Patients and specimens
This study was approved by the Ethics Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, and written informed consent was obtained from all patients or their legal guardians. Traumatic HO was identified using X-ray and CT radiography conducted on 24 patients (14 males and 10 females, previously healthy, age ranging from 24 to 67 years) who had previously suffered an elbow fractures and were treated using internal fixation, or revision after hip/knee arthroplasty (8 patients per group). The muscle tissues were used as baseline controls. Blood samples (5 ml per person) were collected 1 day before clinical surgery and blood samples obtained from 8 healthy individuals were used as baseline controls. All the samples were processed immediately to collect serum, which was then stored in a -80°C freezer. The serum specimens were processed for ELISA.
Male 6 to 8 week old BALB/c mice were housed under specific pathogen-free conditions at the Animal Experimental Center of Shanghai Sixth People's Hospital, and all experiments conducted were approved by the Animal Research Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital. Trauma-induced HO was created using a murine tenotomy model, as we previously described . After anesthesia was induced using an intraperitoneal injection of 1% pentobarbital sodium, a skin incision that was 1 cm in length was made on the lateral aspect of the Achilles tendon to expose its full length. Then, the Achilles tendon was transected precisely at its midpoint using a surgical knife. For the sham operation, the incision was made through the skin without touching the Achilles tendon. The incised skin was closed using absorbable sutures. To assess the effect of clodronate liposomes on HO, the animals were randomly divided into 2 groups: Control group (tenotomy surgery with PBS liposomes) and clodronate group (tenotomy with clodronate). The mice were intraperitoneally injected at doses of 1.4 mg/20 g body weight twice a week from the day of surgery. For the TGF-βblockage experiments, the mice were intraperitoneally injected with TGF-βantibodies (5 mg/kg body weight) daily twice a week before being sacrificed to be used for the micro CT analysis.
Mice were sacrificed using carbon dioxide (CO2) inhalation for an indicated period to be used for histological observations. The ankles with Achilles tendons were dissected and fixed in 4% paraformaldehyde for 24 h. The HO tissues from patients were collected during surgical operation. Then, the tissues were decalcificated using 10% ethylenediaminetetracetic acid (EDTA) solution for 1 month. The decalcified tissues were processed using graded dehydration and were then embedded in paraffin. 5 μm thick histological sections were obtained using a microtome and were subsequently processed for staining.
For SOFG staining, the sections were stained with 0.1% Safranin-O and 0.02% Fast Green (Sigma-Aldrich, Oakville, ON, Canada) by following the manufacturer’s instructions.
Immunohistochemical staining was carried out using primary antibodies against TGF-β, p-Smad2/3, CD68, CD31, and VEGF (Abcam) at a 1:500 dilution of the appropriate secondary antibody. Protein expression was visualized using a DakoCytomation Envision staining kit. The mean density of the positive area was measured using Image-Pro Plus 6.0 (IPP) image analysis software. Three random slides were selected, and the images of five random fields were captured in each sample.
The tenotomy mice were sacrificed at the durations indicated. The hind limbs of the mice in each group were fixed overnight in 4% paraformaldehyde and were analyzed using a high-resolution Micro-CT scanner Skyscan 1176 (Bruker, Kontich, Belgium). The parameter was set at a resolution of 18 μm and 70 kV voltage. The region of interest (ROI) was set as the entire tibia to ensure that all heterotopic bones were included within the ROI. Three-dimensional images were reconstructed and obtained using NRecon software, and HO bone volumes were analyzed using CTAn software, as previously described .
Enzyme-Linked Immunosorbent Assay (ELISA)
The concentrations of TGF-βand VEGF in the serum were determined using the Quantikine ELISA Kit (R&D Systems, Minneapolis, MN), by following the manufacturer’s instructions.
Bone marrow-derived macrophages (BMDMs) were isolated from the bone marrow of 6 to 8 week old mice, as previously described . In brief, both the femur and tibia of the mice were excised and the soft tissue was completely removed. Then, the bone marrow cells were flushed from the marrow cavity using a 26G needle. The harvested cells were used in the experiments described below.
Cell sorting and flow cytometry analysis
After filtration using RBC lysis and washing with 0.1% BSA in PBS, we counted the cells and incubated equal numbers of cells for 45 min at 4°C with the primary antibody. For macrophage identification, we used F4/80, CD115, and CD11b antibodies. For M1 macrophage identification, we used F4/80 and CD86 antibodies. For M2 macrophage identification, F4/80 and CD206 antibodies (BioLegend, San Diego, CA, USA) were used. The stained cells were processed on a BD FACS Calibur flow cytometer and were analyzed using FlowJo software.
The sorted cells were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. To prepare the conditioned medium (CM), the cells were grown to 80% confluence in 5 cm dishes containing DMEM/10% FBS. The medium was discarded, and the cells were further cultured in serum-free DMEM for 24 h. Then, the medium was collected, centrifuged at 1,000 × g for 10 min, and filtered through 0.22-μm filters (Millipore, Billerica, MA).
RNA isolation and real-time PCR
MSC cells (purchased from the Chinese Academy of Sciences, Shanghai, China) were exposed to a macrophage conditioned medium for 48 h. Total RNA was isolated using TRIzol Reagent (Thermo Fisher Scientific), and cDNA was synthesized using the Mix‐X miRNA First-Strand Synthesis Kit (TaKaRa Bio). Subsequently, real-time PCR was performed using SYBR Green Premix Ex Taq (Takara) to quantify the target gene mRNAs. All procedures were performed by following the manufacturer's instructions. The following primer sequences were used: GAPDH, 5’-ATGGGGAAGGTGAAGGTCG-3’ (forward) and 5’-GGGGTCATTGATGGCAACAATA-3’ (reverse); Runx2: forward 5’-CCGCC TCAGTGATTTAGGGC -3’, reverse 5’- GGGTCTGTAATCTGACTCTG TCC -3’. OCN: forward 5’-CCTCAC ACTCCTCGCCCTATT-3’, reverse 5’-CCCTCCTGCTTGGACACAAA-3’. Sox9: forward 5’-CAGCCCCTTCAACCTTCCTC-3’, reverse 5’- TGATGGTCAGCGTAGTCGTATT-3’. Sp7: forward 5’- ATGGCGTCCTCTCTGCTTG-3’, reverse 5’- TGAAAGGTCAGCGTATGGCTT-3’. All of the reactions were performed in triplicate.
GraphPad Prism 8 was used for all statistical analyses of the data obtained. The data are presented as mean ± standard deviation (SD). Comparisons between groups were performed using Student’s t-test, while one-way ANOVA was used for comparisons between multiple groups. All experiments were performed in triplicate, and representative experiments are shown. Statistical significance was set at P<0.05.