2D and 3D culture of bone marrow stem cells (BMSCs)
BMSCs was purchased from Cyagen Biosciences (Guangzhou, China) and were maintained in Dulbecco's Modifed Eagle's medium (DMEM, Gibco, USA) containing 10 % fetal bovine serum (Life Technologies, Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA). Hydroxyapatite scaffold was 3D-printed as described previously (23). BMSCs were then seeded to the 3D hydroxyapatite scaffolds and cultured at 37°C with 5% CO2.
Isolation of BMSC-exos
Medium from 3D and 2D culture of BMSCs was collected and used for exosome isolation. Conditioned medium was centrifuged at 3000 g for 15 min and 20,000 g for 45 min. Supernatants were then filtered with a 0.22-μm filter and centrifuged for 70 min at 110,000 g at 4°C to pellet exosomes. The exosomes were washed with PBS and centrifuged for another 70 min at 110,000 g at 4°C.
Transmission electron microscopy
Isolated exosomes were resuspended in PBS. Exosomes were dropped on a copper grid and air-dried. Exosomes were fixed with 3% glutaraldehyde for 2 h and stained with 2% uranyl acetate for 30 s. Then exosomes were visualized under a transmission electron microscope.
Exosome uptake assay
Exosomes were pre-stained with PKH67 using a PKH67 Green Fluorescent Cell Linker Kit (Sigma, USA). Human umbilical vein endothelial cells (HUVECs, 2 × 105/well) were seeded in 6-well plates and 2 µg of PKH67-labeled exosomes was added to the culture. After incubation for 24 h, cells were fixes with 4% paraformaldehyde and DAPI was used to stain the nuclei. The location of exosomes was observed under a confocal microscope (Carl Zeiss, Germany).
Western blot analysis
Exosomes were collected and lysed using RIPA and separated by 12% SDS-PAGE gels for 45 min. Then samples were transferred to PVDF membrane. The membrane was blocked with 5% BSA and incubated with primary antibodies, such as CD9 (ab59479), CD81 (ab79559), and TSG101 (ab125011), at 4°C overnight. After washing with TBS-Tween, the membrane was incubated with secondary antibody for 1 h at room temperature. The protein bands were detected by chemiluminescence substrate.
Cell counting kit-8 assay
HUVECs (2 × 103 cells/well) were placed in 96-well plates and treated with 10 μg/ml exosomes from 3D or 2D culture of BMSCs. The proliferation of HUVECs was determined using the Cell Counting Kit-8 assay kit (Dojindo Molecular Technologies Inc., Kumamoto, Japan). The absorbance at 450 nm was measured every 24 h. To access the effect of 3D-exos released from scaffolds on cell growth, HUVECs were seeded in 48-well plates containing 3D-exo/hydroxyapatite (3D-exo/HA) scaffolds. After cultured for 1, 3, 7 days, the viability of HUVECs was determined by CCK-8 assay.
Transwell migration assay
The effect of exosome on the migration of HUVEC cells was detected using the Transwell migration assay. HUVECs (1 × 105 cells) in serum-free medium was added into the upper chambers of Transwell (Corning, USA). A total of 5 μg exosomes in 600 μl RPMI-1640 medium containing 1% FBS were added to the lower chamber. After incubating at 37°C for 24 h, HUVECs transferred to the bottom of the insert filter were fixed with methanol and stained with 0.1% crystal violet.
Tube formation assay
Matrigel (300 μl/well) was plated to a 24-well plate and allowed to solidify for 30 min at 37°C. HUVEC cells (1.5 × 104 cells/well) were seeded on top of matrigel and treated with 10 μg/ml exosomes derived from 3D or 2D culture of BMSCs. The number of tubes was counted with three random filed under a light microscope.
Quantitative RT-PCR analysis
After treatment with exosomes (10 μg/ml), total RNA of HUVEC cells was extracted using Trizol reagent (Life science, USA). RNA was transcribed to be cDNA using PrimeScript RT-PCR kit (TaKaRa, Dalian, China). RT-qPCR were performed using SYBR Green (Tiangen Biotech, Beijing, China). The reaction conditions were as follows: 95°C for 3 min, 40 cycles of 95°C for 3 s, 60°C for 30 s. GAPDH was used as an internal control. The relative gene expression was calculated by the 2–ΔΔCT method.
DNA plasmid transfection and siRNA interference
The pcDNA3.1-HMGB1 was constructed by Genecopoeia (Guangzhou, China). siRNA targeting HMGB1 and control siRNA were synthesized by Shanghai GenePharma (Hangzhou, China). HUVEC cells were seeded in 6-well plates and grown to 70% (for DNA transfection) or 30~50% confluence (for siRNA transfection). DNA plasmid and siRNA transfections were performed using Lipofectamine 2000.
Release of exosomes from scaffolds
The release rate of exosomes from hydroxyapatite scaffolds was determined by BCA analysis. 3D-exo/HA scaffolds were plated into the 48-well plate with 200 μl PBS. At different time points（3h,6h,12h,24h,48h), the supernatant was collected and 200 μl fresh PBS was added. The exosomes in PBS were counted by BCA analysis.
After mice were anesthetized with pentobarbital sodium (30 mg/kg), the scaffolds (5 mm in diameter) were subcutaneously implanted into mice to study their angiogenesis. Mice were randomly divided in three groups (n=5), including the control scaffold, 2D-exo scaffolds, and 3D-exo scaffolds. For preparation of 2D-exo/HA scaffolds and 3D-exo/HA scaffolds, the dried scaffolds were incubated in DOPA/Tris-HCl solution for 24 h at 37°C and transferred into 10 μg/ml 2D-exo or 3D-exo PBS solution. After 4 weeks of implantation, scaffold samples were collected and fixed with 4% formaldehyde and embedded in paraffin. The samples were stained by hematoxylin and eosin (HE) and masson, and immunohistochemically stained with anti-VEGF to detect the VEGF expression. Real-time qPCR and Western blotting were also performed to determine the mRNA and protein expression of VEGF and CD31. The animal experiments were approved by XXX.
Data were presented as the mean ± SD. Statistical analyses were performed with SPSS 19.0 (SPSS Inc. USA). Comparisons of multiple groups were performed with one-way multivariate analysis of variance (ANOVA) followed by Turkey's test. P value < 0.05 was considered statistically significant.