All animal experiments were performed by senior attending physicians and approved by the Institutional Ethics Committee of Quanzhou First Hospital Affiliated to Fujian Medical University. Procedures were carried out in accordance with a protocol approved by the Animal Care and Use Committee of Fujian Medical University.

The Achilles tendon injury model used in this study was based on previously reported methods [7]. Specifically, our model was optimized to better simulate the clinical scenario of TMO. Male Sprague-Dawley (SD) rats (n = 60) purchased from the Animal Experiment Center of Shandong University were housed individually with free access to water and chow under a constant 12-h light-dark cycle in specific pathogen-free transparent plastic cages. All animals were acclimatized for 1 week before experimentation. At completion of the study, rats were euthanized in a chamber using 100% CO₂ gas at a flow rate of 20–30% chamber volume per minute [8].

The rats were randomly divided into two batches (5-week and 10-week). Each batch was further subdivided into three groups: the sham-operated group (control group), which underwent the same surgical procedure but without tendon injury; the injury-saline group (model group), which underwent tendon injury and received daily gavage with normal saline; and the celecoxib group, which underwent tendon injury and received celecoxib treatment via gavage (n = 10 per group). To further investigate the effect of celecoxib, we sometimes included an additional sham-operated group that also received celecoxib, designated as the Celecoxib group (Control); for clearer distinction, the group that received tendon injury plus celecoxib gavage was correspondingly designated as the Celecoxib group (Model). Following isoflurane anesthesia (induction: 4%; maintenance: 2%; Attane vet, 1000 mg/g, Piramal Healthcare, Grangemouth, UK), rats were immobilized on the operating table and the surgical area was shaved and disinfected with iodine. A 1-cm longitudinal incision was made over the right Achilles tendon using a sterile surgical blade. The distal portion of the Achilles tendon was exposed and transected laterally. The incision was then sutured closed.

Starting from the first postoperative day, the celecoxib group received 10 mg/kg/day of celecoxib (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) via gavage for either 5 or 10 weeks. The model and control groups received 2 mL of normal saline via daily gavage. Animals were monitored every other day during the first week and weekly thereafter.

Skin temperature and the swelling of limbs for the rats in each group were observed at the 5th or 10th postoperative week, and the formation of heterotopic ossification was observed by taking X-ray under anesthesia. Swelling at the injury point was counted as 1 point, at the knee joint as 2 points, above the knee joint as 3 points, and no swelling as 0 point. The swelling scoring system employed in this study is consistent with that described in prior research, wherein the degree of swelling was quantified to assess the severity of the inflammatory response and associated tissue damage. Similarly, the extent of ossification (or calcification) in the rat Achilles tendon was evaluated following a previously established classification method for grading the degree of ossification [7]. After completion of the X-ray, the bilateral Achilles tendon tissues of rats in different groups were compared for color, thickness, elasticity, and the number and location of ectopic bone. X-ray imaging was performed using a digital X-ray machine (Model: KODAK DR 7500, Carestream Health, Rochester, NY, USA). At the 5th and 10th weeks, rats were fully anesthetized and placed in a supine position on the X-ray examination table, with the target area (operated humerus) positioned at the center of the detector. After adjusting the appropriate parameters (tube voltage 70 kV, tube current 7 mA, exposure time 10 ms), X-ray exposure was performed to obtain the images. These were subsequently processed and stored using the relevant software.

To further evaluate the extent and structure of heterotopic ossification, we performed µCT analysis on the heterotopic ossification tissue in the TMO model. At 10-week post-surgery, 5 rats were randomly selected from each group, anesthetized, and euthanized. The heterotopic ossification tissue from the right Achilles tendon area was carefully dissected, washed with saline, and fixed in 4% paraformaldehyde for 24 h. Samples were then scanned using a Skyscan 1176 µCT scanner (Bruker, Kontich, Belgium) with the following parameters: voltage 50 kV, current 500 µA, exposure time 900 ms, and resolution 9µm. The acquired images were reconstructed using NRecon software (version 1.6.8.0, Bruker, Kontich, Belgium) and analyzed using CTAn software (version 1.18.8, Bruker, Kontich, Belgium).

The following bone morphometric parameters were assessed: bone volume fraction (BV/TV, %), trabecular number (Tb.N, 1/mm), trabecular thickness (Tb.Th, mm), and trabecular separation (Tb.Sp, mm). Additionally, three-dimensional images of the heterotopic ossification tissue were constructed using CTVol software (version 2.3.2.0, Bruker, Kontich, Belgium) to visually present its structural characteristics. Statistical analysis was performed using SPSS 22.0 software (version 22.0, IBM, Armonk, NY, USA). Intergroup comparisons were conducted using one-way ANOVA, with p 0.05 considered statistically significant.

The Achilles tendon was cut off at the junction between the tendon and the gastrocnemius muscle, and the junction with the calcaneus bone. The Achilles tendon specimens were removed completely, together with other tissues. After washing with normal saline, the Achilles tendon was divided into three parts along its sagittal axis. The ossification site tissues were milled with liquid nitrogen, and RNA was extracted with Trizol (cat. no. 15596018, Invitrogen, Waltham, MA, USA). Complementary DNA (cDNA) was synthesized by Super Script III reverse transcriptase (cat. no. 18080044, Life Technologies, Carlsbad, CA, USA), and gene expression levels for Bmp4, Bmp2, Bmp7 and Il2 were quantitatively analyzed using Power SYBR Green PCR Master Mix and StepOnePlus Real Time PCR System (cat. no. 4376598, Applied Biosystems, Singapore). Actb was used as the housekeeping gene for relative quantification analysis. The primer sequences used are shown in Table 1.

At the 5th or 10th week after surgery, a portion of the Achilles tendon tissues was cut in RIPA lysate at low temperature and incubated on ice for 30 min. The samples were then centrifuged for 30 min at 12,000 r/min and 4 °C to obtain the supernatant. A BCA kit (cat. no. P0012, Beyotime, Biotechnology, Shanghai, China) was used for protein quantification. An appropriate volume of 5$\times$SDS sample buffer (including $\beta$-mercaptoethanol) was added to the protein samples, mixed well, and then boiled for 10 min to achieve full denaturation. The expression of BMP proteins was detected by Western blotting using primary antibodies against BMP-2, BMP-4, and BMP-7 (each 1:500; #ab284387, #ab39937, and #ab129156, respectively, Abcam, Waltham, MA, USA). Primary antibodies against Osterix (1:500, #ab209484, Abcam, Waltham, MA, USA), Osteocalcin (1:500, #ab133612, Abcam, Waltham, MA, USA) and $\beta$-actin (1:1000, cat. no. AA128, Beyotime Biotechnology, Shanghai, China) were also used. The secondary antibodies used were anti-rabbit IgG HRP-linked antibody (1:2000, cat. no. 7074, Cell Signaling Technology, Danvers, MA, USA) and anti-mouse IgG HRP-linked antibody (1:2000, cat. no. 7076, Cell Signaling Technology, Danvers, MA, USA).

For IHC examination, tissues were fixed with 4% paraformaldehyde at 4 °C for 24 h, embedded in paraffin, and dewaxed for 5 min at 60 °C in a baking machine before cutting sections. Tissue sections were washed twice with PBS, soaked in a dye box filled with citrate buffer (cat. no. AR0024, Boster Biological Technology, Pleasanton, CA, USA), heated in the microwave for 30 minutes, cooled to room temperature, and finally washed three times with PBS. After blocking with 5% donkey serum (cat. no. 017-000-121, Jackson ImmunoResearch Laboratories, West Grove, PA, USA), the sections were incubated with the primary antibody overnight at 4 °C. Following incubation with the secondary antibody for 1 h at room temperature, the sections were observed by microscopy. BMP-4 protein expression was quantified using ImageJ software (version 1.53t, National Institutes of Health, Bethesda, MD, USA). For each section, three representative fields of view (200$\times$ magnification) of the Achilles tendon tissue were selected as regions of interest (ROI). Positive staining intensity was measured using the “Color Deconvolution” plugin (ImageJ, National Institutes of Health, Bethesda, MD, USA) to separate the DAB-stained brown color from the background. The DAB substrate kit (cat. no. 10006913, Thermo Fisher Scientific, Waltham, MA, USA) was used for chromogenic detection. The integrated optical density (IOD) of the positive staining was calculated for each ROI, with the results expressed as the mean IOD $\pm$ standard deviation (SD) from three fields of view per section. The relative expression levels of BMP-4 were compared between different groups.

Venous blood (2 mL) was extracted from rats at the 5th and 10th week after operation, and the serum was separated by centrifugation. ELISA was used to determine the levels of IL-2, IL-6, and TNF-$\alpha$ in the serum according to the kit instructions (BOSTER, Wuhan, China).

MSCs were a kind gift from Dr. Cheng. The cells were seeded in gelatin-coated dishes and cultured for 3 weeks with Oricell® human bone marrow MSC osteogenic differentiation medium (cat. no. HUXMX-90021, Cyagen Biosciences (Guangzhou) Inc., Guangzhou, Guangdong, China) with or without celecoxib, as recommended by the manufacturer. After induction, the cells were fixed and stained with alizarin red. For the in vitro MSC experiments, cells were divided into the following groups: the Control group (cultured in standard growth medium without osteogenic induction), the Model group (cultured in osteogenic differentiation medium without celecoxib), and the Celecoxibtreated groups (cultured in osteogenic differentiation medium supplemented with 50 µM or 100 µM celecoxib).

The SPSS 22.0 statistical software package was used for analysis. Measurement data were expressed as the mean $\pm$ SD. One-way ANOVA was used for comparisons between the control group, model group, and celecoxib group. Statistical significance was defined as p 0.05.

No significant changes were observed in the control group during the entire experimental period. At the 5th week after surgery, the skin temperature of rats in the celecoxib group was lower than in the model group (Table 2). Moreover, the affected side of the rat in the celecoxib group showed significantly less swelling compared to the model group (p = 0.002) (Table 3 and Fig. 1). At the 10th week after operation, the skin temperature and swelling in the celecoxib group were both significantly lower compared with the model group (p 0.05) (Tables 2,3; Fig. 1).

Fig. 1.

Swelling of the Achilles tendon at the 5th and 10th week after operation (n = 10 biological replicates per group). General contrast between the left and the right Achilles tendons of the model and celecoxib groups at 5 and 10 weeks after operation. At the 10th week, the right Achilles tendons of rats in the model group showed obvious heterotopic ossification.

At the 5th week after surgery in the model group, the Achilles tendon had repaired itself, and adhesion, congestion, and edema were visible on the affected side. Compared to the uninjured side, the affected side had a thickened diameter with decreased elasticity. A transparent fusiform expansion could be seen at the anastomosis with poor activity. X-ray results suggested heterotopic ossification (Level II). Compared with the model group, the celecoxib group had less congestion and edema, and the anastomosis was connected by tendon tissue. The sliding ability of the Achilles tendon in the celecoxib group was better than that of the model group, and the X-rays showed heterotopic ossification (Level I). At the 10th week after surgery in the model group, hard bone-like nodules were visible at the Achilles tendon, the activity was poor, and there was obvious swelling compared to the contralateral side. At the distal end of the Achilles tendon, the X-rays all indicated heterotopic ossification (Level III). In the celecoxib group, the degree of hemorrhage and edema was significantly reduced, and there was obvious adhesion of the sputum. The sliding ability of the Achilles tendon was better than that of the control group. Three rats in the celecoxib group were classified as heterotopic ossification grade III, and the remainder as heterotopic ossification grade II. With the longer experimental period (10-weeks), the Achilles tendon in both the model and celecoxib groups gradually thickened, and ectopic bone formation was visible. However, the overall rate of osteogenesis in the model group was earlier and faster than in the celecoxib group (p 0.05).

Notably, all rats in our study developed heterotopic ossification following the Achilles tendon injury, achieving a 100% success rate for the induction of TMO. Representative images of the TMO model are shown in Fig. 1, X-ray images in Fig. 2, and statistical results in Table 4. While all rats developed heterotopic ossification, the celecoxib group showed a later onset, milder ossification grade, and slower overall bone formation rate.

Fig. 2.

Formation of ectopic ossification at the 10th week after the operation (n = 10 biological replicates per group). (A) Representative X-ray images of the Achilles tendon region. From left to right: sham-operated control group (no ossification), injury + saline model group (ossification present), and injury + celecoxib group (ossification present). (B) Representative three-dimensional micro-computed tomography (µCT) reconstructions of heterotopic bone (scale bar: 500 µm) and quantitative analysis of bone morphometric parameters: bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp). Data are presented as mean $\pm$ standard deviation (SD). Statistical comparisons were performed between the model group and the celecoxib group using one-way ANOVA. *p 0.05, **p 0.01, vs. model group.

BMPs were activated in our TMO model. At the 5th week after the operation, the mRNA expression of Bmp in the right Achilles tendon was significantly higher than in the left Achilles tendon. Bmp mRNA expression in the right Achilles tendon of the celecoxib group was significantly lower than in the model group (Fig. 3A). After 10 weeks, the expression levels of Bmp genes in the Achilles tendon tissue of rats in the celecoxib group were significantly lower than in the model group (Fig. 3B). These results indicate that celecoxib can significantly inhibit the expression of Bmp mRNA in the TMO rat model. We also examined the expression of BMP proteins in tissues by Western blotting. The results showed that BMP protein levels in the ossified tissue of the celecoxib group were lower than in the model group (Fig. 3C,D).

Fig. 3.

Celecoxib inhibits the expression of bone morphogenetic protein genes and proteins (n = 10 biological replicates per group). (A,B) RT-qPCR results for mRNA expression of Bmp genes at the 5th (A) and 10th (B) weeks postoperatively in the heterotopic ossification model in rats. (C,D) Western blot results for BMP protein expression in the Achilles tendon tissues at the 5th (C) and 10th (D) weeks postoperatively. $\beta$-actin was used as the loading control. The gray scale of bands was analyzed by Image J software. All data represent the mean $\pm$ SD. *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001, ns (not significant) by one-way ANOVA.

The role of celecoxib in osteogenesis was further studied during the MSC osteogenesis process. Alizarin red staining in the celecoxib group was lighter than in the model group (Fig. 4A). The expression of BMP-2 and BMP-4 was greatly increased in osteogenic MSCs, and this increase was significantly inhibited by celecoxib at both 50 µM and 100 µM. BMP-7 expression was also elevated in the model group, but was significantly inhibited only at the 100 µM concentration of celecoxib (Fig. 4B). These results indicate that celecoxib can effectively inhibit the expression of key osteogenic BMPs, with BMP-2 and BMP-4 showing greater sensitivity than BMP-7 in this model.

Fig. 4.

Celecoxib reduced osteogenic differentiation and BMP protein expression in mesenchymal stem cells (MSCs). (A) After 3 weeks of induction, MSC osteogenesis was seen using alizarin red staining (200$\times$) (n = 3 independent biological replicates). Control, Model: 10 µm; Celecoxib: 50 µM, 100 µM. (B) BMP protein expression in the model and celecoxib groups (n = 3 independent biological replicates). $\beta$-actin was used as the loading control. The grayscale intensity of protein bands was quantified using ImageJ software. All data are presented as the mean $\pm$ SD. Statistical significance was determined by one-way ANOVA: *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001, ns (not significant).

We next focused on the evaluation of BMP-4 due to its importance in TMO. At the 5th week after operation, BMP-4 protein expression at the surgical site in the model group was significantly higher compared to the contralateral side (p 0.005). BMP-4 expression in the celecoxib group was significantly reduced compared to the model group (p 0.001). These results demonstrate that celecoxib inhibited ossification in the rat TMO model by reducing the expression of BMP-4 protein (Fig. 5A,B).

Fig. 5.

BMP-4 protein expression in rat Achilles tendon tissue at 5 weeks post-operation detected by immunohistochemistry (IHC). (A) The expression of BMP-4 protein in rat Achilles tendon tissues was detected at the 5th week after operation by immunohistochemistry (200$\times$). The brown color indicates positive staining for BMP-4 protein. scale bar: 20 µm. (B) BMP-4 protein positivity was analyzed using ImageJ software. The integrated optical density (IOD) of BMP-4 staining was measured in three representative fields of view per section (200$\times$ magnification). The results are expressed as the mean IOD $\pm$ SD. ***p 0.001 by one-way ANOVA.

We next used ELISA to evaluate the concentrations of IL-2, IL-6, and TNF-$\alpha$ in the serum of rats. The levels of these inflammatory factors in the model group were clearly higher at the 5th week after the operation. In contrast, their levels in the celecoxib group were significantly decreased (Fig. 6A,C,D). We also analyzed Il2 gene expression in the Achilles tendon tissue by RT-qPCR. Il2 expression was significantly increased in the model group, but significantly lower in the celecoxib group (Fig. 6B). This difference was more pronounced at the 10th week. The above results indicated that celecoxib may affect ossification in a rat model of TMO by inhibiting the expression of inflammatory factors.

Fig. 6.

Collectively, our findings show that celecoxib slows osteogenesis in the traumatic myositis ossificans (TMO) model by modulating the expression of BMP-4 and associated factors via a COX-2-dependent mechanism, and by decreasing inflammatory effects. (A) The level of IL-2 in the blood of rats at the 5th and 10th week after surgery, as determined by ELISA. (B) Il2 gene expression in the Achilles tendon tissue of rats in the heterotopic ossification model at the 5th and 10th week after surgery, as determined by RT-qPCR. (C) TNF-$\alpha$ levels in rat blood at the 5th and 10th week after surgery, as determined by ELISA. (D) IL-6 levels in rat blood at the 5th and 10th weeks after surgery, as determined by ELISA. All data represent the mean $\pm$ SD. *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001 by one-way ANOVA.