Animal experiment
All animal experiments were performed by senior attending physicians, were approved by the Institutional Ethics Committee of Quanzhou First Hospital Affiliated to Fujian Medical University and were performed in accordance with a protocol approved by the Animal Care and Use Committee of Fujian Medical University. 60 male SD rats purchased from Animal Experiment Center of Shandong University were housed individually with free access to water and chow with constant 12-hour light-dark cycle in transparent plastic cages that were free of specific pathogens. They were all acclimatized for 1 week before the experiment began. At the end of the study, all SD rats used in this study were placed in the euthanasia chamber and then euthanized by introducing 100% CO2 gas at a flow rate of 20% to 30% of the chamber volume per minute.
They were randomly divided into two batches, 5th-week batch and 10th-week batch, which was divided into the control group, the model group and the celecoxib group in each batch. There were 10 SD rats in each group. After anesthetized with isoflurane (Attane vet, 1000 mg/g, Oiramal Healthcare, UK), the rats were fixed on the operating table. After hair removal on the surgery area and disinfection with iodine, the skin of the rat right Achilles tendon was cut lengthwise through a 1 cm incision by a sterile surgical blade, revealing the end of the Achilles tendon. The Achilles tendon nearly the end was cut laterally. The whole incision was sutured postoperatively. Starting from the first day after the operation, the rats in the celecoxib group were given celecoxib (Gibco, USA) 10 mg/ (kg/d) by gavage. The first batch was given gavage for 5 weeks, while the second batch was given gavage for 10 weeks. The model group and the control group were given 2 mL normal saline by gavage. The groups were given a gavage once a day. Observation was made once every other day after the operation, and observation was made once a week after the first week.
Observation and X-ray results of TMO model in rats
Skin temperature and swelling of limbs of rats in each group were observed at 5th or 10th postoperative week, and the formation of heterotopic ossification was observed by taking films under anesthesia. Swelling at the injury point was counted as 1 point, at knee joint was counted as 2 points, above knee joint was counted as 3 points, and no swelling was counted as 0 point. The ossification (or calcification) of rat Achilles tendon was evaluated by referring to the classification method of ossification degree by Weiliang Chen et al[6]. After the completion of the film, bilateral Achilles tendon tissues were revealed again, and the color, thickness, elasticity, number and location of ectopic bone of the Achilles tendon of the rats were compared and observed.
Real-time quantitative PCR(RT-qPCR)
The Achilles tendon was cut off at the junction between the tendon and gastrocnemius muscle and the junction with the calcaneus bone. The Achilles tendon specimens were removed completely and other tissues were removed. After washing with normal saline, the rat Achilles tendon was divided into three parts along the sagittal axis of the Achilles tendon. The ossification site tissues were milled with liquid nitrogen. RNA was extracted with Trizol (Invitrogen, USA), complementary cDNA was synthesized by Super Script III reverse transcriptase (Life Technologies, USA), and BMP-4 gene and IL-2 gene expression was quantitatively analyzed using Power SYBR Green PCR Master Mix and StepOnePlus Real Time PCR System (Thermo Fisher Scientific, USA). The primer sequences used are shown in table 1.
Western blot analysis
After surgery 5th or 10th week, some Achilles tendon tissues were cut in RPA lysate at low temperature, incubated on ice for 30 min, centrifuged at 12000 r/min at 4℃ for 30 min, and then the supernatant was obtained. BCA kit (Beyotime, China) was used for protein quantification. The protein samples were added appropriate volume of 5×SDS sample buffer (including β-mercaptoethanol), mixt well, boiling water bath for 10 min, and made fully denatured. The expression of BMP-4 protein was detected by Western blotting. The antibody anti-BMP-4(1:500, #ab39937, abcam, USA)and anti-β-actin (1:1000, #AA128, Beyotime, China) were as primary antibody. Anti-rabbit IgG HRP-linked antibody (1:2000, #7074, CST, USA) and anti-mouse IgG HRP-linked antibody (1:2000, #7076, CST, USA) were as secondary antibody.
Immunohistochemical examination
After fixed with 4% paraformaldehyde fixative at room temperature and embedded in paraffin, the part of the tissue was sliced. After dewaxed for 5 min at 60℃ in a baking machine, the slices were washed with PBS twice. Subsequently, the slices were soaked in a dye box filled with citrate buffer, heated in the microwave for 30 minutes, cooled to room temperature, and washed with PBS 3 times. After sealed with 5% donkey serum, the slices were incubated with primary antibody overnight at 4℃. After incubated the secondary antibody, the slices were observed under microscope.
Determination of IL-2 in serum
2 ml of venous blood was extracted from rats at 5th and 10th week after the operation, and the serum was separated by centrifugation. The determination of IL-2 in serum was performed in accordance with the kit instructions (EK0399, BOSTER, China).
Statistical analysis
SPSS13.0 statistical software package was used for analysis. The measurement data were expressed as mean±standard deviation, and one-way ANOVA was used for comparison between groups. The statistical differences were detected among control group, model group and celecoxib group. Statistical significance was accepted at P < 0.05.