1. Ethics statement
The study was approved by the Ethical Committee of Chongqing Medical University(027), written informed consent was obtained from all the participants.All collected tissue samples were processed in accordance with the Declaration of Helsinki.
2. Manufacturing of the exosome-loaded GNPs (GNP-Exos) hydrogel
We prepared the exosome-loading hydrogel according to the methods in our previous study. Briefly, 1 ml of PBS containing 0.8 mg of ADSC-Exos was immediately loaded into a Luer-lock medical syringe and subsequently mixed with the contents of another Luer-lock syringe containing 0.1 g of freeze-dried GNPs powder by repetitive extrusion of the mixtures to obtain the GNP-Exos hydrogel.
3.Characterization: zeta potential, surface topography, and exosome distribution
In order to evaluate the electrostatic assembly of GNPs and exosomes, a Zetasizer Nano ZSP (Malvern Panalytical, China) was used to evaluate the zeta potential values of the GNPs, ADSC-Exos and GNP-Exos. The surface topographies of the GNP-Exos samples were investigated with field-emission scanning electron microscopy (FE-SEM), while laser confocal microscopy (LSCM, TCS.SP8, Leica, Germany) was used to detect the exosomes distribution.
We used a Discovery Hybrid Rheometer (Anton Paar, Austria) to test the rheologic properties of the GNP-Exos. The operating gap distance was set to 1000 μm. The viscoelastic properties of the GNP-Exos were characterized by oscillatory frequency sweep (0.1 to 10 rad/s at a constant strain of 0.5%) and strain sweep (0.1 to 100% strain at a constant frequency of 1 Hz). The self-healing capacity of the GNP-Exos was assessed according to the recovery of G’ and G’’ of the gel network.
5. Release behaviour of ADSC-Exos
After fluorescent labelling of the exosomes, 1 ml of PBS containing 0.8 mg of labelled ADSC-Exos was mixed with 0.1 g of GNPs using a Luer-lock syringe. Then the hydrogel was placed in 5 ml of PBS at 4 °C. All the samples were protected from light. Each day, 1 ml of PBS containing released ADSC-Exos was collected, after which 1 ml of fresh PBS was added into the release system. The fluorescence intensity (585/601 nm) of the collected samples was measured using an EnSpire Multimode Plate Reader (PerkinElmer, USA) and the actual released volume was calculated based on the standard curve for ADSC-Exos.
6. Animal and surgical protocols
The animal experiments were performed following the ARRIVE guidelines, and all Sprague-Dawley (SD) rats were purchased from the Experimental Animal Center of Chongqing Medical University. The animals were provided unlimited access to food and water and were housed under a 12 h light/dark cycle at 23±2 °C. In our experiment, we used injectable GNPs hydrogel-loaded exosomes into skull defects in rats. The surgery was carried out under sterilized conditions, and 8×104 IU/day penicillin was administered to each animal postoperatively. 36 SD rats were divided into three groups: the control, GNPs, and GNP-Exos groups. All rats received anaesthesia by sodium pentobarbital injection (30 mg/kg), and a linear incision was made on the median area of the calvaria in the anterior-posterior direction. The periosteum was detached in opposite directions, followed by the creation of 5-mm-diameter bone defects on each side of the parietal region using a trepan. Afterwards, pure gelatine-based hydrogel was injected and shaped in the skull defects of rats in the GNPs group, while hydrogel-loaded exosomes were implanted in the skull defects of rats in the GNP-Exos group. For the control group, nothing was administered to the defect sites, and the wound was sutured directly.
7. Microcomputed tomography (micro-CT)
After animal sacrifice at 4 and 8 weeks, the calvarial bone of rats was collected and fixed in 4% paraformaldehyde overnight. Then, micro-CT (SANCO Medical AG, Switzerland) was used to evaluate new bone formation in defect sites. The parameters were determined to be 70 kV and 112 μA with a thickness of 0.048 mm per slice in a medium-resolution mode 1024 reconstruction matrix and 200 ms integration time. A total of 315-543 Hounsfield units were selected as the threshold values of bone. In addition, the bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and bone formation rate were calculated using ImageJ, and the volume of interest (VOI) was defined as the cylinder at the centre of each defect.
8. Histological staining
To further evaluate osteogenesis in calvarial bone defects with implanted exosome-loaded GNPs, standard haematoxylin and eosin (H&E) staining and aniline blue staining were performed. After paraffin embedding, the fixed samples were sliced into sections at 8 μm. For aniline blue staining, the sections were successively placed into a 5% phosphotungstic acid solution and 1% aniline blue solution.
9. Immunofluorescence staining
To evaluate the immunomodulation of GNP-Exos in vivo, double immunofluorescence staining was carried out with primary antibodies against F4/80 (macrophage marker) and iNOS (M1 marker) or CD206 (M2 marker). Three nonadjacent slices of each sample were analysed with 3 samples in each group.
10. Cell culture
The human monocyte cell line U937 was purchased from the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI 1640 medium (HyClone, USA) supplemented with 1% penicillin-streptomycin and 10% FBS (Biological Industries, Israel). U937 cells were stimulated with 100 ng/ml phorbol 12-myristate-13-acetate (Sigma-Aldrich) for 24 h. To induce the M1 phenotype, the cells were further incubated with 2 µg/ml lipopolysaccharide (LPS) (Sigma-Aldrich) and 50 ng/ml IFN-γ (Sinobiological, China) in fresh medium. Subcutaneous adipose tissue was collected from four patients during the surgical procedure and immediately immersed in sterile PBS. Adipose tissue collection has been informed and agreed by the patient.The collection process has been informed by the patient.ADSCs were isolated and cultured as previously described . Briefly, the collected adipose tissue was cleaned to remove the surrounding fascia and blood vessels, cut into small pieces and treated with 0.1% collagenase I (Sigma-Aldrich, St. Louis, MO, USA) for 60 min at 37 °C. The digested tissue was washed with α-MEM (HyClone, USA) containing 10% FBS (Bioind, Biological Industry, Israel) and then centrifuged at 1200×g for 5 min. The cell pellets were washed, resuspended in α-MEM (HyClone, USA) supplemented with 10% FBS and 1% penicillin/streptomycin solution, and cultured at 37 °C and 5% CO2.
11. Identification of ADSCs
To evaluate the multilineage differentiation potential of ADSCs, ADSCs were subjected to flow cytometry to assess their phenotypic characteristics. CD44, CD105, CD90, CD31, CD19, CD34 and HLA-DR levels were detected. ADSCs, as a kind of MSCs with multi-differentiation potential, were cultured in adipogenic differentiation medium, stained using oil red O after 14 days or cultured in osteogenic differentiation medium and stained with alkaline phosphatase after 9 days.
12. Isolation of ADSC-conditioned medium (CM) and exosomes
CM from ADSCs was collected as follows: ADSCs were seeded in 10 cm culture dishes at 37 °C with 5% CO2 until reaching 80%-90% confluence. Then, the culture medium was removed from each dish, and the dishes were washed with PBS, after which ADSCs were cultured in serum-free medium for 48 h. The supernatant was collected and centrifuged at 1500 rpm for 10 min to remove cell debris, and ADSC-CM was finally obtained. Exosomes were isolated using ultracentrifugation as follows: after ADSCs were cultured in serum-free medium for 48 h, culture supernatants collected from ADSCs were centrifuged at increasing speeds of 300×g for 10 min, 2000×g for 10 min, and 10,000×g for 30 min to remove cell debris, after which the collected supernatants were ultracentrifuged at 100,000×g for 70 min in an ultracentrifuge (Hitachi, CP100NX, Japan) to obtain primary exosomes. Finally, the pellet was washed with PBS and centrifuged at 100,000×g for 70 min to purify the exosomes. We resuspended the exosome pellet in 100 µl sterile PBS and stored it at -80 °C.
13. Characterization of ADSC-Exos
For particle size determination, nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) were performed in accordance with the protocols. Briefly, the collected exosomes were fixed with 1% glutaraldehyde at 4 °C overnight. After washing, the vesicles were loaded onto formvar/carbon-coated nickel TEM grids and incubated for 30 min. After removing the excess fluid, our samples were then stained with aqueous phosphotungstic acid for 60 s and finally imaged by TEM. The size and concentration of vesicles were determined by a NanoSight tracking analysis system (Brookhaven Instruments Corp, USA).
14. Treatment of macrophages with ADSC-CM and ADSC-Exos
U937 cells were stimulated with 100 ng/ml phorbol 12-myristate-13-acetate (Sigma-Aldrich) for 24 h to obtain macrophages. To induce the M1 phenotype, the cells were further incubated with 2 µg/ml lipopolysaccharide (LPS) (Sigma-Aldrich) and 50 ng/ml IFN-γ (Sinobiological, China) in fresh medium. Macrophages were pretreated with ADSC-CM (diluted 1:1 with α-MEM containing 10% foetal bovine serum) and ADSC-Exos (200 µg/ml) for 2 h followed by stimulation with LPS and IFN-γ. For gene expression analysis, samples were harvested after 24 h; for protein expression analysis, samples were harvested at 48 h; and for the phosphokinase array, samples were harvested at 30 min.
15. Fluorescent labelling of exosomes: endocytosis experiments
The collected exosomes were labelled with a PKH67 Linker Kit (Sigma-Aldrich, USA) according to the manufacturer’s protocol, and PBS was used to replace exosomes as a control. According to the above experimental steps, after 24 h of cell seeding, the labelled exosomes and the control (PBS) were added to the culture medium and cultured at 37 °C for 0.5 h and 1 h; after removing the original medium, the cells were washed three times with PBS, 10 min each time, and fixed in 4% neutral buffered formalin. Nuclei were stained with 4 ′,6-diamino-2 ′-phenylindole (DAPI). Finally, images were captured using an Olympus microscope (Olympus, Japan).
16. Real-time RT-qPCR
For gene expression analysis, the cells were harvested and lysed in RNAiso plus (TaKaRa, Japan), after which RNA was quantified with NanoDrop (Thermo Scientific, USA) and reverse-transcribed using the PrimeScript™ RT Reagent Kit (TaKaRa, Japan) to obtain cDNA. For miR-451a expression analysis, after RNA isolation, first-strand cDNA was synthesized via a miRNA First Strand Synthesis Kit (Sangon Biotech, China) according to the manufacturer’s instructions. RT-PCR was performed with TB Green to quantify mRNA levels on a Bio-Rad real-time PCR system (CFXConnect, USA), which were calculated by the 2−ΔΔCt method. The expression levels of target genes were normalized to the expression levels of the control housekeeping gene β-actin. MiR-100 expression was normalized to that of U6. The primers for target genes are included in Supplementary Table S1.
17. Western blot assay
Cells were lysed and centrifuged at 12,000 rpm at 4 °C following the addition of a protease inhibitor. Protein concentrations in purified exosomes and cells were quantified using the Enhanced BCA Protein Assay Kit (Beyotime, China). For western blot analysis, 30 μg of cell lysates or SHED-Exo lysates were loaded for each sample, separated by 10% SDS-PAGE and transferred to PVDF membranes. The primary antibodies used to assay protein expression were against CD9 (1:800, ProteinTech, USA), CD63 (1:1000, Abcam, USA), CD86 (1:800, Bioss, China), CD206 (1:1000, Abcam, USA) and iNOS (1:1000, Abcam, USA), and β-actin (1:5000, Bioss, China) was used as the loading control (Bioss, China). Images were captured and analysed with a computer programme (ImageJ, USA).
18. Transfection of miRNA-451a
After U937 cells were stimulated with 100 ng/ml phorbol 12-myristate-13-acetate (Sigma-Aldrich) for 24 h, the obtained macrophages were seeded on culture slides. The macrophages were transfected with 50 nM miR-451a mimic, 100 nM miR-451a inhibitor, or the corresponding control oligonucleotide (miR-451a mimic negative control (NC) or miR-451a inhibitor NC) using EntransterTM-R4000 transfection reagent (Engreen Biosystem Co., Ltd., China) in 2 ml of RPMI 1640 medium according to the manufacturer’s instructions. Six hours post incubation, the medium was removed, and the cells were washed with PBS and transfected for 24-48 h. The sequences were as follows: hsa-miR-451a mimic (sense, 5’-AAACCGUUACCAUUACUGAGUU-3′; antisense, 5′-CACAAGUUCGGAUCUACGGGUU-3′), hsa-miR-451a inhibitor (sense, 5’-AACUCAGUAAUGGUAACGGUUU-3′), mimic NC (sense, 5’UUGUACUACACAAAAGUACUG-3′; antisense, 5′-GUACUUUUGUGUAGUACAAUU-3′), and inhibitor NC (sense, 5’-CAGUACUUUUGUGUAGUACAA-3′). All oligos were synthesized by Sangon Biotech (Shanghai, China).
19. Treatment with a macrophage migration inhibitory factor (MIF) inhibitor
Induced cells were incubated with the MIF inhibitor ISO-1 (10 µM) (Selleck, USA) for 2 h and then stimulated with LPS and INF-γ for 24 h followed by mRNA expression analysis.
20. Statistical analysis
GraphPad Prism 7.0 (GraphPad Software, USA) was employed to performed statistical analyses. Data are expressed as the mean ± SEM. Statistical differences were evaluated using Student’s t test or one-way ANOVA with Tukey’s post hoc test for multigroup comparisons, and P values < 0.05 were considered significant.