Rat models

Male Sprague‒Dawley rats (6 weeks old, weighing 150–170 g) were used in the experiments. After being anesthetized with isoflurane, the rats were peritoneally injected with sodium pentobarbital (40 mg/kg). To construct the rat model of achillotenotomy, the rats were deeply anesthetized and then underwent bilateral midpoint achillotenotomy through a posterior approach under aseptic conditions. To construct the controlled cortical impact (CCI) rat model, the anesthetized rats were positioned in a stereotaxic frame. Then, we cut the skin of the head, performed a craniotomy using a drill (4 mm in diameter) over the right parietal-temporal cortex, exposed the dura, induced moderate brain injury by using an eCCI (electronic craniocerebral trauma instrument) and set the impact tip at a velocity of 3.0 m/s and 3.0 mm depth to hit the cerebral cortex. All incisions were closed with interrupted 4-0 silk sutures. Rats in the sham group underwent the same skin incision and craniotomy but not achillotenotomy or CCI. Rats in the HO group underwent achillotenotomy and craniotomy but not CCI. Rats in TBI group underwent achillotenotomy and CCI. All rats received analgesics to relieve pain after surgery and were allowed to move freely in their cages.

Micro-CT

After the rats were euthanized, the crus was fixed in 4% paraformaldehyde and analyzed on a Micro-CT scanner (Inveon Micro-CT system, Siemens AG, Germany; 80 keV and 500 mA; 10 μm isotropic). We calculated the length of the tendon as the distance between the heel bone (tendon-bone junction) and the muscle (tendon-muscle junction, the sudden enlargement of the tendon). Then, the tendon was selected by threshold segmentation of the value used for the soft tissue. Total tendon volume was calculated as the sum of the gray pixels in the posterior tibial region.68 Three-dimensional reconstruction and morphometry were performed to calculate bone microarchitectural parameters, including bone mineral density (BMD), bone volume (BV), bone trabecular thickness (Tb.Th) and trabecular number (Tb.N).

Histological evaluation

After fixation, the tissues were decalcified with 10% EDTA (6381-92-6; Solarbio, CN) solution with gentle shaking for 1 month. After decalcification, the samples were embedded in paraffin and cut into 6-µm-thick sections. After being dewaxed and hydrated, the sections were stained with hematoxylin-eosin (G1120, Solarbio, CN) and Safranin O and Fast Green (S8884, Sigma-Aldrich) and observed by microscopy (DM4, Leica, Germany). Bone trabecular thickness (Tb.Th) and trabecular number (Tb.N) were calculated by ImageJ software (National Institute of Health, Bethesda, USA). The sections were stained with alizarin red S (40 mmol/L, pH = 4.2; MilliporeSigma, Burlington, USA) for 20 min. The nuclei were counterstained with DAPI (Invitrogen). The relative fluorescence intensity was analyzed by ImageJ software (National Institute of Health, USA).

Immunohistochemistry

Before immunohistochemical and immunofluorescence staining, the paraffin-embedded sections were dewaxed, hydrated and subjected to heat-induced epitope retrieval. For cell samples, we used 4% paraformaldehyde to fix the cells for further staining. For immunohistochemical staining, we diluted the following primary antibodies in QuickBlock™ Primary Antibody Dilution Buffer (P0262; Beyotime, CN): NLRP3 (DF15549; Affinity, USA; 1:200 dilution) and CASPASE-1 p20 (AF4005; Affinity, USA; 1:200 dilution). Hydrogen peroxide (0.3%) was used to block endogenous peroxidase activity. After being blocked with goat serum (C0265, Beyotime, CN) and incubated with the primary antibody at 4 °C overnight, the sections were washed with PBS (P1020, Beyotime, CN), followed by incubation with the anti-rabbit IgG secondary antibody (Kit-5010, MXB, CN) for 1 h at 37 °C. Color was developed with DAB solution (DAB-4033, MXB, CN), and the sections were counterstained with hematoxylin. For immunofluorescence analysis, we diluted primary antibodies against alpha-SMA (BF9212, Affinity, USA; 1:300 dilution), N-GSDMD (DF13758, Affinity, USA; 1:300 dilution), L1CAM (bs-1996R, Bioss, CN; 1:300 dilution), CD63 (67605-1-Ig, Proteintech, USA; 1:300 dilution), F4/80 (DF2789, Affinity, USA; 1:300 dilution), CD4 (DF16080, Affinity, USA; 1:300 dilution), and Ly6G (orb322983, Biorbyt, CN; 1:300 dilution). For secondary reactions, we used species-matched Alexa Fluor 488 AffiniPure Donkey Anti-Mouse IgG (H+L) (PC-80014; PlantChemMed Biology Co., Ltd., ShangHai, China) and Alexa Fluor 594 AffiniPure Donkey Anti-Rabbit IgG (H+L) (PC-80009; PlantChemMed Biology Co.) antibodies at 37 °C in the dark. The sections were mounted with prolonged antifade mountant containing 4′,6-diamidino-2-phenylindole (DAPI) (S2110; Solarbio, CN). TUNEL staining (C1091, Beyotime, CN) and Hoechst/PI staining (CA1120, Beyotime, CN) were performed according to the protocols provided by the manufacturers. We observed the sections with a confocal microscope (FV1000, Olympus, Japan). Finally, the data were analyzed with ImageJ software (National Institute of Health, USA). For all the immunohistochemical and immunofluorescence staining experiments, there were three biological replicates.

Transmission electron microscopy (TEM)

Glutaraldehyde (2.5%) in PBS was used to fix the tendons (0.01 mol/L, pH = 7.4). The specimens were then fixed in 1% osmium tetroxide for 1 h and dehydrated in an ascending series of ethanol. The dehydrated specimens were immersed in propylene oxide and embedded in epoxy resin. After being dissected and prepared, 90-nm-thick sections were cut and stained with uranyl acetate and lead citrate. A JEM-123 transmission electron microscope (TEM, JEOL, Tokyo, Japan) was used to observe the sections at 110 kV.

Flow cytometry

After isolating EVs from the plasma of HO rats and NHO rats, 200 μL of PBS was used to resuspend each kind of EV. Then, these EVs were incubated with an anti-L1CAM antibody (diluted 1:150; ab272733; Abcam, USA) for 30 min. After the mixture was centrifuged at 100 000 r/min for 1 h, the EVs were resuspended in PBS. The EVs were incubated with secondary antibodies (diluted 1:300; SA00014-9; Proteintech, USA) after the addition of 2% serum albumin. After total EVs were washed with 2% bovine serum albumin in PBS, L1CAM+ BEVs were detected by a flow cytometer (CytoFLEX, Beckman Coulter, California, USA).

Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy

Glutaraldehyde (2.5%) in phosphate buffer (0.01 mol/L, pH = 7.4) was used to fix the tendons. The specimens were then dehydrated with an ascending series of ethanol and treated with hexamethyldisilane (Electron Microscopy Sciences, Hatfield, PA, USA). A field-emission scanning electron microscope (FE-SEM, S-4800, Hitachi, Tokyo, Japan) operating at 5 kV was used to observe the samples. The mineral elemental composition of the tendons was characterized by using energy-dispersive X-ray spectroscopy (Element EDS System, Ametek, Berwyn, PA, USA).

Plasma BEVs isolation

After the rats were anesthetized, peripheral blood was collected in tubes containing EDTA as an anticoagulant. We centrifuged the samples (1 200 × g, 10 min, 4 °C) immediately to separate the plasma from the peripheral blood. The plasma was carefully collected and transferred to a new tube without disturbing the intermediate buffy coat layer. After the samples were incubated for 10 min at room temperature, the plasma was centrifuged (3 000 × g, 20 min, 4 °C), and the supernatant was collected after an additional centrifugation step (11 000 × g, 30 min, 4 °C). The supernatant was subsequently transferred to a new tube and centrifuged at 18 000 × g for another 30 min at 4 °C. Finally, we collected the supernatant in a new tube and centrifuged it at 100 000 × g for 2 h at 4 °C to precipitate total EVs in the plasma. Total plasma EVs were resuspended in 200 μL of PBS with inhibitor cocktails, and 100 µL of L1CAM biotinylated antibody (mouse anti-rat) (bs-1996R-Bio, Bioss, Bioss, CN) in 50 µL of 3% BSA (bovine serum albumin) was added and mixed. The mixture was incubated for 1 h at 4 °C. After that, we added 10 μL of streptavidin-agarose UltraLink Resin (20512ES08, Yeasen, CN) and 40 µL of 3% BSA, followed by incubation for 30 min at 4 °C. After centrifugation (400 × g, 15 min, 4 °C), each pellet of agarose settled to the bottom of the tube, and BEVs were adsorbed on the surface of the pellets. The supernatant contained BEVs-depleted EVs. Each agarose pellet was resuspended in 100 µL of 0.05 mol/L glycine-HCl (pH 1.0) to release the BEVs from the surface of the pellets. Then, the mixture was centrifuged at 4 000 × g for 10 min at 4 °C. The final supernatant contained BEVs. Finally, we added cocktails of protease and phosphatase inhibitors (78440; Thermo Fisher Scientific, USA) at the recommended concentrations to each tube, and the suspensions were stored at –80 °C until further use.

Brain BEVs isolation

We dissected and separated rat brains after they were sacrificed. Five hundred milligrams of brain tissue was cut and added to culture medium supplemented with lyophilized papain (P4762; Sigma, USA; 20 U/mL). The brain tissue was crushed and incubated at 35 °C for half an hour. Then, the mixture was centrifuged, and the BEVs were separated from the obtained supernatant according to the aforementioned separation steps.

Characterization of BEVs

A total of 10 μL of each BEV suspension was placed on a copper mesh and incubated for 20 min at room temperature. Then, the copper mesh was dried by placing it sideways on filter paper, after which it was immersed in saturated uranyl acetate for 3 min and immersed in pure water for 5 min. The samples were subsequently dried and observed by transmission electron microscopy (TEM; JEOL, Tokyo, Japan). Prior to analyzing particle size distribution, the samples were diluted in PBS to obtain a range of 60%-70% transmittance and then analyzed by NanoSight NTA Software (Litesizer500, Anton Paar, GER). The concentration of BEVs was then determined by a BCA protein assay (PC0020, Solarbio, CN) according to the manufacturer’s instructions. We performed Western blot analysis according to standard protocols. The EVs were subjected to lysis buffer (P0013F; Beyotime, CN), separated by SDS‒polyacrylamide gel electrophoresis (#1610183; Bio-Rad Laboratories, UK) with running buffer (B0002; Thermo Fisher Scientific, USA), and transferred to polyvinylidene fluoride (PVDF) membranes (IEVH85R; Merck, USA) in an ice bath. The membrane was blocked with 5% BSA for 2 h and then incubated overnight with primary antibodies at 4 °C. Primary antibodies against TSG101 (Ab125011, Abcam, USA, 1:1 000 dilution), CD9 (Ab92726, Abcam, USA, 1:1 000 dilution), CD63 (Sc-5275, Santa Cruz, Thermo Fisher Scientific, USA, 1:1 000 dilution), L1CAM (BS-1996R, Bioss, CN, 1:1 000 dilution) and calnexin (10427-2-AP, Proteintech, USA, 1:1 000 dilution) were used. The secondary antibodies used for western blotting were anti-rabbit IgG (cat# 7074; Cell Signaling Technology, USA; dilution, 1:2 000 dilution) and anti-mouse IgG (cat# A9044; Sigma-Aldrich, USA; dilution, 1:2 000 dilution). Finally, the membrane was incubated with an enhanced chemiluminescence HRP substrate (WBKLS0100, Millipore, USA) for 3 min and observed with an imaging system (Azure 600, Azure Biosystems, CN).

Cell culture

L929 cells (a mouse fibroblast line) purchased from Procell were seeded in DMEM (PM150421, Pricella, CN) supplemented with 10% fetal bovine serum (PC-00001; PlantChemMed Biology Co.) and 1% penicillin streptomycin solution (100X) (PC-86115; PlantChemMed Biology Co.) in 5% CO2 at 37 °C.

Exosome labeling and cellular uptake assay

BEVs were labeled with the red fluorescent agent PKH26 (PKH26GL-1KT; Sigma-Aldrich, USA) and centrifuged at 100 000 × g for 70 min to remove unbound dye. Then, the BEVs were suspended in PBS. We cocultured PKH26-labeled BEVs (500 μg/dish) with L929 cells. At the indicated times, we stained the cytoskeleton with phalloidine (49409-10NMOL; Sigma-Aldrich, USA; 5 μg/mL) and observed the cells by confocal microscopy (FV1000; Olympus, Tokyo, Japan). After being cultured for 3 days, the cells were stained with N-GSDMD and Hoechst 33342/PI according to the instructions. To further assess the effect of BEVs on pyroptosis and calcification, we added EVs (500 μg per dish), BEVs-depleted EVs (500 μg per dish) and BEVs (500 μg per dish) combined with a Caspase-1 inhibitor (Ac-YVAD-cmk, HY-16990, MedChemExpress, USA; 1 mg/mL dilution) to the cell culture system for 3 or 7 days. We assessed pyroptosis in the coculture system by performing green immunofluorescence staining for N-GSDMD after 3 days. To assess the effect of the BEVs on calcification, the system was maintained for 7 days. After being fixed with 4% paraformaldehyde, the cells were stained with Alizarin Red S (50 mmol/L, pH = 7.2). We removed the excess dye by washing the samples with water for 30 min. A Zeiss microscope (Thorn-wood, NY, USA) was used to image the plates. The stained areas were measured with ImageJ software.

Western blot analysis of fibroblast pyroptosis in response to BEVs

After fibroblasts were cocultured with BEVs, we lysed the cells in RIPA buffer (P0013B, Beyotime, CN). We separated the proteins by SDS‒polyacrylamide gel electrophoresis (#1610183; Bio-Rad Laboratories, UK) and blocked the samples with BSA. After the proteins were blotted onto polyvinylidene fluoride (PVDF) membranes, the membranes were incubated with N-GSDMD (#DF12275; Affinity, USA; 1:300 dilution), HMGB1 (#AF7020; Affinity, USA; 1:300 dilution) and β-actin (#4970S; Cell Signaling Technology, USA; 1:1 000 dilution). The secondary antibodies used for western blotting were anti-rabbit IgG (cat# 7074; Cell Signaling Technology, USA; dilution 1:2 000) and anti-mouse IgG (cat# A9044; Sigma-Aldrich, USA; dilution 1:2 000 dilution). An enhanced chemiluminescence HRP substrate (WBKLS0100, Millipore, USA) was used to visualize and analyze the membrane.

Mass spectrometry analysis of BEVs

BEVs were isolated and treated with RIPA lysis buffer. After trypsin digestion, the mixture was injected into a mass spectrometer (Orbitrap Eclipse Tribrid Mass Spectrometer, Thermo Fisher Scientific, USA). After calibrating the system with the standard compounds, the mass spectrometer was operated in the data-dependent mode. In this mode, the mass spectrometer cycled between full MS scans with m/z 100–1 800. Only proteins with high protein FDR confidence were considered for further analysis.

Detection of phosphorus and calcium concentrations in the supernatant of fibroblasts

Dynamic monitoring of phosphorus and calcium concentrations in the supernatant of fibroblasts was performed at different times after being cocultured with BEVs filtered through a 0.22-mm filter. Detection was performed by inductively coupled plasma‒optical emission spectrometry (ICP–OES, Agilent 5900, Agilent, USA). The analysis was repeated three times under the same conditions.

Fluorescence imaging analysis of BEV distribution

To track BEVs in vivo, we injected PKH26-labeled BEVs into rats that had undergone achillotenotomy. One hundred microliters (intravenously, 6 µg/µL) of BEVs were injected into each rat. Then, the rats were killed 3 days after the injection, and the tendons were harvested. The specimens were fixed in 4% paraformaldehyde for 24 h and 30% sucrose for 3 days. After that, optimal cutting temperature compound (Leica, Wetzlar, Germany) was used to embed the specimens. Then, the specimens were stored at –80 °C. Five-micron-thick sections were cut from the specimens and stored for further experiments. The BEVs in the Achilles tendon were imaged by confocal microscopy (FV1000, Olympus, Tokyo, Japan).

GW4869 administration

To reduce the concentration of BEVs in plasma, we used GW4869 (D1692; Sigma-Aldrich, USA), which is a compound that can inhibit the secretion of BEVs from the central nervous system. GW4869 was diluted to a final concentration of 0.25 mg/mL in 0.9% normal saline and then intravenously injected into rats (250 µg/100 g body weight, twice per week).

Ac-YVAD-cmk Injections

To ameliorate pyroptosis in fibroblasts in the Achilles tendon after TBI, Ac-YVAD-cmk (178603-78-6; Sigma-Aldrich, USA) was dissolved in normal saline and injected into rats at a dose of 1 μg/rat three times per week. An equal volume of normal saline was injected into the mice in the sham group.

Indomethacin administration

To explore the inhibitory effects of nonsteroidal anti-inflammatory drugs on NHO, indomethacin, which is a common clinical nonsteroidal anti-inflammatory drug, was administered to rats in the NHO group. Indomethacin (CAS:53-86-1; Solarbio, Beijing, China) was diluted to a concentration of 3 mg/kg, after which the rats in the NHO group were orally administered indomethacin by syringe feeding after surgery.

Parallel experiment

A traumatic brain injury rat model was constructed in which the Achilles tendons of the left leg was sham-treated (sham) and those of the right leg were injured (injured). All rats received analgesics to relieve pain after surgery and were sacrificed 3 days postinjury for immunofluorescence staining and 3 weeks postinjury for micro-CT and H&E staining.

Statistical analysis

Analyses were performed using GraphPad Prism 8.0 (GraphPad Software, USA). All the data are presented as the means ± standard deviations. The Shapiro‒Wilk test and modified Leven test were used to test the normality and homoscedasticity assumptions of the corresponding datasets, respectively. Student’s t-test and one-factor or two-factor analysis of variance followed by Holm–Šidák multiple comparison tests were used to evaluate the differences among groups. For all tests, statistical significance was set at α = 0.05.