Synovial fluid collection and enzyme-linked immunosorbent assay (ELISA).
Synovial fluids were obtained from 40 temporomandibular disorder patients, and Visual Analog Scales (VSA) scores were recorded according to their own temporomandibular joint pain. The 40 patients were divided into two groups based on their clinical symptoms and imaging findings. More specifically, they differed from the position of the TMJ discs and the destruction of the condyle which was much clear through the Cone beam Computer Tomography (CBCT). The two groups respectively are the articular disk displacement (ADD) group (n = 24) and the osteoarthritis (OA) group (n = 16). The method to collect synovial fluid was described previously.35,36 A totals of 2 ml of diluted synovial fluid was extracted after normal saline was injected into the TMJ cavity with a 5 ml syringe containing 2ml of normal saline, which was irrigated three times. Informed consent was obtained from the patients, and the protocol was approved by the Ethics Committee, School and Hospital of Stomatology, Wuhan University. The content of Netrin-1 in the synovial fluid was measured by a human ELISA kit (HM11241; Bioswamp, Wuhan, China). The method was performed according to the manufacturer’s protocol.
Mouse TMJ OA model.
40 eight-week-old male C57BL/6 mice were divided into the control group and the MIA group. After intraperitoneal injection of pentobarbital sodium, the preauricular area of the mice was palpated to feel the zygomatic arch; under this was the TMJ cavity. After the preauricular area and zygomatic arch were disinfected with 70% ethyl alcohol, the needle was injected into the upper front of the junction of the zygomatic arch and the temporal bone with a 1-ml syringe, and the needle was slowly injected backward and downward so that it could be judged that the needle had been inserted into the joint cavity.23 For the MIA group, the mouse was given a unilateral TMJ injection of 20 µL of MIA with a concentration of 1 mg/50 µL.27,37 The same amount of saline was injected into the unilateral temporomandibular joint cavity in the control group. Finally, euthanize the mice by intraperitoneal injection of an overdose of sodium pentobarbital solution. The protocol was approved by the Ethics Committee, School and Hospital of Stomatology, Wuhan University.
Von Frey test.
The Von Frey test was used to measure the pain experienced by the mice. 38 The hard-plastic tip was used to stimulate the midpoint of the connection between the eyes and ears of the mouse's head and face. In the process of the experimental operation, the mechanical stimulation intensity increased, and in turn, mouse behavior was observed at the same time. When reactions such as rubbing the mouth or scratching the head were observed, the stimulus intensity (g) was recorded. The operation was repeated five times every 30 s, and the head withdrawal threshold was recorded. Then, the average value was taken as the TMJ pain threshold.
Every 10 mice which consist of 5 from the control group and 5 from the MIA group were euthanized 1, 2, 3, and 4 weeks after the injection. All condylar specimens were immediately fixed with 4% paraformaldehyde for 24 hours. Take four specimens from each group for micro-CT detection after flushing overnight. And then, all the specimens were decalcified in 10% EDTA for two months, dehydrated and embedded in paraffin. All 40 specimens were sliced and performed histological staining immunohistochemistry staining.
The condyles were collected and fixed with paraformaldehyde for 24 h. After flushing overnight, the condyles were analyzed by micro-CT (SkyScan1176, Germany). The camera pixel size was 12.59 µm at 50 kV and 500 µA. The image pixel size was 9 µm. Three-dimensional reconstruction of the temporomandibular joint condylar head was performed to observe the bone changes, and BV/TV, Tb.Th and Tb.N values were also analyzed to compare the destruction of bone structures.
The specimens were cut into 4-µm sections. Hematoxylin & eosin (H&E) staining and toluidine blue (T.B) staining were performed. Cartilage thickness, the number of chondrocyte cells and the proteoglycan area were measured as previously reported.39 The Mankin score assesses the integrity of the cartilage surface shown by HE staining, cellularity and the condition of cartilage matrix shown by toluidine blue staining.
Immunohistochemistry of Netrin-1.
After de-paraffinized, rehydrated and washed, sections were antigen-retrieved by microwave oven for about 25 minutes. And then, sections were incubated peroxidase blocking solution at 37℃ for 30 minutes to inactivated cell endogenous peroxidase. Bovine serum albumin was used at 37℃ for 30 minutes to block unspecific ligations. After that, the sections were incubated the polyclonal goat IgG anti-Netrin-1 antibody (1:200; AF1109-SP, R&D system, Minnesota, USA) overnight at 4°C. The next day, the sections were incubated with secondary antibody and peroxidase at 37℃ for 30 minutes in sequence. At last, use diaminobenzidine (DAB) for color development and observe the sections under the microscope. The reagents were from the UltraSensitive S-P goat kit (KIT-9709/9719, Maixin, Fuzhou, China).
TRAP staining and cellular immunofluorescence.
The raw264.7 cell line was placed in a medium containing 10% FBS and 90% DMEM, incubated and subcultured in an incubator at 37°C, 95% O2, 5% CO2. Add 10ng/ml RANKL to the culture medium of the newly passaged cells, and incubate the differentiation in a 37°C, 95% O2, 5% CO2 incubator. When performing TRAP staining, configure the cell fixation solution according to the steps of the TRAP kit (Sigma-Aldrich, USA) and add it to the dish for 30 seconds; then, in the dark, drop the prepared staining solution into the dish, and incubate in a 37°C incubator for one hour; finally, wash the dish three times for three minutes each time and observe under a microscope.
For the cellular immunofluorescence, first, the induced osteoclasts were fixed with 4% paraformaldehyde for 15 minutes, and then blocked with 1% BSA for 30 minutes. Next, the osteoclasts were incubated with the polyclonal goat IgG anti-Netrin-1 antibody (1:200; AF1109-SP, R&D system, Minnesota, USA), the Unc5B rabbit monoclonal antibody (1:200; #13851, Cell Signaling Technology, Massachusetts, USA) and the DCC rabbit polyclonal antibody (1:200, 19123-1-AP, Proteintech, Wuhan, China) overnight at 4℃. The next day, in the dark, add the secondary antibody and incubate for one hour at 37℃. Finally, DAPI was added to stain the nucleus.
All data are expressed as the mean ± SD. GraphPad Prism 8 was used to analyze the dates unless otherwise indicated. Ordinary one-way analysis of variance (ANOVA), two-way ANOVA and unpaired t- test were performed. A P-value < 0.05 indicated a statistically significant difference. For the correlation analysis, Excel was used. We performed correlation analysis and regression analysis, and a linear fit. At last, we used variance analysis to test whether this linear correlation is statistically significant. A P-value < 0.05 indicated a statistically significant difference.
Written informed consent was obtained from all participants. All methods were performed in accordance with relevant guidelines and regulations. Methods of sample collection, storage and experimental protocols for human study were approved by the Ethics Committee, School and Hospital of Stomatology, Wuhan University and informed consent was obtained from the patients. All methods were performed in accordance with ARRIVE guidelines. For the animal study, excessive sodium pentobarbital solution was injected intraperitoneally into mice for the euthanization of mice and all the protocols were approved by the Ethics Committee, School and Hospital of Stomatology, Wuhan University.