All the experimental procedures were performed with the approval of the Experimental Research Institute of Nanjing University of Chinese Medicine and followed the guidelines of the Institutional Animal Care and Use Committee.
Animal Experiment In Vivo
A total of 59 rats (four-week-old Sprague-Dawley (SD) rat, male, 70-100 g) were used for in vivo experiments. Five rats were used in BMSCs extraction and 54 rats were used in rotator cuff reconstruction model building. Rotator cuff reconstruction model included two groups: phosphate buffer saline (PBS) group and BMSC-Exos group, and each group included 27 rats. Eighteen rats were used in tissue section analysis (PBS group, 9; BMSC-Exos group, 9). Eighteen rats were used in angiogenesis analysis (PBS group, 9; BMSC-Exos group, 9). Eighteen rats were used in biomechanical test analysis (PBS group, 9; BMSC-Exos group, 9). The rats were sacrificed after tissue obtained.
Cells and cell culture
The U937 cells and HUVECs were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). The U937 cells were cultured in 1640 medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, HyClone, Utah, USA), 1% double-antibiotics (streptomycin + penicillin; Gibco). HUVECs (ATCC, Rockville, MD, USA) were cultured with Dulbecco’s Modified Eagle Medium (DMEM; Gibco) containing 10% FBS and 1% double-antibiotics (streptomycin + penicillin; Gibco). Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air.
Five rats were selected, and the BMSCs inside their femurs and tibiae were obtained after anesthesia. Isolated BMSCs were incubated with standard media comprising DMEM (Gibco) supplemented with 10% FBS and 1% double-antibiotics (streptomycin + penicillin; Gibco). After 3–5 days of incubation (at 80% confluence), cells were re-plated in 10 cm Petri dishes and maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air (Fig. S1, Fig. S2).
The model of U937-M0/M1 macrophages
U937 cells were induced into macrophages according to reference . U937 cells were collected by centrifugation and inoculated in a 6-well plate at 8 × 105 cells/well. U937-M0 (inactive type) was obtained using phorbol myristate acetate (PMA, 5 ng/mL, Biyuntian Company). PMA was added in the dark and the cells cultured for 48 h at 37 °C with 5% CO2. The medium containing PMA was then replaced, and 20 ng/mL interferon-gamma (IFN-γ , PeproTech, Rocky Hill, NJ, USA) and 100 ng/mL lipopolysaccharide (LPS, Sigma, St. Louis, MO, USA) were added for 24 h to induce and achieve U937-M1 cells (classic activated type).
Extraction of exosomes
After BMSCs had grown to about 80% fusion, the culture medium was replaced by exosome-depleted FBS-containing medium (EXO-FBS-250A-1; System Biosciences, Mountain View, CA, USA). The cells were then cultured a further 48 h. The medium was collected and centrifuged at 4 °C at 300 × g for 10 min and at 2000 × g for 10 min. After centrifugation, 0.22 µm SteritopTM (Millipore, Billerica, MA, USA) was used to remove the cells and cell debris. After that, the supernatant was transferred to the upper layer of an Amicon ultra-15 spinning Filter Unit (Millipore), and the supernatant liquid was centrifuged at 4000 × g at 4 °C to about 200 µL. The supernatant was cleaned with PBS twice and then ultra-filtered to 200 µL. The liquid was placed on a 30% sucrose/D2O buffer and placed in a sterile Ultra-ClearTM tube (Beckman Coulter, Brea, CA, USA) and centrifuged at 100,000 × g for 60 min at 4 °C (Sorvall, Avanti J-26XP, fixed-angle rotor; Beckman Coulter). The fraction containing the BMSC-Exos was recovered using an 18-G needle, diluted in PBS, and centrifuged at 4000 × g at 4 °C in centrifugal filter units until the final volume reached 200 µL. BMSC-Exos were stored at −80 °C. The protein content of BMSC-Exos was measured by the bicinchoninic acid assay (BCA; Thermo Fisher, Waltham, MA, USA). A microplate reader (ELx800, BioTek, USA) was used to measure the absorbance at a wavelength of 562 nm.
Immunofluorescence and immunohistochemistry
For the immunofluorescence assays, briefly, the paraffin sections were de-waxed, dehydrated, and incubated overnight at 4 °C with anti-CD31 and anti-endomucin (diluted 1:100; Abcam, Cambridge, UK). The secondary antibody (diluted 1:100; Jackson, West Grove, PA, USA) was applied, and the cells were incubated for 1 h in the dark. Finally, the nuclei were counterstained with DAPI for 15 min. The stained cells were photographed using a fluorescence microscope.
For the cell immunofluorescence (Carl Zeiss Microscopy GmbH, Jena, Germany) assays, HUVECs were cultured in a 24-well plate. The cells were fixed with 4% paraformaldehyde and incubated with 0.5% Triton X-100 (Sigma) and then blocked with goat serum for 1 h. The cells were then incubated overnight at 4 °C with the primary antibody (anti-YAP1, diluted 1:100; Abcam). The secondary antibody (diluted 1:200; Jackson, West Grove, PA, USA) was applied, and the cells were incubated for 1 h in the dark. Finally, the nuclei were counterstained with DAPI for 15 min. The stained cells were photographed using a fluorescence microscope.
For the immunohistochemistry assays, the paraffin sections were de-waxed, dehydrated, and incubated overnight at 4 °C with anti- type I collagen (Col I), anti- type II collagen (Col II), and anti-CD86 (diluted 1:100; Abcam). After the primary antibody was removed, the secondary antibody (diluted 1:100, Thermo Fischer) was added, and the sections were incubated for 1 h at room temperature. The stained cells were developed with diaminobenzidine and counterstained with hematoxylin.
Safranin and Fast Green staining
After the shoulder joint was fixed and decalcified, the shoulder was embedded in paraffin, sliced at 5 μm in the coronal position, and hydrated after dewaxing. For Fast Green staining, the slices were placed in the Fast Green dye liquid for 5–10 min. Excess dye was washed with water until the cartilage became colorless. The slices were then slightly soaked in the differentiation fluid and then washed with tap water. For staining with Safranin, the slices were placed in Safranin dye liquid for 15–30 s and then quickly dehydrated with three cylinders of anhydrous ethanol. For the transparent seal, sections were cleared with xylene for 5 min and then sealed with a neutral gum seal. The cartilage stained red or orange with a green background.
Western blot analysis
Cells were placed on ice immediately following treatment and washed with ice-cold Hank’s balanced salt solution. Nuclear proteins were prepared using the CellLytic NuCLEAR extraction kit (Sigma-Aldrich). All the wash buffers and final resuspension buffer included a 1× protease inhibitor cocktail (Pierce, Rockford, IL, USA), NaF (5 mM), and Na3VO4 (200 mM). The protein concentration of the lysate was measured using the BCA protein assay kit (Thermo Fischer). Nuclear or total cell proteins were resolved on 8% to 12% SDS-PAGE and transferred by electroblotting to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked in 5% bovine serum albumin reagent (Beyotime, Jiangsu, China) and incubated overnight at 4 °C with primary antibody dilution buffer (the dilution followed the specification; Abcam) and then incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:5000) for 2 h. Afterward, the membranes were developed using the enhanced chemiluminescence substrate LumiGLO (Millipore, Bedford, MA, USA) and exposed to X-ray film. The bands were analyzed with Gel-Pro Analyzer 4.0 (Bio-Rad, Hercules, CA, USA).
RNA isolation and quantitative real-time polymerase chain reaction
Primers for quantitative real-time polymerase chain reaction (qRT-PCR) were designed using Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). Total RNA from cells was isolated with Trizol® Reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized by a first-strand cDNA synthesis kit (Thermo Fischer) according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using iQ5 optical system software (Bio-Rad) with FastStart Universal SYBR Green Master (Recho, Basel, Switzerland) for mRNA quantitation of all referred genes. Relative expression was calculated using the 2−ΔΔCt method normalized to GAPDH (endogenous loading control). The following primers were used: GAPDH, sense (5¢-GGAGCGAGATCCCTCCAAAAT-3¢), anti-sense (5¢-GGCTGTTGTCATACTTCTCATGG-3¢); interleukin (IL)-1β, sense (5¢-ATGATGGCTTATTACAGTGGCAA-3¢), anti-sense (5¢-GTCGGAGATTCGTAGCTGGA-3¢); TNF-α, sense (5¢-CCTCTCTCTAATCAGCCCTCTG-3¢), anti-sense (5¢-GAGGACCTGGGAGTAGATGAG-3¢); IL-6, sense (5¢-ACTCACCTCTTCAGAACGAATTG-3¢), anti-sense (5¢-CCATCTTTGGAAGGTTCAGGTTG-3¢); IL-8, sense (5¢-ACTGAGAGTGATTGAGAGTGGAC-3¢), anti-sense (5¢-AACCCTCTGCACCCAGTTTTC-3¢); nitric oxide synthase 2 (NOS 2), sense (5¢-AGGGACAAGCCTACCCCTC-3¢), anti-sense (5¢-CTCATCTCCCGTCAGTTGGT-3¢).
U937-M0 cells were incubated with IFN-γ + LPS and BMSC-Exos for 24 h. Cells (1 × 106/tube) were washed twice with PBS and then resuspended in 100 μL PBS before 1 μL FITC-labeled CD86 antibody (BD Biosciences, San Jose, CA, USA) was added. The cells were incubated for 30 min at 4 °C in the dark. Any unbound antibody was washed away by PBS before the cells were resuspended in 300 μL PBS. Flow cytometry (FACSCalibur, BD Biosciences, USA) was used to detect the expression of CD86 on the cell surface.
Enzyme-linked immunosorbent assay
TNF-α, IL-1β, IL-6, and IL-8 were determined by enzyme-linked immunosorbent assay (ELISA) using a commercially available ELISA set (R&D Systems Inc., Minneapolis, MN, USA). ELISA was performed according to the manufacturer’s instructions. All samples and standards were measured in duplicate.
Cell viability assay
Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) was used to evaluate the effects of BMSC-Exos on HUVEC proliferation. BMSC-Exos (100 μg/mL) or the same volume of PBS was added into the culture medium, and the cells were incubated for 0, 24, 48, 72, and 96 h. After incubation, PBS was used to wash cells for 3 times and CCK-8 solution (10 µL; 1:10 diluted) was added into the fresh culture medium on 37 ° C for 2 hours. Finally, the optical absorbance for each sample was measured at 450 nm using an absorbance microplate reader (ELx800, BioTek).
Cell proliferation was also measured using the 5-ethynyl-29-deoxyuridine (EdU) assay kit (RiboBio, Guangzhou, China) according to the manufacturer’s instructions. Briefly, cells were seeded into 24-well plates at a density of 2.0 × 104 cells/well and cultured for 24 h before the administration of EDU (50 mM). After, Apollo and DNA stains were added. Finally, proliferation images were acquired and analyzed by fluorescence microscopy (Carl Zeiss Microscopy GmbH, Jena, Germany).
Tube formation assay
To evaluate angiogenesis induced by BMSC-Exos, HUVECs were inoculated on Matrigel-coated 96-well plates (Matrigel, BD biosciences, CA, USA) at 2 × 104 cells/well. Matrigel was dissolved overnight at 4 °C in advance and placed in each hole upon ice. BMSC-Exos (100 μg/mL) or PBS was added to each hole, and then the cells were cultured for 30 min at 37 °C. The polygonal structures of HUVECs were observed with an optical microscope 6 h after the cells had been placed on the Matrigel. The tube-forming ability was evaluated by measuring the total length and number of tube branches.
Transwell migration was used to measure the effect of BMSC-Exos on HUVEC migration. HUVECs (5 × 104 cells/hole) were added into the upper chamber of the transwell chamber (Corning, NY, USA; Pore size: 8 µm). BMSC-Exos (100 μg/mL) or PBS was added in the lower chamber for a 24 h culture. The cells that did not migrate above the filter membrane were gently wiping off using a cotton swab. The cells that migrated to the bottom of the filter membrane were fixed and stained with hematoxylin staining. Five fields were randomly selected under the microscope to calculate the number of migratory cells.
The scratch wound assay was used to evaluate the effect of BMSC-Exos on HUVEC migration. HUVECs were inoculated in a 24-well plate (2 × 105 cells/hole), and after the cells had grown to 90% confluence, a sterile 200 µL pipette tip was used to scratch the cells in the hole vertically. The scratch in each hole was required to be straight, and the scratch in each hole was the same. The removed cells were washed 2 times with PBS, and then 0 or 100 µg/mL BMSC-Exos were added. The cell migration images were recorded 0, 12, and 24 h after the scratch.
Nitric oxide assay
U937 or M1 macrophages (2 × 105 cells/mL) were placed in the cell culture plate. To observe the nitric oxide (NO) release during the induction of U937-M1 macrophages, BMSC-Exos (100 μg /mL) or equivalent PBS was added to the culture medium 1 h before the addition of IFN-γ and LPS. NO release was analyzed in the supernatant every 24 h for four consecutive days. The Griess reagent (Promega, USA) mixed with an equivalent volume of culture supernatant was incubated at room temperature for 10 min. The absorbance was determined by spectrophotometry at 540 nm. The sodium nitrite (NaNO2) standard curve was used to determine the concentration of NO.
RCT and reconstruction model building
After successful anesthesia with an intraperitoneal injection of 10% chloral hydrate (0.3 mL/100 g), incised the shoulder joint laterally and pulled the trapezius muscle away. The insertion to the humeral head of the supraspinatus was exposed. The supraspinatus insertion was resected. Approximately 2 mm of the distal tendon of the supraspinatus was cut off. Then, the skin was sutured. An RCT in rats is considered to simulate the chronic RCT that frequently occurs in humans . Four weeks later, a 3-0 nonabsorbable Propathene line was used to fix the supraspinatus tendon to the humeral head through bone tunnels. The subjects were divided into two groups. In the PBS group, PBS was injected into the tail vein, and in the BMSC-Exos group, BMSC-Exos (200 µg of total protein of BMSC-Exos precipitated in 200 µL of PBS) was injected into the tail immediately after suturing.
Micro-CT analysis of angiogenesis at the reconstruction sites
Micro-CT system (Siemens Inveon PET/CT, Berlin, German) was used to assess vascularity. Vascular networks around the shoulder junction were examined using micro-CT analyses combined with perfusion of a contrast agent. Briefly, blood vessels were first rinsed with normal saline containing heparin and 4% paraformaldehyde (PFA). Then, using MICROFIL® injection compound (Flow Tech, Inc., Carver, Massachusetts) contrast media, a radio-opaque silicone rubber compound containing lead chromate was perfused via the heart. After perfusion, the reconstruction shoulder was removed and scanned using the micro-CT system. The samples were subsequently decalcified for 30 days using a 10% EDTA solution. After complete decalcification, the samples were scanned again to visualize only the vascularization within the callus tissue. Three-D reconstructions were made using Inveon research workplace (2.0, Berlin, German).
Biomechanical tests were performed 4 and 8 weeks after the surgical reconstruction. Mutilation was performed at the lower end of the humerus. The supraspinatus and its insertion were carefully retained; the other muscles, except for the supraspinatus, were removed. The supraspinatus-humerus structure was obtained. The specimen was immediately placed in 4% paraformaldehyde solution, and the shoulder joint tissue was firmly fixed on an INSTRON biomechanical tester (Instron, Boston, MA, USA). The instrument was loaded, and a displacement velocity of 5 mm/min was applied to test the maximum tensile load of the supraspinatus tissue. The loading load when the supraspinatus tendon was broken was observed and recorded in detail as the maximum breaking load (N). The linear slope of the load-displacement curve was taken as the stiffness.
SPSS 19.0 statistical software (SPSS, Inc., Chicago, IL, USA) was used for all statistical analyses. The measurements are presented as the mean ± standard deviation. The data were analyzed using a one-way analysis of variance followed by Bonferroni’s post-hoc test for multiple comparisons. p < 0.05 was considered to indicate a statistically significant difference.