Animals
Male Sprague Dawley rats weighing 180–230 g at 8–10 weeks of age were provided by the Experiment Center of Harbin Medical University (Harbin, Heilongjiang, China). All animals were treated according to the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the protocol was approved by the corresponding ethics committee (no. Ky2018-135).
Isolation and identification of TSCs and ADSCs
The isolation methods of ADSCs and TSCs were as performed in previous studies [18,19]. In brief, TSCs were isolated from rat tendon and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) (Biological Industries, Kibbutz Beit-Haemek, Israel) and 1% penicillin-streptomycin (Beyotime, Haimen, China). The multilineage differentiation potential of TSCs, as well as the identification of surface markers (CD90- and CD105-positive, and CD106- and CD11b-negative), were demonstrated in our previous study [19]. ADSCs were isolated from subcutaneous fat of rats and cultured in DMEM/F12 (Invitrogen) containing 10% FBS and 1% penicillin-streptomycin. Flow cytometry was used to identify surface markers. The adipogenic, osteogenic and chondrogenic differentiation of ADSCs was induced in differentiation medium (Cyagen, Santa Clara, CA, USA) to identify their differentiation potential.
Isolation and identification of ADSC-Exos
At 80% confluence, the culture medium of the ADSCs was changed to exosome-depleted medium (DMEM/F12 containing 10% exosome-depleted FBS (Biological Industries) and 1% penicillin-streptomycin) and incubated for 24 h. Then, the culture medium was collected without ADSCs and centrifuged at 300 × g for 10 min, 3000 × g for 10 min, 10,000 × g for 30 min, and 100,000 × g for 2 h to isolate exosomes. Exosomes attached to the bottom of the centrifuge tube were diluted with phosphate-buffered saline. Nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blotting were used to identify and evaluate the collected exosomes.
Cellular internalization of ADSC-Exos
ADSC-Exos were incubated with 1 µM PKH26 (Sigma-Aldrich, St. Louis, MO, USA) in Diluent C (Sigma-Aldrich) for 5 min and excess dye was removed by ultracentrifugation. The labeled exosomes were subsequently added to serum-free medium of TSC cultures and incubated overnight. The nuclei were labeled with Hoechst 33342 (UE, China) and photos were taken with an inverted fluorescence microscope (Leica, Wetzlar, Germany).
Treatment of TSCs with ADSC-Exos
For determining the effect of ADSC-Exo treatment, 1 × 106 TSCs were seeded into six-well culture plates for 24 h and divided randomly into four groups. ADSC-Exos were added to exosome-free medium at 0, 25, 50, or 100 μg/mL and used to replace the TSC culture medium. For additional analyses, 10 nM of the TGF-β/SMAD2/3 inhibitor, SB431542, or 10 nM of the BMP/SMAD1/5/9 inhibitor, dorsomorphin, (MedChemExpress, Monmouth Junction, NJ, USA) were added to the TSCs 30 min prior to addition of the ADSC-Exos. TSCs treated with various concentrations of ADSC-Exos, with or without inhibitors, were incubated for 30 min or 24 h, then collected for analyses.
EdU assay
For the cell proliferation analysis, TSCs were incubated with 50 μM 5-ethynyl-2′-deoxyuridine (EdU) from an EdU Assay Kit (UE) for 4 h. The TSCs were then fixed with 4% paraformaldehyde and stained using the same EdU assay kit. Nuclei were labeled with Hoechst 33342 and photos were taken with an inverted fluorescence microscope.
Scratch assay
TSCs at 2 × 105 cells/well were inoculated into a 6-well plate for overnight culture. A straight line wound was made in the cultured cells using a sterile 200-μL pipette tip. Serum-free medium with ADSC-Exos was then added into each well. Images were obtained at 0 and 24 h after ADSC-Exo treatment using an inverted microscope with an Axiocam 506 camera and ZEN 2011 software (Zeiss, Oberkochen, Germany).
Transwell assay
TSCs at 1 × 105 cell/well were inoculated into the Transwell upper chamber, and ADSC-Exos were added into the lower compartment. After culturing for 24 h, the TSCs were fixed with absolute ethanol, then stained with crystal violet. Images were obtained under a light microscope.
Western blot analyses
Experimental protocols and surgical procedures
A total of 63 Sprague-Dawley rats were divided into three groups of 21: (1) Control: animals that underwent surgery for partial resection of the patellar tendon; (2) Gelatin methacryloyl (GelMA): animals that underwent surgery for patellar tendon partial resection and were inoculated with 30 μL GelMA (EFL-GM-60, 10% w/v) over the tendon defect; and (3) ADSC-Exos: animals for which the injured patellar tendon was treated with 30 μL GelMA containing 200 μg of ADSC-Exos. The exosome content was determined according to previous studies [19]. Rats were anesthetized with 0.3% sodium pentobarbital (30 mg/kg). The right patellar tendon was surgically exposed and the central 1/3 of the tendon tissue removed as in previous studies [20]. GelMA was then inoculated into the lesion and cross-linked into a gel state by ultraviolet light. The skin incision was closed using 4-0 sutures. The modeling process is shown in Figure 1. Animals were sacrificed on days 7, 14, or 28 post-surgery and the tendons were collected.
Histopathological and immunohistochemical analyses
Paraffin-embedded tendon tissues were sectioned at a thickness of 4 μm. The tissues were then stained with H&E for histopathological analysis. The stained patellar tendons were evaluated using light microscopy.
For immunohistochemical analyses, paraffin sections of tendon tissues were incubated with Immuno-Block reagent for 30 min after being deparaffinized and rehydrated. The sections were then incubated with the rabbit primary antibodies (Abcam,): anti-CD146 (monoclonal; 1:250), anti-TNMD (polyclonal; 1:100), anti-collagen I (polyclonal; 1:100), anti-SCXA (polyclonal; 1:100), anti-ALP (polyclonal; 1:200), and anti-Runx2 (monoclonal; 1:1000). Horseradish peroxidase–conjugated goat anti-rabbit IgG (1:500; Jackson ImmunoResearch, Ely, UK) was used as the secondary antibody. After counterstaining with hematoxylin, the sections were dehydrated and fixed.
For immunofluorescence analyses, sections of tendon tissues were incubated with the rabbit primary antibodies (Abcam): anti-CCR7 (monoclonal; 1:200), anti-CD163 (monoclonal; 1:100), anti–IL-6 (monoclonal; 1:100), and anti-IL-10 (monoclonal; 1:100). The sections were then incubated with secondary antibodies (1:200, Proteintech, Rosemount, IL, USA) for 1 h. Nuclei were labeled with 4',6-diamidino-2-phenylindole and photos were taken with a DM4 B microscope (Leica). Three fields per section were selected randomly for statistical analysis.
Biomechanical testing
Two bony ends of a healing tendon were fixed on a universal material testing machine (Zwick, Roell, Germany). The tissues were investigated using a standard failure test with a testing speed of 5 mm/min. Failure load (N) and stiffness (N/mm) were obtained by the software of the testing machine. Young's modulus (N × 103/mm2) was calculated after measuring the cross-sectional area (mm2) of the tendon with a vernier caliper.
Statistical analyses
All values are expressed as means ± standard deviation. Quantitative data for each group were analyzed by a one-way analysis of variance followed by the Tukey–Kramer test. P < 0.05 was considered statistically significant.