ACLT-induced OA mice
The mice (C57BL/6J) were obtained from Dashuo Biological Institute (Chengdu, China) and all experimental procedures were approved and conducted by the Institutional Animal Care and Use Committees of Sichuan University.All experiments followed the guidelines for the ARRIVE guidelines. Anterior cruciate ligament transection (ACLT) surgery was operated to establish OA model in 4-week-old mice. A total of 20 male mice were randomly divided into Sham group (n = 10) and ACLT group (n = 10). In sham group, joint cavity was opened at right knee joint without cutting the anterior cruciate ligament, while ACLT surgery was performed on the right knee joint as ACLT group. Mice were sacrificed by CO2 inhalation at 4 weeks after surgery, and samples of hind legs were dissected and fixed in 4% paraformaldehyde for 24 hours at room temperature, and then stored at 4℃.
Micro-computed tomography (µCT 50, Scanco Medical, Bassersdorf, Switzerland) was used to observe and evaluate the knee joint morphology of mice. Samples were scanned at 10 µm voxel size and then analyzed using the evaluation software provided by the manufacturer (Scanco Medical, Bassersdorf, Switzerland).
Hematoxylin and Eosin Staining
The acquired tissues were decalcified in 10% EDTA for 8 weeks. After the tissues were dehydrated and embedded in paraffin, paraffin tissue slices were sectioned at 8μm thickness by rotary microtome. The tissue slices were deparaffinized in xylene for 15 minutes, immersed in gradient ethanol for 3 minutes in each concentration, and hydrated in double distilled water (ddH2O) for 10 minutes. The procedure above was repeated in all staining methods at the beginning of specific staining. In hematoxylin and eosin (H&E) staining, hematoxylin was applied for 5 min, followed by water rinse, differentiation with 1% hydrochloric acid ethanol and blue in ammonia-H2O. Eosin was stained for 1 min. After dehydrating in gradient ethanol, the slices were cleared in xylene for 5 minutes, and mounted in resinene.
Safranin O/Fast green Staining
The hydrated tissue slices were stained with Weigert’s iron hematoxylin for 5 minutes and differentiated in 1% hydrochloric acid ethanol for 10 seconds. Fast green was applied to stain the background for 2 minutes. Double distilled water slightly washed the slices, and Safranin O was placed on the tissue slices for 15minutes. At last, the slices were processed by dehydration, vitrification, and sealed with resinene.
Masson’s Trichrome Staining
Masson’s trichrome staining, known as a three-color staining protocol, was operated strictly according to the official specification (Solarbio Life Sciences, Beijing, China). After the appropriate staining was observed under an optical microscope, the slices were dried, cleared and mounted in resinene.
Tartrate Resistant Acid Phosphatase Staining
Tartrate resistant acid phosphatase (TRAP)staining, for analyzing osteoclasts, was performed by the official protocal (Wako Pure Chemical Industries, Osaka, Japan). The hydrated tissue slices were stained with TRAP staining solution for 1h at room temperature and rinsed in double distilled water for 5 minutes. Alcian blue was applied to stain the background for 5 minutes. In the end, the slices were dehydrated, cleared and mounted in resinene.
Quantitative real-time PCR
Tissues and Cells in TRIzol (Invitrogen, Life Technologies, Grand Island, NY, USA) were disrupted using mortar with liquid nitrogen. Total RNA was isolated using the mRNA Selective PCR Kit (Takara, Japan). Complementary DNA was synthesized using SuperScript® III Reverse Transcriptase (Invitrogen, USA). Quantitative real-time PCR was performed in quadruplicates on a 7900HT sequence detector (Applied Biosystems, Palo Alto, CA, USA) using TaqMan Assay-on-Demand primers supplied by Applied Biosystems. Gene of interest cycle thresholds were normalized to TATA-box binding protein (Tbp) house-keeper levels by the ΔΔCt method and displayed as relative copies per Tbp or relative expression normalized to experimental control groups.
Immunofluorescence was carried out using Vector kits DI1788 for green and DI1794 for red for polyclonal antibodies generated from rabbit (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions. The fluorescence secondary antibody was incubated (1:200) for 2 hours, and the slices were kept out of light from this step. PBS was used to rinse the slices for 10 minutes, and 40,6-diamidino2-phenylindole (DAPI) was applied for 10 minutes to show the cell nuclear. The images were collected by confocal laser scanning microscope (CLSM) after the slices were sealed by 50% glycerine.
The data was collected from at least three individual experiments, and then statistically analyzed via one-way analysis of variance. The difference was statistically significant when P<0.05.