This reseach was approved by Animal Ethics Committee of Third Hospital of Hebei Medical University(approval number: Z2019-005-1). Female Sprague-Dawley rats (50–80g) aged 4 weeks were provided by the Laboratory Animal Center of Hebei Medical University. 120 rats were randomly divided into two groups: PI group (n=60) and Control group (n=60). Each group was investigated and sacrificed by overdose anesthesia at 4, 8 and 12 weeks post-operatively. According to animal euthanasia guidelines [22, 23], rats were intraperitoneally injected with an overdose of pentobarbital sodium (200 mg/kg) for euthanasia. Rats were placed in a controlled environment (60% ± 5% humidity, 12/12 h light/dark cycle, 22 °C ± 3 °C) and were free to obtain food and water. In order to make animals accustomed to the laboratory environment, an adaptation period of one week was given before the experiment began.
Anesthesia/analgesia
The injectable anesthetic pentobarbital sodium (30 mg/kg) was chosen instead of surgical anesthesia. It was injected into the proximal thigh (quadriceps femoris) and the needle was pointed back to avoid sciatic nerve injury. For post-operative care, animals were provided with 24-hour quiet recovery time in warm and dry areas. They were also provided with food to promote gastrointestinal peristalsis and to prevent stagnation after they are fully awake.
Surgical technique
Before the operation, the left knee of the rats was placed in an extended state. After the surgical area is shaved, the surgery is prepared aseptically. A midline skin incision was performed; after the skin and subcutaneous tissue were separated, the capsule was exposed through the medial approach of the knee. Similar methods of PI induced by surgery have been reported in our previous studies [24, 25]. In this study, the medial retinaculum of knee joint was cut off, PI could be seen during the operation. The incision was then closed without reconstruction of MPFL, retinaculum and capsules. When the surgery is over, dressing was applied.
Micro-computed tomography (Micro-CT) assessment
In order to assess changes in the subchondral bone microstructure caused by PI, the femoral trochlear was carefully harvested and imaged using a micro-CT (SkyScan model 1076, Skyscan, Belgium). All scans were performed with a voxel size of 10 μm at 50kV and 800μA.The regions of interest (ROIs) of the trochlear were located transversely below the subchondral bone plate, excluding the cortex. Based on previous studies, the following representative parameters were selected to assess changes in subchondral bone [7]: bone volume to total volume fraction (BV/TV fraction), bone mineral density (BMD), trabecular (TB) thickness, the TB separation (SP),the structure model index (SMI), TB pattern factor (PF), TB number, and degree of anisotropy (DA) were calculated. CT scan was then performed 4, 8 and 12 weeks after surgery.
Histological
The articular cartilage of femoral trochlear was separated at different stages, fixed with 4% polyformaldehyde, decalcified in 10% EDTA until completely demineralized and embedded in paraffin. 5μm sections were cut along the femoral axis to get the transverse images of the trochlea sulcus. The glycosaminoglycans in the cartilage were evaluated using Safranin O, and the protein was subjected to fast green counter staining to evaluate cartilage degradation and scored using a semi-quantitative histopathological grading system based on the OARSI score [26,27]. Immunohistochemical
The slices were deparaffinized in xylene and rehydrated. We washed three times with phosphate buffered saline at room temperature for 5 minutes each time. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide (H2O2) for 10 minutes.The antigen of slide was repaired by microwave treatment in 10 mm sodium citrate (pH 6.0) for 10 minutes. Incubation of slices overnight at 4℃ with anti-collagenX (BoAoSen,BeiJing, China), anti-MMP-13(Servicebio,WuHan,China) anti-TNF-a(abclonal, WuHan, China), anti-NFKP65 (Servicebio, Wu Han, China) at a dilution of 1:50. The primary antibody was omitted from the negative control reaction. Thereafter, by the magnification of 20×100 of objective lens, at least per unit area in five randomly selected areas of each slice in each group for photographing. Try to make the organization fill the entire field of view when taking pictures, and ensure that the background light of each photo is the same. Image-Pro Plus 6.0 software was used to select the same brown color as the unified standard for judging all photos. Each photo was analyzed to obtain the cumulative optical density (IOD) of each photo and the area of the tissue (AREA). And determine the areal density, areal density=IOD/AREA, the greater the areal density value indicates the higher the positive expression level.
Real-time polymerase chain reaction (RT-PCR)
At 4, 8 and 12 weeks after surgery, cartilage from trochlear was detected by RT-PCR for mRNA expression (n=36 knees, each group=18 knees). RNA from chondrocytes and cartilage was extracted using Trizol reagent (Servicebio, Wuhan, China). mRNA was translated into cDNA by the RevertAid™ first strand cDNA synthesis kit (Thermo, #K1622) according to the instruction of manufacturer. MMP-13, Collagen X, TNF-a and NF-κB p65 were the target genes, all genes were analyzed by applying sequence detection system, and the expression of related mRNA of these genes was standardized by relative to the GAPDH gene and calculated with the formula 2-ΔΔCt (cycle threshold) method. All primers used are shown in (Table 1). We performed each experiment three times and obtained the average values.
Statistical analysis
Descriptive statistical analysis was performed with mean values and standard deviations. Data were analyzed with statistical software GraphPad Prism (GraphPad software V7, La Jolla, CA, USA) and Shapiro-Wilks test for each variable to evaluate normality, Levene’s test evaluated the homogeneity of variance. Two-way analysis of variance (ANOVA) analysis the parametric data between the two groups at each time point. p < 0.05 was considered statistically significant. According to the result of preliminary experiments, each group and each time point needs at least six rats, with a confidence of 90% and an efficacy of 80% (1-β) [28].