Animals and management
Male, 4 months old Lewis rats (Charles River Laboratories, Portage, MI, USA) weighing roughly 350 g at the beginning of the experiments were used in this study. All the in vivo procedures were approved by the Institutional Animal Care and Use Committee (IACUC) and were performed in accordance with the Guide for the Care and Use of Laboratory Animals and with the US National Institutes of Health (NIH) Publication No. 85-23, revised 1996 [29]. The rats were pair housed in ventilated Innovive cages in a temperature- and humidity-controlled room on a regular 12 h light/dark cycle. Irradiated LabDiet™ 5053 (Purina, Richmond, IN, USA) and water were provided ad libitum. The animals were acclimated for 1 week prior to use in the 10-week study. Since all rats were of the same age (4 months) we randomization into study groups was based on their body weight the day before surgery. A group of 12 rats received sham surgery (Group 1), whereas 12 rats underwent MMT surgery (Group 2). Fluorochrome labels were administered to label actively mineralizing bone surfaces for analysis of new bone formation. Calcein (Sigma-Aldrich Cat# C-0875, St. Louis, MO, USA) was administered subcutaneously and intraperitoneally at 10 mg/kg on days 56 and 57 of the study. Alizarin (Sigma-Aldrich Cat# A-5533, St. Louis, MO, USA) was dosed intraperitoneally at 30 mg/kg on day 67 of the study.
Surgery
The rats were induced and maintained under anesthesia using isoflurane. One dose of carprofen (Pfizer Animal Health, New York, NY, USA) and sustained-release buprenorphine (Zoopharm, Windsor, CO, USA) were administered prior to surgery for analgesic coverage. The right knee was shaved, aseptically prepared, and draped for surgery. In the sham group, a surgical approach to the medial collateral ligament on the right hind limb was completed, and the surgical incision was closed in 2 layers using absorbable sutures. In the surgery groups, medial meniscal tear (MMT) surgery was performed by transecting both the medial collateral ligament and medial meniscus of the right hind limb, followed by closure in 2 layers using absorbable sutures [7].
Body weight sample collection and serum analyses
Body weight was recorded twice weekly throughout the study. At the end of the 10-week study the entire right hind limb was carefully harvested, skinned, and cleaned of the soft tissue, with care taken not to disrupt the knee joint. The limbs were wrapped in saline-soaked gauze and frozen at −20 °C for ex vivo imaging and histological analyses of the tibial articular cartilage and bone. Blood was collected at 5 and 10 weeks post-surgery by jugular venipuncture under isoflurane anesthesia. Serum was harvested and stored for serum chemistry and analyses of biomarkers of bone formation and bone and cartilage degradation. Osteocalcin was assessed by rat EIA kit (Cat# BT-490, Biomedical Technologies, Stoughton, MA, USA) and P1NP was quantified in serum samples by LC/MS method [30]. CTX was analyzed by rat LAPS™ Assay (Cat# AC-06F1), TRAP5b by Rat TRAP™ Assay (Cat# SB-TR102), and CTX II by CartiLaps™ Assay (Cat# AC08F1) all produced by Immunodiagnostics Systems (Scottsdale, AZ).
Dynamic weight bearing
Dynamic weight-bearing (DWB) measurements were obtained before surgery, at week 5 and before euthanasia to assess the effects of surgery on the weight-bearing capacity of the hind and front legs. The DWB system (Bioseb, Boulogne, France) is a non-invasive method used to obtain the weight and surface area of all four feet in a freely moving animal [14, 31]. The data were analyzed using the DWB software, version 1.3. Zone parameters were set for the analysis as follows: ≥4 g for one sensor or a minimum of 3 adjacent sensors ≥2 g (to be considered a valid zone). For each time segment that was stable for more than 1 s, zones that meet the above criteria were validated and assigned as either right or left and front or rear. For each testing period, the animals were placed into the chamber and allowed 20–30 s to explore prior to data collection for a total of 3 min. Besides the body weight, the following parameters were measured: weight (g), percentage of weight (% weight) and surface area (mm [2] ) placed on the front left leg, front right leg, both front legs combined, rear left leg, rear right leg and both rear legs combined.
Radiology
All the knee joints were X-rayed with the Faxitron Model MX20 specimen scanner (Faxitron Bioptics LLC., Tucson, AZ, USA) using the settings recommended by manufacturer; exposure time of 12–18 s at 31–35 kV. All the samples were imaged at 3× magnification and were positioned horizontally, with the center of the beam at the knee joint. Radiographic images were used to assess the gross anatomy of the region of interest to be evaluated by µCT and to inspect the bone samples for the presence of fractures or other bone abnormalities.
μCT and EPIC μCT measurements
μCT was conducted on the right knee joint, utilizing a MicroCT 100® computed tomography system (Scanco Medical, Bassersdorf, Switzerland) to obtain a scout 3D image of the knee and ensure that the samples were reproducibly scanned and analyzed exactly at the same region of interest (ROI) in each specimen and that the size of the ROI that we selected allowed for meaningful analysis of bone structures at the proximal tibial epiphysis and metaphysis.
Following imaging of the entire knee, the femur and tibia were carefully separated to ensure that the articular cartilage and meniscus of the joint were not disrupted. The tibia was then cut above the tibiofibular junction and the proximal tibia was then placed in a plastic custom-made positioning device to ensure consistent scanning. Pre-contrast scans of all the tibias were obtained using the MicroCT 100® with the following parameters: 800 slices, a 10 µm resolution, a total scanned area of 8.0 mm [2], and source energy of 70 kVp, 115 µA at 8 W to capture the entire proximal tibia section.
Following pre-contrast μCT scans, 1.2 mL of Hexabrix (ioxaglate meglumine 39.3 % and ioxaglate sodium 19.6 %, Mallinckrodt, St. Louis, MO, USA) was added to a 15 mL conical tube and diluted with 1.8 mL of 0.1 M phosphate-buffered saline containing protease inhibitors (1 % Protease Inhibitor Cocktail Set I, CalBiochem, San Diego, CA, USA), yielding a 40 % solution of Hexabrix [23]. The tibia was then placed in this Hexabrix solution and was capped and incubated in a covered, rocking water bath at 37 °C for 3 h based on the quality of contrast scans obtained in the pilot study. After incubation period, the sample was removed, patted dry and placed in the plastic positioning device within a μCT holder, containing a small amount of saline to help maintain sample hydration during scanning with the MicroCT 100®. Post-soak scanning of the right tibia was performed in the same manner as described above, except that the parameters were set differently to better visualize the cartilage, with source energy of 55 kVp, 145 µA at 8 W and an average scan time of 42 min per sample [23].
Proximal tibial metaphysis
The cancellous bone compartment of the metaphysis was analyzed 1 mm below the growth plate and extending 3 mm distally to include the metaphyseal secondary spongiosa. Cancellous bone was evaluated in a ROI drawn on 100 consecutive slices with a thickness of 1.0 mm that best represented the central segment of the tibia [32]. Cancellous bone parameters included bone mineral density, tissue volume (bone and bone marrow), bone volume, bone volume/tissue volume ratio, bone surface, bone surface/bone volume, trabecular number, trabecular thickness, trabecular separation, connectivity diameter, and structural model index.
Subchondral bone (medial tibial plateau)
A 2.0 mm × 0.5 mm ROI was drawn on the pre-contrast images to include the cortical and cancellous subchondral bone underlying the articular cartilage. This region was drawn on the same 100 consecutive slices (1.0 mm total thickness) used for the metaphyseal and epiphyseal regions, which best represented the central segment of the tibia.
For zonal analysis of the subchondral bone at the medial tibial plateau, the lengths of tibial plateau for the sham and MMT rats were measured to average 2.4 and 3.0 mm, respectively. Longer plateau in MMT rats was due to formation of the osteophytes on the outside of the medial edge of the joint. Based on the pilot data, the medial tibial plateau was divided into 3 equal zones of 0.8 mm each starting from the inside of the plateau adjacent to the central collateral ligaments (Zone 3). Very edge of the plateau containing small part of the osteophytes were exclude from the analysis due to variation in size and shape, but also because excluded pieces of the plateau does not contain cartilage defect and therefore were not deemed relevant for evaluation of the subchondral bone and cartilage. Based on the pilot data and taking into account irregular geometry of the proximal tibia, we kept the depth (0.6 mm) and width (1.0 mm) of ROI similar for both sham and MMT rats since chosen size allows for reproducible measurements of the central cartilage and subchondral bone to always be captured and analyzed. Zone 1 (z1) was designated as the outside of the medial edge of the joint, Zone 2 (z2) was designated as the central zone, and Zone 3 (z3) was designated as the inside of the tibial plateau adjacent to the central collateral ligaments. The parameters of cortical and cancellous bone included bone volume and bone mineral density.
Articular cartilage (medial tibial plateau)
Using the post-contrast scans, contour lines were drawn around a ROI that included the cartilage overlying the medial tibial plateau. The ROI was purposely drawn to include a small amount of bone and soft tissue around the cartilage to ensure that an appropriate threshold was selected to segment the cartilage from bone tissue and soft tissue according to histographic analysis of the tissues [18, 23, 24]. The lower threshold was determined to be 70, and the upper threshold was 400 (Gauss filter parameters: sigma = 1.2, support = 2). Contour lines were manually drawn with semi-automatic contouring applied every 3–10 slices over a total of 300 slices (3 mm) to capture most of the articular surface. The 3D morphology of the entire articular cartilage layer drawn was then visualized and quantified in terms of average cartilage thickness, volume and surface area using direct distance transformation algorithms [22–24, 33].
Other ROIs were drawn and analyzed on this central midpoint of the articular surface, corresponding to standard histological evaluation techniques for the articular cartilage [17]. The length of the medial articular cartilage was measured and divided into 3 zones of equal length as already described for the subchondral bone. The parameters of articular cartilage included cartilage volume (Car.V) and cartilage thickness (Car.Th).
Histology
After the completion of EPIC μCT imaging of the articular cartilage, six tibias were randomly chosen and placed in 10 % neutral buffered formalin for 72 h prior to demineralization in Immunocal (Decal Chemical Corp. Tillman, NY, USA). The tibias were then processed into paraffin and were serially sectioned at ~200 µm intervals into 5 μm-thick sections for staining. The slides were stained with hematoxylin and eosin (H & E) and toluidine blue for general structural evaluation and with safranin O for the evaluation of cartilage damage. General cartilage degeneration included chondrocyte death/loss, proteoglycan loss, or fibrillation [7]. Thickness and degeneration of the articular cartilage at the medial tibial plateau were determined on three longitudinal sections of the proximal tibia using an ocular micrometer. Cartilage thickness was measured separately on each of three zones, as suggested in the literature [34, 35]. In addition, scoring of the osteophytes and categorization into small (up to 300 µm), medium (300–450 µm), and large (450–600 µm) was undertaken with the ocular micrometer. Marginal zone proliferative changes had to be ≥200 µm to be designated as osteophytes.
To assess active bone formation the remaining six tibias were embedded in methylmethacrylate and cut into three consecutive 8 μm-thick longitudinal sections using a polycut sliding microtome (Leica Biosystems, Nussloch, Germany) and one 20 μm-thick sections using a bone cutting and grinding system (Exakt Norderstedt, Germany). Unstained sections were used to assess new bone formation around osteophytes and at metaphysis.
Statistical analysis
Data are reported as means ± standard deviations (SDs). Differences were tested for significance by Student’s t test for unpaired observations when comparing various parameters between the sham and MMT groups. Results were considered statistically significant if the p value was ≤0.05. The statistical analyses were performed using Sigma Plot software (version 12.2, Systat Software, Chicago, IL, USA).