Protective effects of total fraction of avocado/soybean unsaponifiables on the structural changes in experimental dog osteoarthritis: inhibition of nitric oxide synthase and matrix metalloproteinase-13

Sixteen adult crossbred dogs (aged 2 to 3 years), each weighing 20 to 25 kg, were used in this study. They were housed in a large kennel in individual galvanized steel cages (1 m [width] × 1.75 m [length] × 2.4 m [height]), each separated by a panel. All cages were equipped with an automatic watering system. Dogs were selected after complete physical and musculoskeletal evaluations by a veterinarian. Haematological and biochemical analyses were conducted in the animals before their inclusion in the study. Surgical sectioning of the anterior cruciate ligament (ACL) of the right knee was performed on all dogs, as previously described [13] but with modifications. This model was created by sectioning of the ACL after joint capsule opening under general anaesthesia with pentobarbital sodium (25 mg/kg). All dogs received a multimodal and pre-emptive analgesic protocol based on opioid (fentanyl patch 75 μg [Janssen-Ortho, Markham, ON, Canada] and meperidine 4 mg/kg subcutaneous injection [Sandoz, Montreal, QC, Canada]) and intra-articular analgesia (marcaine 1 mg/kg [Hospira, Quebec, QC, Canada]). During the postoperative period, if needed, dogs were treated with fentanyl patch, oxymorphone (0.1 mg/kg SC; Sandoz) or meperidine SC, repeated as necessary. After surgery, the dogs were housed on a farm where they were free to exercise in a large enclosure. All dogs actively exercised in exterior runs (1.35 m [width] × 9.15 m [length]) for a 2-hour period, 5 days a week, under the supervision of an animal care technician.

The OA dogs were randomly assigned to two treatment groups, to which the animal care personnel were blinded. Dogs assigned to group 1 (n = 8) received placebo treatment (encapsulated methylcellulose) and those assigned to group 2 (n = 8) received ASU (Piascledine™; Expanscience, Courbevoie, France) orally once a day, every day including weekends, at a dosage of 10 mg/kg per day, which corresponds to twice the recommended daily dosage for the treatment of patients with knee or hip OA. The dosage was established based on the recent FDA guidelines [14]. Drug treatment was initiated immediately after surgery and continued until the dogs were euthanized 8 weeks later. The study protocol was approved by the institutional ethics committee and conducted in accordance with the Canadian Council on Animal Care guidelines.

Immediately after sacrifice, the right knee of each dog was placed on ice and dissected. Each knee was examined for gross morphological changes, as previously described, by two independent observers who were blinded to treatment group allocation [13].

The degree of osteophyte formation was graded by measuring the maximal width (mm) of the spurs on the medial and lateral femoral condyles using a digital caliper (Digimatic Caliper; Mitutoyo Corporation, Kawasaki, Japan). These two values, recorded for each dog, were considered separately for the purposes of statistical analysis.

The medial and lateral menisci of each knee were also scored macroscopically at the time of dissection as intact, fibrillated or torn, based on a method modified from that reported by Cook and coworkers [15].

The macroscopic lesion sizes at the cartilage surface were measured (in mm²) as previously described [13]. Overall scores were obtained for the femoral condyles and tibial plateaus separately by summing the score for each region recorded.

Full thickness cartilage sections were removed from the weight-bearing lesional areas of the femoral condyles and tibial plateaus, allowing standardization of sampling as recommended by the OA Research Society International guidelines [15]. Histological evaluation was performed on sagittal sections of cartilage removed from each femoral condyle and tibial plateau specimen [16]. Specimens were dissected, fixed in TissuFix #2 (Laboratoires Gilles Chaput, Montreal, QC, Canada) and embedded in paraffin for histological evaluation. Serial sections (5 μm) were stained with Safranin-O. Two independent observers (CB and JC), who were blinded to treatment group allocation, graded the severity (consensus score) of the OA lesions in each cartilage section, which was divided into three subregions [15], on a scale of 0 to 29, modified from that reported by Sakakibara and colleagues [17]. This scale was used to evaluate the severity of OA lesions based on the loss of Safranin-O staining (scale 0 to 4), cellular changes (scale 0 to 12), structural changes (scale 0 to 10, where 0 = normal cartilage structure and 10 = complete disorganization) and pannus formation (scale 0 to 3). The final score (range 0 to 87) corresponds to the sum of the final scores for the three subregions of each specimen from the femoral condyle or tibial plateau.

Synovial membrane was removed and processed as described above for histological analysis. Samples were stained with haematoxylin-phloxine-saffron. The severity of synovitis was graded on a scale of 0 to 10 [13] by two blinded and independent observers (CB and JC, consensus score), who added the scores of histologic criteria: synovial cell hyperplasia (scale 0 to 2), villous hyperplasia (scale 0 to 3), and mononuclear (scale 0 to 4) and polymorphonuclear (scale 0 to 1) cell infiltration; 0 indicates normal structure.

Specimens of full-thickness sections, which included the calcified cartilage and subchondral bone, were removed from the OA knee of all dogs and were placed in 70% ethanol and further decalcified with rapid bone decalcifier (RDO; Apex Engineering, Aurora, IL, USA). Specimens were embedded in paraffin for the purpose of histomorphometric analysis.

Sections (5 μm) of each specimen were subjected to hematoxylin/eosin staining. A Leitz Diaplan microscope (Leica Microsystems, Wetzlar, Germany) connected to a personal computer (Pentium IV based, using OSTEO II Image Analysis Software [Bioquant, Nashville, TN, USA]) was used to conduct bone histomorphometry analysis.

Subchondral bone histomorphometry was performed on three nonconsecutive sections of each specimen using our previously published method [18], modified from that of Matsui and coworkers [19]. The calcified cartilage/subchondral bone junction was used as the upper limit of each field. The depth was measured from the upper limit to 2,000 μm. Measurement of the bone surface (% of tissue surface) and trabecular thickness (μm) followed standard conventions, as previously described [18]. The measurement of the fields was then averaged for each section. Values for each section were considered separately for the purposes of statistical analysis.

Calcified cartilage histomorphometry was also done on three nonconsecutive sections of each specimen, as previously described [18]. From each section, three representative fields of 1,000 μm length (original magnification ×60) were selected. The tidemark/cartilage and calcified cartilage/bone junctions were used as upper and lower limits. The mean thickness (mm) of the calcified cartilage was calculated. The measurement made in the three fields was then averaged for each section and the value of each section was considered separately.

Cartilage specimens from the condyles and plateaus were processed for immunohistochemical analysis, as previously described [16, 20], fixed in TissuFix #2 (Laboratoires Gilles Chaput) for 24 hours, and then embedded in paraffin. Serial sections (5 μm) of paraffin-embedded specimens were placed on Superfrost Plus slides (Fisher Scientific, Nepean, ON, Canada), de-paraffinized in toluene, rehydrated in a reverse graded series of ethanol and pre-incubated with 0.25 units/ml chondroitinase ABC (Sigma-Aldrich Canada, Oakville, ON, Canada) in phosphate-buffered saline (PBS; pH 8.0) for 60 minutes at 37°C. The specimens were subsequently washed in PBS, incubated in 0.3% Triton X-100 for 20 minutes, and then placed in 3% hydrogen peroxide/PBS for 15 minutes. Slides were further incubated with a blocking serum (Vectastain ABC kit; Vector Laboratories, Inc., Burlingame, CA, USA) for 60 minutes, after which they were blotted and then overlaid with the primary antibody against the following: inducible nitric oxide synthase (iNOS; 1/200, rabbit polyclonal; Santa Cruz Biotechnology Inc. [ref. #SC-650], Santa Cruz, CA, USA), matrix metalloproteinase (MMP)-1 (1/40 dilution, mouse monoclonal; Calbiochem ref. #444209; EMD Biosciences, Darmstadt, Germany), MMP-13 (1/6, goat polyclonal antibody; R&D Systems, Minneapolis, MN, USA), a disintegrin and metalloproteinase domain with thrombospondin motifs (ADAMTS)5 (1/50, rabbit polyclonal; Cedarlane [ref. #CL1-ADAMTS5], Hornby, ON, Canada) and ADAMTS4 (1/40, rabbit polyclonal; Cedarlane [ref. #CL1-ADAMTS4]) for 18 hours at 4°C in a humidified chamber.

Each slide was washed three times in PBS (pH 7.4), stained using the avidin-biotin complex method (Vectastain ABC kit), and incubated in the presence of the biotin-conjugated secondary antibody for 45 minutes at room temperature followed by the addition of the avidin-biotin-peroxidase complex for 45 minutes. All incubations were carried out in a humidified chamber at room temperature and the colour was developed with 3,3'-diaminobenzidine (DAKO Diagnostics Canada Inc., Mississauga, ON, Canada) containing hydrogen peroxide. The slides were counterstained with haematoxylin/eosin.

To determine the specificity of staining, three control procedures were employed in accordance with the same experimental protocol: omission of the primary antibody; substitution of the primary antibody with an autologous preimmune serum; and immunoadsorption with immunizing peptide for iNOS (Santa Cruz Biotechnology Inc.) and ADAMTS4 and ADAMTS5 (Cedarlane) or MMP-1 and MMP-13 recombinant protein (R&D Systems). As previously demonstrated, all controls exhibited only background staining (data not shown) [16]. Each section was examined under a light microscope (Leitz Orthoplan; Leica Inc., St. Laurent, QC, Canada) and photographed using a CoolSNAP cf Photometrics camera (Roper Scientific, Rochester, NY, USA).

The presence of the antigen in the cartilage was quantified using our previously reported method [13, 16, 21]. and estimated by determining the number of chondrocytes that stained positive throughout the entire thickness of the cartilage. Three sections from each femoral condyle and tibial plateau were examined, and each one was separately scored. The resulting data were integrated as a mean for each specimen. The cartilage was divided into six microscopic fields – three in the superficial zone and three in the deep zone (×40; Leica Inc.) – and the results were averaged for each zone. Before evaluation, it was ensured that an intact cartilage surface for each OA specimen could be detected and used as a marker for validation of the morphometric analysis. The superficial zone of cartilage corresponds to the superficial and the upper intermediate layers, and the deep zone to the lower intermediate and deep layers. The total number of chondrocytes and those staining positive in each zone for the specific antigen were determined. The final results were expressed as the percentage of chondrocytes staining positive for the antigen (cell score), with the maximum score being 100%. Each slide was subjected to two independent readers who were blinded to treatment group allocation. The final score was a consensus between the two observers (CB and JC).

For the purposes of statistical analysis, the data obtained from the femoral condyles and tibial plateaus were considered separately. iNOS, MMP-1, ADAMTS4 and ADAMTS5 were quantified in the superficial zone of cartilage because the staining for these antigens was found to be negligible in the deep zone, whereas MMP-13 was quantified in the deep zone, which is its preferential zone of expression [22].

Macroscopic and histologic data are expressed as mean ± standard error of the mean. Immunohistochemical and histomorphometric results are expressed as the median (range). Statistical analysis, unless otherwise specified, was performed using the Mann-Whitney U-test or Student's unpaired t-test. P values of less than or equal to 0.05 were considered statistically significant.

Bone surface was 75.9% (50.8% to 86.2%; median [range]) of the tissue surface in the placebo group compared with 79.3% (66.0% to 90.8%; P < 0.05) in the ASU-treated group (Figure 3). Trabecular thickness did not differ between the ASU (132.3 μm [102.8 μm to 200.5 μm]) and placebo (129.1 μm [90.0 μm to 156.0 μm]) groups.

Chondrocytes staining positive for iNOS were found preferentially located in the superficial zone of the OA cartilage. The percentage of positive cells was found to be significantly decreased in the ASU-treated group (21.4% [19.3% to 27.6%]) compared with the placebo-treated group (25.6% [21.2% to 30.1%]; P = 0.02; Figure 4).

In contrast to iNOS, MMP-13 was detected preferentially in the deep zone of OA cartilage. Dogs treated with ASU exhibited a significant reduction in the level of MMP-13 (8.6% [4.8% to 13.10%]) compared with the placebo-treated group (16.3% [9.0% to 24.8%]; P = 0.01; Figure 5).

The levels of other mediators were also studied and found to be similar in the two groups: MMP-1, 28.0% (23.2% to 30.9%) of chondrocytes in the placebo group versus 29.6% (27.8% to 30.7%) in the ASU-treated group; ADAMTS4, 23.9% (22.0% to 28.2%) versus 26.9% (22.9% to 28.9%); and ADAMTS5, 24.6% (20.7% to 29.8%) versus 26.05% (17.9% to 29.5%).