2.1 Cell culture
The umbilical cord from full-term neonates delivered abdominally was collected in sterile condition. Mesenchymal stem cells (MSCs) were obtained by enzyme digestion and cultured in Dulbecco’s Modified Eagle Medium (DMEM)/F12 medium. The MSCs (Passages 3–6) were expanded using (DMEM)/F12 medium with 10% bovine fetal bovine serum (FBS) and 100 U/mL penicillin-streptomycin. Human umbilical vein endothelial cells (HUVECs) were purchased from ATCC (Manassas, VA) and cultured using Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS and 100 U/mL penicillin-streptomycin. The basal medium, FBS, and phosphate-buffered saline (PBS) used in cell culture experiments were obtained from Gibco (Grand Island, USA) and were ultracentrifuged before use at 160 000 g for 8 h to remove vesicle analogs.
2.2 Characterization of umbilical cord-MSCs (UC-MSCs)
To assess the surface antigen of umbilical cord-MSCs (UC-MSCs), flow cytometry analysis was performed. The MSCs were first incubated with PE-conjugated antibodies against CD90, CD105, CD34, and CD45 for 20 min and then collected after centrifugation for 5 min at 1000 g. The cells were washed twice with PBS and finally resuspended in PBS for flow cytometry analysis (BD Biosciences, USA). All antibodies used in our experiment were purchased from BD Biosciences.
2.3 Multidirectional identification of UC-MSCs
The UC-MSCs (Passage 6) were seeded in 6-well plates and cultured in an appropriate differentiation medium according to the manufacturer’s instructions. For adipogenic differentiation, the entire process lasted 14 days. After differentiation, the cells were stained with oil red O. Osteogenic differentiated cells were stained with alizarin red on day 21, while the chondrogenic differentiated cells were stained with alcian blue and safranin O on day 21, separately [24]. All reagents were obtained from StemCell Technologies.
2.4 Enrichment of MSC-derived extracellular vesicles
To isolate EVs, a large amount of culture medium was required, which was collected by seeding MSCs at a density of 8000 cells/cm2 in the Cell Factory System. Once the cells reached 80–90% confluency, the existing medium was collected [25] and stored at − 80°C, which was pooled together to make up a volume of 6 L. The collected medium was then centrifuged at 16 000 g for 30 min at 4°C to remove the whole cells and excess cellular debris. To enrich the EVs in the supernatant, we used the description of Rider et al. [26] with slight modifications and added PEG 6000 to the medium to achieve a final PEG concentration of 8%. Samples were mixed thoroughly and incubated for 12 h at 4°C. After which, the samples were centrifuged at 12 000 g for 1 h at 4°C. The resultant pellet was thoroughly dissolved in 10 mL of PBS and ultracentrifuged at 120 000 g for 60 min to obtain EVs. Later, EV solutions were stored at − 80°C for further processing.
2.5 Transmission electron microscopy (TEM)
Transmission electron microscopy (TEM) was used to observe the morphologies of EVs. EV samples were dropped onto a carbon grid and adsorbed for 20 min. Next, the EVs were fixed in 1% glutaraldehyde for 5 min, washed with PBS, and visualized using a transmission electron microscope (Hitachi, Japan) [27].
2.6 Western Blot Analysis
An equal amount of protein from both EVs and MSC samples was loaded on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto poly vinylidene fluoride (PVDF) membranes. The membranes were then incubated with primary antibodies CD9, CD63, CD81, and GM130 overnight at 4°C. Later, the membranes were developed with horseradish peroxidase-conjugated secondary antibody and visualized. The western blot results were used to analyze the expression of exosomal markers in the isolated EVs.
2.7 High-sensitivity flow cytometry analysis
To measure accurate particle concentration in high-sensitivity flow cytometry analysis (Flow NanoAnalyzer U30, NanoFCM), the EVs suspension was diluted to 1:1000 with filtered PBS. For immunofluorescence staining analysis, PE-conjugated antibodies specific to CD9, CD63, or CD81 were added to the EV suspension while to investigate the co-expression of CD9, CD63, and CD81 in individual vesicles, PE-conjugated antibodies (for CD9 and CD63) and PerCP Cy5.5-conjugated antibodies (for CD63 and CD81) were added to EV suspensions and determined using double immunization fluorescent staining analysis. All antibodies were purchased from BD Biosciences.
2.8 Preparation and Characterization of chitosan hydrogel
Chitosan hydrogel was prepared as per the previous report [28]. A stock solution of 2% chitosan was prepared by dissolving chitosan powder (Hidebei, China) in 0.1 M acetic acid. Similarly, a 50% β-glycerophosphate (β-GP) solution was prepared in sterile water. Both solutions were then sterilized by filtration and stored at 4°C until subsequent applications. For the experiment, both 50% β-GP and 2% chitosan solution were stirred in the water bath at a volume ratio of 5:1 at 37°C to form a chitosan hydrogel. Later, the EVs (1 × 1010 particles) were mixed well with 5 volumes of 2% chitosan hydrogel by stirring in an ice bath to obtain the chitosan hydrogel-incorporated EVs (CS-EVs). Finally, the parallel plate rheometer was used to measure the elastic moduli (G’) and viscous moduli (G”) to investigate the rheological properties of chitosan hydrogel and CS-EVs at different temperatures ranging between 4°C and 37°C.
2.9 In vitro study of the release dynamics of CS-EVs
The release behavior of EVs leaching from the chitosan hydrogel in vitro was evaluated using the Transwell method. The chitosan hydrogel was first mixed with EVs (1 × 1011 particles) to form the CS-EVs, as mentioned above. The CS-EVs solution was then loaded onto the upper chamber of the Transwell (approximately 200 µL per well) and mounted on a 24-well plate, followed by gelatinization for 10 min at 37°C to form CS-EVs. Next, the CS-EVs were submerged in 1.5 mL PBS at 37°C for different time points (up to 3 days). Subsequently, the supernatants were collected for high-sensitivity flow cytometer (HSFCM) analysis to evaluate the release ratio of EVs. Meanwhile, the release behaviors of EVs at lower doses were also studied by similarly processing the chitosan hydrogel and EVs (1 × 1010 particles).
2.10 In vitro study of the uptake dynamics of CS-EVs
The EVs were stained with PKH26 (Sigma-Aldrich, USA) as per the manufacturer’s instructions. The uptake behavior of EVs leaching from CS-EVs in HUVEC cells was further determined using the method described above. The preparation of CS-EVs (1 × 1011 particles) was similar to the above description, i.e., the CS-EV solution was loaded on the upper chamber of the Transwell, and then the HUVEC were seeded in a 24-well plate at a density of 6000 cells/cm2. After 12 h, HUVECs were gently washed with PBS and fixed in 4% paraformaldehyde for 15 min. The washing step was repeated, and the cells were stained with FITC Phalloidin and DAPI (Solarbio, China). The signals from the stained cells were detected using laser scanning confocal microscopy LSM780 (Zeiss, GRE).
2.11 Migration tests
A scratch wound healing assay was performed to determine the beneficial effects of CS-EVs on the promotion of cell migration. Upon HUVECs reaching 90% confluency, a pipette tip was used to create a scratch in the confluent cell layer. Next, both chitosan hydrogel and CS-EVs (1 × 1011 particles) were loaded on the upper chamber of the Transwell, and the cells were cultured at 37°C for an additional 22 h. The migration distance of cells at 6 h and 22 h was observed under the microscope and photographed.
2.12 Tube formation assay
We performed an in vitro tube formation assay to test the proangiogenic effect of leached EVs. Matrigel obtained from BD Biosciences was thawed at 4°C and added to a 24-well plate (approximately 300 µL per well). HUVECs were seeded on the Matrigel-coated plate and treated with an FBS-free medium. Next, the chitosan hydrogel and CS-EVs (1 × 1011 particles) were loaded on the upper chamber of the Transwell and incubated for 8 h. After which, images were acquired using a microscope with a camera system.
2.13 In vivo animal experiments
Healthy Sprague-Dawley (SD) rats, weighing 240 ± 20 g, were purchased from SPF Biotechnology Co., Ltd. For the construction of the diabetic rat model, the rats were first fed a high-sucrose and high-fat diet for 10 weeks and then administered an intraperitoneal injection of STZ (35 mg/kg, Sigma) at the 10th and 11th weeks. The criterion for successful model construction was a value of fasting blood glucose > 11 mmol/L for more than 4 weeks.
Next, we anesthetized twenty-four rats and randomly divided them into four groups of six animals. Dorsal hairs of rats were shaved to create a full-thickness wound of a 10 mm diameter. Four groups were used to evaluate CS-EVs in promoting wound healing. The wounds were then covered with hydrogel without EVs, CS-EVs (1 × 1010 particles), EVs (1 × 1010 particles), or PBS. Later, the wound was covered with gauze and 3 M Tegaderm film to prevent infection. After 5, 10, or 15 days of surgery, the wound area was photographed, and the dressing was changed in every group. Finally, all rats were sacrificed after 15 days of surgery, and the skin of the wound was collected for hematoxylin and eosin (H&E) staining.
2.14 Statistical analysis
All experiments were performed in triplicates and also repeated three times at least. The SPSS 18.0 software was used for statistical analysis. Data were represented as mean ± SD, where P < 0.05 was considered significant.