A total of 75 male C57BL/6J mice (8 weeks, 20±2g) were purchased from JOINN Laboratories (Suzhou), Inc. (License No. SCXK(Su)2018–0006, Suzhou, China). Following arrival at the facility, mice were allowed at least 1 week to acclimatize . Experimental animals (3–5 mice/cage) were housed in a room at 23 ± 2°C, with 55±10% humidity and lighting was maintained at a 12-hours-on–12-hours-off schedule (light on from 7:00 a.m. to 19:00 p.m.). Food and water were provided ad libitum. Maintenance, use, and treatment of all animals were in accordance with accepted standards of the Ethics Committee of Soochow University (ECSU–2019000158).
Destabilization of the medial meniscus (DMM) surgery
After 1 week of adaptive feeding, the medial meniscal ligament (MMLT) in the right knee joint was severed to establish an OA model as described previously [27, 28]. In brief, after isoflurane inhalation anesthesia in mice, a medial incision of the right knee joint was created to slightly shift the extensor muscle of the knee joint without transecting the patellar ligament and expose the right knee joint. The MMLT was cut, so that the medial meniscus could be moved to the medial side. After repositioning the knee extensor, the medial incision was sutured and the skin closed. For mice that underwent sham surgery, a similar surgical approach was used, however, the knee joint was operated without MMTL transection. After surgery, mice were had access to food and tap water ad libitum.
Grouping, drug medications, and treatment protocols
Two days after DMM surgery, mice were randomly and equally divided into five groups (n = 15 in each group), including a sham-operated control group (Sham group), an osteoarthritis (OA) group (DMM group), an OA treated with Etoricoxib 5mg/kg (DMM+E5) group, an OA treated with Etoricoxib 10mg/kg (DMM+E10) group, and an OA treated with Etoricoxib 20mg/kg (DMM+E20) group.
Etoricoxib was purchased from Jiangsu argon krypton xenon material technology co. LTD (Suzhou, China). Etoricoxib and the vehicle (40% ethyl alcohol–saline solution) were freshly prepared before each injection, Etoricoxib was dissolved in the vehicle solution and placed in a warm bath until completely dissolved. According to the grouping scheme mentioned above, mice were injected intraperitoneally, 3 times a week for 4 weeks with an injection dose of 0.1mL per mouse. Mice in the Sham group and DMM group were injected with a similar dose of vehicle (Figure 1).
Micro computed tomography measurements
Four weeks after a 2-days recovery period, mice were euthanized by continuous CO2 inhalation, the right knee joints were harvested, and fixed in 10% neutral formalin. After fixation for 48 hours, the specimens were transferred to 70% ethanol for high-resolution micro computed tomography (Micro-CT) (SkyScan1176, Aartselaar, Belgium) . The scanner was set at a gamma-ray voltage of 50KV and a current of 200uA, the filter was 0.5mm Al, and a resolution of 9 μm per pixel was used. The standardized parameters and thresholds of grey values for all samples were 0–0.075. The NRecon software and NReconServer software were used for the reconstruction of two-dimensional images, and Dataview software was applied to adjust the XYZ axes. Images were analyzed using the CTan software, and sagittal and coronal images of the tibial subchondral bone were applied to evaluate changes. The subchondral bone of the medial tibial plateau with 15 consecutive layers in the regions of interest (ROI) in the recombinant images of mice was selected for three-dimensional reconstruction and quantitative analysis. Specific indicators included bone volume fraction (BV/TV) and trabecular thickness (Tb.Th). Three-dimensional images were acquired by Mimics software.
Atomic force microscopy analysis and nano-mechanical testing
Fresh knee joints were dehydrated in 20% and 30% sucrose solution for 12 hours, respectively. After dehydration, knee joints were embedded with optimal cutting temperature compound and cut into 20–30 µm thick sections by a section microtome (CM3050S, Leica, Nussloch, Germany). From each group, four slides were selected, and five areas were taken from each slide, which were scanned by atomic force microscopy (AFM) (Dimension ICON, Bruker, USA). AFM tests were carried out at room temperature (RT). First, the force constant k (40N/m) of the probe (Brooke, Germany) and the curvature radius R (5nm) of the tip was calibrated. Then, we performed tests in the ROI of the subchondral bone, obtained the mechanical curve, and calculated the elastic modulus by using the formula (1). In this study, the Hertz model, which is the most commonly used in the AFM test of biological tissue, is used to calculate the compression modulus of the elastic modulus. The formula (1) is as follows:
F, E, ν, R and δ represent pressure, Young’s modulus, Poisson’s ratio, indentation radius, and indentation depth, respectively.
Scanning electron microscopy analysis
After excess tissue was removed from fresh knee joints, knee joints were digested with a mixed enzyme solution comprised of type I and type II collagenase (4% type I collagenase mixed with 4% type II collagenase). The subchondral bone was obtained by placing the knee joints in an incubator at 37 ℃ and by changing the digestive solution every day until the cartilage and surrounding soft tissues were completely digested. After digesting, the specimens were washed with phosphate buffered solution, then fixed with 4% glutaraldehyde for 2.5 hours at RT, rinsed in phosphate buffered solution, and dehydrated in graded ethanol series. After the residual moisture was further removed by the critical point dryer, the specimens were fixed on a metal platform through conductive glue, then sprayed with gold by ion spatter to enhance its conductivity. Finally, specimens were observed by scanning electron microscopy (SEM) (FEI Quanta 250, Hillsboro, USA).
After Mirco-CT imaging, samples were decalcified in 10% ethylenediamine tetraacetic acid (EDTA) (pH = 7.4) on a shaker at RT for 14 days. Following decalcification, and trimming away excess soft tissues, knee joints were dehydrated in graded ethanol series. Then, samples were transferred to N-butyl alcohol for 8 hours and infiltration with paraffin was performed for 7 hours. Finally, paraffin-embedded tissues were cut into 6-µm-thick sections using a rotary microtome. Hematoxylin-eosin (HE) staining was adopted to evaluate pathological changes of cartilage of medial tibial and subchondral bone of medial tibial and synovial tissues, and cartilage, subchondral bone, and synovium were evaluated by the modified Mankin score, the empty/total osteocyte ratio, and the synovialitis-score, respectively [30–33]. Safranin O-Fast Green staining was performed on sagittal sections to determine changes in proteoglycans, and cartilage destruction was scored using the Osteoarthritis Research Society International (OARSI) OA cartilage histopathology assessment system as described .
Statistical analysis was performed using SPSS 23.0 (SPSS Inc., Chicago, IL, USA) and GraphPad prism 7.0 (GraphPad Software, La Jolla, CA, USA). Normal distribution and homogeneity of variance of data were evaluated by Shapiro-Wilk test and Levene’s test, respectively. Data were presented as the mean ± standard deviation (SD). Significant differences between study groups were obtained using a one-way analysis of variance (ANOVA) with Tukey’s post-hoc test. Statistical significance was set at p＜0.05.