.026, respectively) increased, compared with the value on day 0.
On days 70 and 77, lameness was present during 3 of the 4 maneuvers (Figure 4); compared with the value on day 0, the mean vector sum was significantly greater when horses were on the treadmill (P < 0.01 for both days 70 and 77), circling to the left (P < 0.01 for both days 70 and 77), and moving on a straight line after right distal forelimb joint flexion (P = 0.01 and P = 0.03 for days 70 and 77, respectively). Lameness was evident when horses were moving on a straight line before right distal forelimb joint flexion on days 70 and 77. On day 70, the mean vector sum was significantly (P = 0.04) greater than that on day 0, but the value on day 77 was not significantly (P = 0.053) different. Pressure-plate analysis did not detect any relevant asymmetry in limb loading between left and right forelimbs.
Synovial fluid sample examination
Compared with findings on day 0, a significant increase in IL-6 (P = 0.01) and PGE2 (P < 0.001) concentrations in synovial fluid samples was detected on day 35 (Figure 5). In addition, on day 36, IL-1 receptor antagonist protein concentration was significantly increased (P = 0.02) and remained elevated on day 49 (P = 0.03). Peaks of both synovial fluid HA and PGE2 concentrations were detected on day 49 (P = 0.01 and P < 0.001, respectively) and again on day 77 (P = 0.02 and P = 0.009, respectively). In contrast, both IL-10 and TNF-α concentrations decreased significantly, compared with findings on day 0, on days 77 (P = 0.02) and 63 (P = 0.04), respectively. After a detectable peak on day 35, synovial fluid IL-6 concentration was also decreased significantly (P = 0.046) on day 63. Other synovial fluid variables including serum amyloid A, interferon-γ, transforming growth factor-β3, GAGs, and matrix metalloproteinase 13 concentrations remained stable overtime.
Mean ± SEM concentrations of TNF-α(A), IL-6 (B), IL-10 (C), PGE2 (D), HA (E), and IL-1 receptor antagonist protein (IRAP; F) in synovial fluid samples collected from the right MCP joint that underwent an osteochondral fragment-groove procedure in each of 6 study horses before surgery on day 0 and at various time points after surgery. *Value is significantly (P < 0.05) different from that determined on day 0.
Citation: American Journal of Veterinary Research 80, 3; 10.2460/ajvr.80.3.246
Postmortem macroscopic examination of MCP joints
All left sham-operated MCP joints were assigned scores of 0 for all macroscopic variables. Among the right MCP joints that underwent an osteochondral fragment-groove procedure, a mean score of 2 was assigned on day 77 for wear lines (Table 2), which corresponded to 3 to 5 partial-thickness or 1 or 2 full-thickness wear lines/joint surface (Figure 6). The groove created on the third metacarpal bone was clearly visible in all right MCP joints. The created osteochondral fragments were reattached with fibrous tissue to the dorsomedial aspect of the PP. In 3 of the 6 horses, partial-thickness erosions < 5 mm in diameter and covering < 25% of the cartilage joint surface and mild synovial villi thickening were present. In all right MCP joints, mild to moderate synovitis and hyperemia were present. No right MCP joints had evidence of palmar arthrosis, covering of subchondral bone with fibrocartilage, or synovial petechiation.
Day-77 postmortem macroscopic and histologic scores (see appendix) assigned to the right MCP joint that underwent an osteochondral fragment–groove procedure on day 0 in the 6 horses.
| Horse | ||||||
|---|---|---|---|---|---|---|
| Variable | 1 | 2 | 3 | 4 | 5 | 6 |
| Macroscopic score | ||||||
| Wear lines | 3 | 2 | 1 | 2 | 3 | 1 |
| Erosions | 0 | 0 | 1 | 0 | 1 | 1 |
| Extent of erosions | 0 | 0 | 1 | 0 | 1 | 1 |
| Synovitis | 1 | 2 | 1 | 1 | 1 | 2 |
| Synovial hyperemia | 1 | 2 | 1 | 1 | 1 | 2 |
| Increase in villi density or thickening | 0 | 1 | 0 | 0 | 1 | 1 |
| Histologic score | ||||||
| Cluster (complex chondrone formation) in the cartilage on the PP joint surface | 0 | 0 | 4 | 0 | 0 | 0 |
| Cluster (complex chondrone formation) in the cartilage on the third metacarpal bone | 0 | 0 | 2 | 0 | 0 | 3 |
| Focal cell loss in the cartilage of the third metacarpal bone | 0 | 0 | 0 | 0 | 0 | 2 |
| Cellular infiltration of the synovium | 0 | 0 | 2 | 2 | 0 | 1 |
| Vascularity of the synovium | 0 | 2 | 2 | 0 | 0 | 1 |
Scores for macroscopic variables ranged from 0–3 with the exception of the extent of erosions, which ranged from 0–4, and covering of subchondral bone with fibrocartilage, which ranged from 0–2. Scores for histologic variables ranged from 0–4. Both the right (treated) and left (sham-operated) MCP joints were evaluated macroscopically; all sham-operated MCP joints were assigned scores of 0 for all macroscopic variables. No right MCP joints had evidence of chondrocyte necrosis on the PP joint surface or the third metacarpal bone, palmar arthrosis, covering of subchondral bone with fibrocartilage, synovial petechiation, fibrillation or fissuring of the cartilage of the PP joint surface or third metacarpal bone, or focal cell loss in the articular cartilage adjacent to the osteochondral fragment. Regarding the histologic findings for samples of synovia, no joints that underwent an osteochondral fragment–groove procedure had evidence of abnormalities apart from increased vascularity and cellular infiltration (ie, no intimal hyperplasia, no subintimal edema, and normal subintimal fibrosis).
Representative photographs of a right MCP joint that underwent an osteochondral fragment–groove procedure (performed on day 0) from 1 of 6 study horses and was opened for postmortem macroscopic evaluation on day 77. A—View of the articular surface of the third metacarpal bone. Full-thickness wear lines (arrows) are clearly visible on the medial condyle of the third metacarpal bone. The induced groove opposing the osteochondral fragment (outlined area) is visible at the condyle of the third metacarpal bone. B—View of the PP. Full-thickness wear lines are also visible (black arrows). The approximately 8-mm-long osteochondral fragment (outlined area) is attached at the dorsomedial aspect of the PP. Moderate synovitis and hyperemia are present (white arrow).
Citation: American Journal of Veterinary Research 80, 3; 10.2460/ajvr.80.3.246
Postmortem histologic examination of MCP joints
Among the right MCP joints that underwent an osteochondral fragment-groove procedure, no chondrocyte necrosis on the PP joint surface or the third metacarpal bone was identified. Complex chondrone formation (cluster) was present in the articular cartilage adjacent to the osteochondral fragment of 1 joint (horse 3) and in the opposing groove of the joint in each of 2 horses (horses 3 and 6; Table 2). Focal cell loss was present in 1 groove (horse 6). Occasional to moderate cellular infiltration was present in synovial membranes for 3 of 6 right MCP joints. A slight to mild increase in the number and dilatation of blood vessels (ie, vascularity) in focal locations throughout the synovial membrane sections was present in 3 of the 6 horses. No right MCP joints had evidence of fibrillation or fissuring of the cartilage of the PP joint surface or third metacarpal bone or focal cell loss in the articular cartilage adjacent to the osteochondral fragment. Regarding the histologic findings for samples of synovia, no joints that underwent an osteochondral fragment-groove procedure had evidence of abnormalities apart from increased vascularity and cellular infiltration (ie, no intimal hyperplasia, no subintimal edema, and normal subintimal fibrosis) Cartilage samples collected from the area adjacent to the created fragment and at the level of the groove lesion in the right MCP joints and from the left sham-operated MCP joints were stained with Alcian blue stain. Compared with percentage area of Alcian blue staining in the sham-operated joints, there was significantly (P = 0.002) less staining in the right MCP joints that underwent an osteochondral fragment-groove procedure, indicating lower GAG content (Figure 7).
Mean ± SEM percentage area staining for GAGs (A), COMP (B), and collagen type 2 (C) in cartilage samples collected from the region adjacent to the osteochondral fragment (OCF) and the groove in the right MCP joints that underwent an osteochondral fragment–groove procedure (performed on day 0) in 6 study horses and samples collected from the 2 corresponding areas of cartilage in the left sham-operated MCP joints of the same horses. *Under a bar, values for left (sham-operated) and right (treated) MCP joints are significantly (P < 0.05) different.
Citation: American Journal of Veterinary Research 80, 3; 10.2460/ajvr.80.3.246
Immunohistochemical analyses
Cartilage samples collected from the area adjacent to the created fragment and at the level of the groove lesion in the right MCP joints and from the left sham-operated MCP joints underwent immunohistochemical staining for COMP and collagen type II. Compared with findings for the sham-operated joints, the percentage area that stained for COMP or collagen type II was significantly (P = 0.024 and P < 0.001, respectively) less in the articular cartilage adjacent to the osteochondral fragment of the MCP joints that underwent an osteochondral fragment-groove procedure (Figure 7). In addition, the percentage area that stained for collagen type II at the site of the groove in the right MCP joints was also significantly P = 0.020) lower than that for the sham-operated joints.
Discussion
In the present study, an osteochondral fragment–groove procedure was performed in the right MCP joints of nonlame horses, and resultant lameness and morphological changes were assessed. The study findings indicated that the osteochondral fragment–groove procedure resulted in early and progressive lameness with local histologic signs typical of osteoarthritis of the MCP joint (ie, cellular infiltration in the synovial membrane, reduction of GAG and collagen type II contents in the articular cartilage, and presence of chondrones in the articular cartilage). To the authors’ knowledge, this study was the first to determine the usefulness of the osteochondral fragment–groove procedure as a means of experimental induction of MCP joint osteoarthritis within an 11-week period in horses.
In the present study, clinical osteoarthritis was successfully induced, given that lameness was detected under different circumstances with an inertial sensor-based method in all 6 experimental horses. In contrast, lameness could not be detected with pressure plate analysis. It should, however, be noted that the pressure plate analysis was performed after all the other clinical examinations, which probably ameliorated the lameness in much the same manner as lameness is reduced after exercise in horses with naturally occurring osteoarthritis.26,27 Furthermore, the data set obtained by the pressure plate is not as representative as the one obtained with the inertial sensor system. With a pressure plate, lameness is evaluated only as horses move in a straight line; furthermore, owing to the assembly's limited physical dimensions, only 1 stride/trial can be analyzed. In contrast, an unlimited number of consecutive strides can be measured with the sensor system.
In the 6 right MCP joints that underwent the osteochondral fragment–groove procedure, postmortem macroscopic examination revealed synovitis, indicative of arthritis.1,4,21 In the treated joints from the 6 horses, wear lines were present in 6 joints and partial-thickness cartilage erosions were detected in 3 joints. These macroscopic degenerative changes of the cartilage were indicative of successful induction of osteoarthritis.21 Moreover, these results were confirmed histologically by detection of local reduced GAGs, COMP, and collagen type 2 content in the articular cartilage of the treated joints.8,28 The increase in proinflammatory cytokine concentrations in the synovial fluid samples also indicated an ongoing inflammatory process in the joints during the first weeks following the osteochondral fragment-groove procedure.8 However, synovial fluid samples were collected only from the joints in which the osteochondral fragment-groove procedure had been applied; therefore, it is possible that the observed changes in biomarker concentrations were due in part to the arthroscopic procedure. In horses, Rossetti et al29 noted an increase in synovial fluid PGE2 content after tarsocrural arthroscopy, but this value peaked at 12 hours after surgery. However, in contrast, Maninchedda et al13 reported no increase in MCP joint synovial fluid PGE2 concentration after only arthroscopic lavage. In addition, Jones et al30 reported that synovitis of 3 to 12 days’ duration (determined from a literature search) and 28 days’ duration (determined by their own research) was induced in canine stifle joints after arthroscopy. Because synovial fluid samples were analyzed in the present study on days 35, 36, 49, 63, and 77 after surgery on day 0, it appears highly unlikely that the postsurgical increases and decreases in synovial fluid biomarker concentrations were attributable to the arthroscopic procedure alone; hence, comparison of synovial fluid biomarker concentrations at these time points with the baseline day 0 value of the healthy joint is valid in our opinion. Studies involving other well-validated experimental procedures (eg, the canine anterior cruciate ligament model) have included similar baseline comparisons to assess changes in synovial fluid biomarker concentrations.31,32
The lameness induced by the osteochondral fragment–groove procedure in horses of the present study was variable and, in some horses, intermittent over time. This can be viewed as a limitation, but it reinforces the relevance of this procedure as a means of simulating naturally occurring osteoarthritis. Indeed, naturally occurring osteoarthritis is typically associated with considerable variation among individuals with regard to related signs of pain, rate of development, and degree of the degenerative changes.8,33 Moreover, in all horses, lameness increased after the flexion test, which is also typically observed in horses with naturally occurring osteoarthritis.8,33 In clinical circumstances, it is ideal to treat horses that are at an early stage of osteoarthritis, when there is onset of cartilage damage but no radiographic changes or severe lameness, because these horses have a superior prognosis. In the present study, degenerative changes of articular cartilage were identified but there was no radiographic evidence of chronic osteoarthritis. Thus, the osteochondral fragment–groove procedure used in the present study could facilitate evaluation of treatments in horses at an early stage of osteoarthritis when the affected joints are still amenable to interventions.
The osteochondral fragment–groove procedure used in the present study provided similar osteoarthritis induction as that associated with creation of an osteochondral fragment at the distal aspect of the radial carpal bone of a middle carpal joint (a procedure that is most commonly used to experimentally induce osteoarthritis in horses21). In the carpal joint osteochondral fragment procedure, an 8-mm-diameter chip fragment is created on the distal-dorsal aspect of the radial carpal bone, and the fragment bed is debrided to create a 15-mm-wide defect.8 Frisbie et al34 reported a mean lameness score of approximately 1.5 (on a severity scale of 5) for horses that underwent this procedure, which is almost the same as the most common lameness score of 2 among horses at the end of the present study. In addition, mild radiographic changes have been detected in horses 91 days after carpal joint osteochondral fragment procedure.35 In contrast to the carpal joint osteochondral fragment procedure, the osteochondral fragment–groove procedure used in the present study did not induce detectable radiographic joint changes. However, in the previous study,35 postsurgery radiography was performed 2 weeks later than that performed in the present study, which allowed a longer period during which bony changes could become detectable. The radiographic changes related to bone proliferation and osteophyte formation in the previous study35 were assigned scores of approximately 0.4 and 0.25, respectively, on a severity scale of 0 to 3, indicating very mild radiographic changes. Overall, the lack of radiographic changes in the present study indicated an early stage of osteoarthritis, which has the advantage that potential new treatments can be tested at an early disease stage and possibly prevent further development of osteoarthritis.
In previous studies,34,36 macroscopic changes associated with the carpal joint osteochondral fragment procedure that were identified at necropsy included partial- or full-thickness cartilage erosions mostly on the third carpal bone and synovitis around the fragment. Histologically, increases in cellular infiltration and vascularity in the synovium were evident, along with intimal hyperplasia and chondrone formation in the article cartilage.34,36 All of those previous findings are similar to those of the present study. Therefore, similar to the carpal joint osteochondral fragment procedure, the osteochondral fragment–groove procedure used in the present study appeared to provide a solid means for testing new osteoarthritis treatments in horses.
The horses used in the present study received a saline solution injection in the experimentally injured MCP joint on day 35 as part of another investigation. Interestingly, after the injection, a decrease in lameness was detected by the inertial sensor—based system; furthermore, decreases in synovial fluid TNF-α and PGE2 concentrations were detected. The decrease in lameness can possibly be explained by the short rest period (3 days) after the joint injection. However, it has been recently shown in human medicine that intra-articular saline solution injections have a therapeutic effect on osteoarthritic joints,37 which might explain the temporary lameness reduction in the study horses. Similar results were also obtained in a study38 of dogs that had single-limb lameness because of osteoarthritis in a stifle or elbow joint and that received an intra-articular injection of autologous protein solution or saline solution (control group) in the affected joint; dogs in the saline solution–treated control group had improvement in lameness 2 weeks after injection (as determined from owner-completed surveys) but not at 12 weeks after injection. Similarly, in the present study, lameness worsened after horses resumed training. Because experimental procedures to induce osteoarthritis in joints are often developed to evaluate the effect of new treatments, we propose that the saline injection administered to the horses in the present study increases the scientific relevance of the research findings. Indeed, when an osteoarthritis induction procedure is used to test a new treatment, a well-designed study includes a placebo-treated or control group along with the experimental treatment group or groups. For assessments of local treatments, this often entails administration of an intra-articular saline solution injection, as performed in the larger study to evaluate a stem cell–based product in which the horses of the present study were involved. Given that intra-articular saline solution injections are reported37,38 to have a therapeutic effect on osteoarthritis, osteoarthritis induction procedures that do not test the use of a local saline solution injection can possibly provide results of apparently effective osteoarthritis development in an initial study, which cannot be recreated in a placebo group of a study testing a new treatment. Therefore, the intra-articular saline solution injection administered to the horses of the present study has enhanced the procedure's relevance for drug development applications.
Interestingly, in the present study, the HA concentration in the synovial fluid increased, although it is commonly reported that HA concentrations in osteoarthritic joints are lower than those in healthy joints.39,40 Possibly, the HA production is increased in the early stages of osteoarthritis as compensation for the increased breakdown rate, resulting in a fluctuating concentration of synovial fluid HA, as seen in the MCP joints that underwent the osteochondral fragment–groove procedure in the present study. However, to our knowledge, no studies have been performed to assess changes in synovial fluid HA concentration during the early stages of osteoarthritis.
Limitations of the present study included a sample size of 6 horses with 1 treated joint/horse. The sample size was selected on the basis of reports13,24 of similar studies performed in the field. The follow-up period of 11 weeks was relatively short, compared with follow-up periods in studies41,42 designed to evaluate cartilage healing. The time line of the present study was determined by the objective to devise an experimental procedure that could be used to evaluate early osteoarthritis in MCP joints of horses and that would result in simultaneous rapid onset of lameness and cartilage wear. For treatments designed to intervene in the development of osteoarthritis, it is important to have measurable outcomes at points early in the disease process. Regardless of the present study's limitations, performance of the osteochondral fragment-groove procedure in MCP joints followed by a standardized exercise program efficiently induced early, measurable signs of osteoarthritis (as determined by results of lameness evaluation and gross joint examination) within an 11-week period in horses.
Acknowledgments
Funded by a grant (No. 130543) from the Flemish Agency for Innovation and Entrepreneurship (Vlaio) and by Global Stem Cell Technology NV, Evergem, Belgium.
Dr. Spaas declares competing financial interests as a shareholder in Global Stem Cell Technology NV. Drs. Spaas and Broeckx are employed by Global Stem Cell Technology NV and inventors of several patents owned by the company. None of the other authors declare a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.
The authors thank Michelle Dumoulin, Eline Van de Water, Julie Jardin, Patrick Willems, Marjan Steppe, Lore Van Hecke, Hayam Hussein, and Michael David for technical assistance.
ABBREVIATIONS
| COMP | Cartilage oligomeric matrix protein |
| ECVP | European College of Veterinary Pathologists |
| GAG | Glycosaminoglycan |
| HA | Hyaluronic acid |
| IL | Interleukin |
| MCP | Metacarpophalangeal |
| PGE2 | Prostaglandin E2 |
| PP | Proximal phalanx |
| TNF-α | Tumor necrosis factor-α |
Footnotes
Misdom Frank osteotome, 8 mm, Misdom Frank, West Chester, Pa.
Acromionizer, 4.0 mm, mauve, reference No. 7205326, Dyonics/Smith & Nephew, Zaventem, Belgium.
Monosyn, USP 3-0, Ethicon, Norderstedt, Germany.
Penicilline, Kela, Hoogstraten, Belgium.
The Equinosis Q with Lameness Locator software, Equinosis, Columbia, Mo.
RSscan 3D 2m-system, RSscan International, Paal, Belgium.
AMTI BP4602070RS-2K, AMTI, Watertown, Mass.
Abcam, Cambridge, England.
R&D systems, Abingdon, England.
Enzo Life sciences, Brussels, Belgium.
Genorise, Berwyn, Pa.
Corgenix, Broomfield, Colo.
Astarte Biologics, Bothell, Wash.
T61, Intervet, Brussels, Belgium.
LAS, version 4.1, Leica microsystems, Diegem, Belgium.
ab74524, Abcam, Cambridge, England.
ab34712, Abcam, Cambridge, England.
Envision DAB+ kit, Dako, Leuven, Belgium.
SAS version 9.4, SAS Institute Inc, Cary, NC.
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Appendix
Scoring system used in the macroscopic and histologic evaluations of equine MCP joints.
| Lesion | Score | Description |
|---|---|---|
| Macroscopic evaluation | 0 | None |
| Wear lines | 1 | 1 or 2 partial-thickness wear lines/joint surface |
| 2 | 3–5 partial-thickness or 1 or 2 full-thickness wear lines/joint surface | |
| 3 | > 5 partial-thickness or > 5 full-thickness wear lines/joint surface | |
| Erosions | 0 | None |
| 1 | Partial-thickness erosion, ≤ 5 mm in diameter | |
| 2 | Partial-thickness erosion, > 5 mm in diameter | |
| 3 | Full-thickness erosion | |
| Extent of erosion | 0 | None |
| 1 | 25% of the cartilage surface | |
| 2 | 50% of the cartilage surface | |
| 3 | 75% of the cartilage surface | |
| 4 | 100% of the cartilage surface | |
| Palmar arthrosis | 0 | None |
| 1 | Partial-thickness erosion, ≤ 5 mm in diameter | |
| 2 | Partial-thickness erosion, > 5 mm in diameter | |
| 3 | Full-thickness erosion | |
| Covering of subchondral bone with fibrocartilage | 0 | None |
| 1 | Partial | |
| 2 | Complete | |
| Synovitis | 0 | Absent |
| 1 | Mild | |
| 2 | Moderate | |
| 3 | Severe | |
| Synovial hyperemia | 0 | Absent |
| 1 | Mild | |
| 2 | Moderate | |
| 3 | Severe | |
| Synovial petechiation | 0 | Absent |
| 1 | Mild | |
| 2 | Moderate | |
| 3 | Severe | |
| Increase in villi density or thickening | 0 | Absent |
| 1 | Mild | |
| 2 | Moderate | |
| 3 | Severe | |
| Microscopic evaluation of cartilage Chondrocyte necrosis | 0 | Normal appearance of the section without necrosis |
| 1 | No more than 1 necrotic cell located near the articular surface/200× field | |
| 2 | 1 or 2 necrotic cells/200× field | |
| 3 | 2 or 3 necrotic cells/200× field | |
| 4 | 3 or 4 necrotic cells/200× field | |
| Cluster | 0 | No cluster formation throughout the section |
| (complex chondrone formation) | 1 | 2 chondrocytes (doublets) within the same lacuna along superficial aspect of the articular cartilage section |
| 2 | 2 or 3 chondrocytes within the same lacuna | |
| 3 | 3 or 4 chondrocytes within the same lacuna | |
| 4 | > 4 chondrocytes within the same lacunae | |
| Fibrillation or fissuring | 0 | No fibrillation or fissuring of the articular cartilage surface |
| 1 | Fibrillation or fissuring of the articular cartilage restricted to surface and superficial zone | |
| 2 | Fibrillation or fissuring extends to the middle zone | |
| 3 | Fibrillation or fissuring extends to the deep zone | |
| 4 | Fibrillation or fissuring extends to the deepest zone | |
| Focal cell loss | 0 | Normal cell population throughout the section |
| 1 | 0% to 20% acellularity/200× field | |
| 2 | 21% to 40% acellularity/200× field | |
| 3 | 41% to 60% acellularity/200× field | |
| 4 | > 60% acellularity/200× field | |
| Microscopic evaluation of synovium | ||
| Cellular infiltration (lymphocytes and plasma cells) | 0 | No mononuclear cells visible in the section |
| 1 | Occasional small areas of mononuclear cells throughout the section | |
| 2 | Moderate presence of mononuclear cells in 25% of the section | |
| 3 | Moderate presence of mononuclear cells in 26% to 50% of the section | |
| 4 | Marked presence of mononuclear cells in > 50% of the section | |
| Vascularity | 0 | Normal |
| 1 | Slight increase in the number of vessels in focal locations throughout the section | |
| 2 | Mild increase in the number and dilatation of vessels in focal locations throughout the section | |
| 3 | Moderate increase in the number and dilatation of vessels in > 50% of the section | |
| 4 | Marked increase in the number and dilatation of vessels in > 50% of the section | |
| Intimal hyperplasia | 0 | None |
| 1 | Villi with 2–4 rows of intimal cells within the section | |
| 2 | Villi with 4–5 rows of intimal cells over 25% to 50% of the section | |
| 3 | Villi with 4–5 rows of intimal cells over > 50% of the section | |
| 4 | Villi with > 5 rows of intimal cells over 50% of the section | |
| Subintimal edema | 0 | No edema |
| 1 | Slight edema detected within the section | |
| 2 | Mild edema detected within 25% of the section | |
| 3 | Moderate edema detected within 26% to 50% of the section | |
| 4 | Marked edema in > 50% of the section | |
| Subintimal fibrosis | 0 | Normal |
| 1 | Slight increase in fibrosis within the section | |
| 2 | Mild increase in fibrosis within 25% of the section | |
| 3 | Moderate increase in fibrosis within 26% to 50% of the section | |
| 4 | Marked increase in fibrosis in > 50% of the section |
