Forty male Lewis rats (Sprague-Dawley, 200–230 g; Harlan, Nossan Milan, Italy) were maintained in a monitored environment and provided with standard rodent chow and water. The study was authorized by the University of Messina Review Board for the care of animals (Protocol number 8/U-apr16). Animal care was in conformity with Italian regulations for the protection of experimental animals (DM 116192) and with European Economic Community regulations (OJ of EC L 358/1 12/18/1986).
OA was induced by intra-articular injection of MIA in the knee joint . On day 0, rats were anesthetized with 5.0% isoflurane (Baxter International). A volume of 25 μl sterile saline solution + 3 mg MIA was injected into the knee joint through the right infrapatellar ligament. The left knee received an equal volume of 0.9% sterile saline. MIA was prepared in sterile conditions and injected using a 50-μl Hamilton syringe with a 27-gauge needle that was inserted into the joint to about 2–3 mm. On day 21 post-MIA administration, knee joints were inspected in detail to determine histopathological changes. Cartilage was stained to verify the presence of OA or not.
Rats were randomly divided into the following groups:
MIA + vehicle (solvent solution)
Rats were subjected to induction of OA as described above, and received 25 μl of the solvent solution in the infrapatellar area of the right knee at days 3, 7, 14, and 21 (n = 10) by intra-articular injection.
MIA adelmidrol 0.6% + 1.0% sodium hyaluronate
Rats were subjected to induction of OA as described above, and were treated by intra-articular injection of adelmidrol 0.6% + sodium hyaluronate 1.0% (sodium hyaluronate with high molecular weight, between 1.5 and 2.0 million daltons) at a dose of 150 μg/25 μl on days 3, 7, 14, and 21 after MIA induction (n = 10)
MIA adelmidrol 2% + sodium hyaluronate 1.0%
Rats were subjected to induction of OA as described above, and were treated by intra-articular injection of adelmidrol 2% + sodium hyaluronate 1.0% (sodium hyaluronate with high molecular weight, between 1.5 and 2.0 million daltons) at a dose of 150 μg/25 μl on days 3, 7, 14, and 21 after MIA induction (n = 10)
MIA + sodium hyaluronate 1.0%
Rats were subjected to induction of OA as described above, and were treated by intra-articular injection of sodium hyaluronate 1.0% (sodium hyaluronate with high molecular weight, between 1.5 and 2.0 million daltons) at a dose of 150 μg/25 μl on days 3, 7, 14, and 21 after MIA induction (n = 10)
Rats were administered by intra-articular injection with 0.9% saline (25 μl) instead of MIA and were treated with either vehicle or different formulations on days 3, 7, 14, and 21 (n = 10)
Mechanical sensitivity was evaluated using a dynamic plantar aesthesiometer (Ugo Basile, Comerio, Italy). The rats were placed on a metal mesh surface in a chamber in a room with a controlled temperature (22 °C) and they were allowed to adapt for 15 min before the testing began. The touch stimulator part was oriented under the animal. When the aesthesiometer was activated, a plastic monofilament touched the paw in the proximal metatarsal region. The filament exercised a gradually increasing force on the plantar, starting below the threshold of detection and increasing until the stimulus became painful and the rat removed its paw. The force required to produce a paw withdrawal reflex was recorded automatically and measured in grams. A maximum force of 50 g and a ramp speed of 20 s were used for all the aesthesiometry tests.
Analysis of motor function (Walking Track Analysis)
The rat was placed in a walking track with a dark end. White office paper of the appropriate size was placed on the bottom of the track. Hind limbs of the rat were dipped in ink, and the rat was allowed to walk along the track, leaving paw prints on the paper. The test was performed before induction on day 0 and at days 3, 7, 14, and 21 after induction . The functionality index of the sciatic nerve (SFI), calculated by Walking Track Analysis, was evaluated at 60 min after the injection on days 3, 7, 14, and 21: values close to 0 indicate normal functioning, and values tending to –100 indicate an alteration of sciatic nerve functionality .
Micro-computed tomography analysis
In order to evaluate the bone mass and microarchitecture parameters, including the fraction of bone volume, the proximal and distal parts of the right tibiae were scanned using micro-computed tomography (Micro-CT; Skyscan, Belgium) The scan conditions were as follows: an aluminum filter of 0.5 mm, X-ray voltage of 50Kv, X-ray current of 200 mA, and an exposure time of 360 ms. After scanning, the cross-sectional slices were reconstructed and three-dimensional analyses were performed using CTAn SkyScan software.
On day 21 after MIA administration, rats were sacrificed by anesthetic overdose and perfused with 4% paraformaldehyde solution. Tibiofemoral joints were collected and post-fixed in neutral buffered formalin (containing 4% formaldehyde), decalcified in EDTA, and processed as following described. After decalcification, the specimens were embedded in paraffin. Mid-coronal tissue sections (5 μm) were stained for evaluation; all histomorphometric analyses were performed by an observer blinded to the treatment groups. Sections were stained with hematoxylin and eosin and observed by light microscopy (Dialux 22 Leitz; Leica Microsystems SpA, Milan, Italy). Histopathological analysis of the cartilage was assessed by the modified score of Mankin  (score range 0 to 12, from normal to complete disorganization and hypocellularity).
Cartilage degeneration was assessed by staining with toluidine blue and analyzed using the following criteria described by Janusz et al. : 1 = mild into the surface region; 2 = slightly extended in the upper center; 3 = moderate in the median area; 4 = extended area deep; and 5 = severe degeneration.
Mast cell staining
Identification of mast cells was performed as described previously . Knee sections were cut at 5-μm thickness and stained with 0.25% toluidine blue, pH 2.5, for 45 min at room temperature. The sections were then dehydrated and mounted for viewing. Three non-sequential sections were chosen by a block at random from each paw for examination. All sections were evaluated at 200×, while some sections were photographed at 400× using a Nikon inverted microscope. The density of mast cells is expressed as the number of mast cells per unit area of bone tissue.
Measurement of cytokines, metalloproteinases, and nerve growth factor
The levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, nerve growth factor (NGF), and matrix metalloproteinase (MMP)-1, MMP-3, and MMP-9 were measured in serum. Assays were performed using commercial colorimetric enzyme-linked immunosorbent assay (ELISA) kits (TNF-α, IL-1β, and NGF: Thermo Fisher Scientic; MMP-1, MMP-3, and MMP-9: Cusabio).
Unless otherwise stated, all compounds were obtained from Sigma-Aldrich Co. All other chemicals were of the highest commercial grade available. All stock solutions were prepared in non-pyrogenic saline (0.9% NaCl; Baxter Healthcare Ltd., Thetford, Norfolk, UK).
All values in the figures and text are expressed as mean ± standard error of the mean (SEM) of n observations. For in vivo studies, n represents the number of animals studied. In experiments involving histology, the figures shown are representative of at least three experiments performed on different days. The results were analyzed by one-way analysis of variance (ANOVA) followed by a Bonferroni post-hoc test for multiple comparisons. Non-parametric data were analyzed with the Fisher's exact test. A P value less than 0.05 is considered significant.