Animals
All animal experiments concerned on this research met the necessities and requirements of animal experimental ethics (protocol quantity: 2019-N(A)-086). Sprague–Dawley (SD) rats (age: 12–13 weeks; weight: 150–230 g; n = 86) have been bought from the Animal Heart, Anhui Medical College. Earlier than the experiment, 90 feminine rats have been acclimated at 20–25 ℃ for one week with a relative humidity of 35–65%. Feminine and male mice have been bought from the Animal Experimental Heart of Anhui Medical College. Every mouse was aged round six to eight weeks and weighed 30–40 g.
hUCMSC and HUVEC tradition
hUCMSCs have been cultured in an incubator (HERAcell150i, Thermo, China) containing 5% carbon dioxide utilizing α-Minimal Important Medium (MEM) (Hyclone, USA) and recognized by way of stream cytometry (CytoFLEX, Beckman, USA). Within the stream identification course of, the fluorescein antibodies concerned included anti-CD73 (344,015, Biolegend, China), anti-CD105 (323,205, Biolegend, China), anti-CD90 (328,108, Biolegend, China), anti-CD14 (367,115, Biolegend, China), anti-CD19 (363,007, Biolegend, China), anti-HLA-DR (307,605, Biolegend, China), anti-CD34 (343,505, Biolegend, China), and anti-CD45 (368,503, Biolegend, China). Adipose and chondrogenic differentiation media (Gibco, USA) have been used to measure the differentiation capability of hUCMSCs. HUVECs have been recognized utilizing immunofluorescence staining. The antibodies concerned included anti-CD31 (342,553, Biolegend, China) and anti-vWF (371,979, Biolegend, China). HUVECs have been cultured in a endothelial cell medium (ScienCell, USA) supplemented with 5% fetal bovine serum.
Exosome extraction and identification
Exosomes have been obtained by way of speedy centrifugation. First, the cell tradition supernatants of hUCMSCs (cells passaged three – 5 occasions) have been collected and centrifuged at 2,000 × g for 10 min and lifeless cells have been eliminated. For the remaining supernatant, the centrifugal power was elevated to 10,000 × g for 30 min to take away cell particles, and the handled supernatant was then prepared for centrifugation. Crude exosome precipitates have been obtained by way of centrifugation at 100,000 × g for 75 min. Exosomes have been precipitated with PBS once more at 100,000 × g and centrifuged for 75 min, and purified exosomes have been obtained. Lastly, the extracted exosomes have been resuspended utilizing 200 μL of chilly PBS. The morphology of hUCMSC-Exos was noticed by way of TEM (JEM-ARM300F, JEOL, Japan). The dimensions of hUCMSC-Exos and expression of floor markers (CD63, CD9, and CD81) have been decided utilizing a NanoSight (Malvern, England) and thru western blotting, respectively.
Synthesis of GelMA-Exos hydrogel
Primarily based on earlier literature studies, we synthesized GelMA (12). To 50 mL of PBS, 10 g of gelatin and eight mL of methacrylate have been added individually and completely combined. The answer was diluted to terminate the chemical response of gelatin with methacrylate after which dialyzed with double-distilled water (DD-H2O) for 5 days. The answer was then freeze-dried and saved at − 20 °C.
The GelMA-Exos hydrogel was ready as follows: First, an answer of the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) was ready, and GelMA was dissolved in LAP answer at a 25% (w/v) focus and heated in a water tub at 40 ℃ for 20 min. Subsequent, the exosome answer (300 μg of exosome protein suspended in 200 μL of PBS) was added and stirred for 30 s. GelMA-Exos answer was irradiated below ultraviolet mild (wavelength 405 nm) for 40 s and saved at − 20 °C after gelation.
Hydrogel degradation assay
GelMA and GelMA-Exos have been added to five mL of PBS and incubated at 37 °C. At 5 days intervals, the gel was faraway from the PBS, freeze-dried, and weighed utilizing a microbalance (Fixed, China). Feminine and male mice have been bought from the Animal Experimental Heart of Anhui Medical College. Every mouse was aged round six – eight weeks and weighed 30–40 g. After anesthetizing the mice, the pores and skin was sterilized and reduce from the backs of the animals. The GelMA hydrogel and GelMA-Exos have been sterilized and implanted into the pores and skin tissue on the backs of mice. The pores and skin incision was then re-sutured. After 28 d, GelMA and GelMA-Exos have been eliminated and weighed.
Biocompatibility experiments with GelMA-Exos
After domesticating the mice as described beforehand herein, an incision was made within the backs of the animals. The 18 mice have been randomly divided into three teams: management group, GelMA group, and GelMA-Exos group. There have been six mice in every group. GelMA-Exos and GelMA of comparable volumes and weights have been positioned below the pores and skin, whereas within the management group, the pores and skin was solely reduce after which re-sutured. After 28 d, the mice have been euthanized, and their hearts, lungs, livers, spleens, and kidneys have been eliminated for subsequent experiments. HE staining was carried out on the hearts, lungs, livers, spleens, and kidneys of mice (n = 3). Three sections of various elements of every mouse have been taken from completely different organs, and a complete of 45 tissue sections have been analyzed. Then, we used a microscope (X200) (Nikon TS2, Nikon, Japan) to watch the completely different tissue sections. The spleens of the mice have been eliminated individually and rinsed with PBS. A single-cell suspension was ready by putting mouse spleens on a 100-target cell display; they have been then subjected to urgent and grinding on the cell display utilizing a syringe needle core for 30 s, and the display was washed by including 10 mL of sterile PBS primarily based on three batches. Immune cells within the single-cell suspension have been collected by way of centrifugation (200 × g, 10 min). The cells in every group have been precipitated with 0.5 mL of PBS, 3 μL of stream cytometry (CytoFLEX, Beckman, USA) antibodies have been added, and the pattern was incubated at 4 ℃ for 15 min at the hours of darkness. The antibodies used included anti-CD45 (45-0451-80, Invitrogen, USA), anti-CD3 (100,203, Biolegend, China), anti-CD45R (100,203, Biolegend, China), anti-CD11B (17-0118-41, Invitrogen, USA), and anti-F4/80 (12-4801-80, Invitrogen, USA). Stream cytometry (Beckman, Germany) was then carried out.
Characterization of GelMA-Exos and GelMA
To characterize the floor morphology of GelMA and GelMA-Exos, SEM pictures have been obtained utilizing a Sigma 300 area emission scanning electron microscope (EVO10, Zeiss, German). Hydrogel samples have been ready and freeze-dried in a single day. Then, black double-sided tape was used to safe the hydrogel to the work floor. The floor of the pattern was coated with gold by means of argon sputtering for a number of seconds. Pictures have been then captured. GelMA and GelMA-Exos have been adequately dried utilizing a freeze-dryer, and the dried merchandise have been collected and milled right into a powder. Each GelMA and GelMA-Exos have been characterised utilizing Fourier-transform infrared spectrophotometry (Nicolet™ iS50, Thermo, USA). The rheological properties of the GelMA hydrogel, together with the G’ and G’’, have been decided utilizing a rheometer (RheolabQC, Anton Paar, China). The GelMA hydrogel was scanned utilizing an X-ray diffractometer (Smartlab SE, Rigaku, Japan) after which analyzed and mapped utilizing ORIGIN 2019 software program.
Launch kinetics of GelMA-encapsulated hUCMSCs‑Exos
To detect the discharge of hUCMSCs-Exos from GelMA in vitro, we combined 100 mg of hUCMSCs-Exos with 20% GelMA at 4 °C and positioned them within the higher compartment of a transwell, with 100 μL of PBS added to the decrease wells, incubated at 37 °C. The PBS answer was faraway from the decrease compartment 4 days aside, and the BCA technique was used to detect the protein focus in PBS answer and calculate the degrees of launched exosomes.
Uptake of hUCMSC-Exos
Exosomes have been stained and labeled utilizing the Paul Karl Horan (PKH)26 package (Sigma, USA). Exosomes have been fluorescently double-stained with an anti-CD63 (Sigma, USA) antibody. The 25% GelMA was combined with double-labeled exosomes in a water tub. HUVECs have been inoculated in 12-well plates at roughly 105 cells per properly. HUVECs have been continued to co-culture with GelMA-Exos for 12 h. The nucleus of HUVECs was labeled utilizing a 4′,6-diamidino-2-phenylindole (DAPI) (1:1,000) answer. Random imaging and the statement of HUVECs in 12-well plates have been carried out utilizing a confocal microscope (Energy HyD, Leica®, Germany).
Surgical process
The animal vein graft mannequin used on this research was constructed in response to the literature (2). The surgical process used was as follows: SD rats have been anesthetized intraperitoneally and heparinization was induced. The necks of SD rats have been reduce (roughly 1 cm) alongside the route of the trachea, and unilateral veins have been remoted. Subsequent, the jugular vein was grafted onto the carotid artery. After venous transplantation, the rats have been fed warfarin water in response to their physique weight. To check the mixed results of hUCMSC exosomes and GelMA, SD rats have been divided into 4 teams as follows: vein graft, vein graft + GelMA (smeared across the graft), vein graft + exosomes, and vein graft + GelMA-Exos. 4 weeks after transplantation, the surgically remoted venous grafts have been retained, and the rats have been euthanized.
Colour doppler ultrasound
After anesthesia, the hair elimination course of was carried out on the necks of rats, after which, conductive gel was daubed close to the unique incision. An ultrasonic sensor probe (L9-3U; RESONA9, China) was used to seek out and find the graft vein and observe whether or not it was unblocked.
HE staining
Slices have been paraffin-embedded, dewaxed, hydrated, and baked in an oven at 62 ℃ for 60 min. They have been then soaked in xylene (10,023,418, China) 3 times, for about 10 min every time. Then, they have been soaked in 100% ethanol twice for one minute every time after which soaked in 95%, 90%, 85%, 80%, 70%, and 50% ethanol for one minute every. A drop of hematoxylin staining answer (BASO, China) was added, and the pattern was rinsed with faucet water for 5 minutes; the blue colour returned after soaking the pattern in faucet water for 15–30 min. The slices have been immersed in a 1% hydrochloric acid ethanol answer for about 5–30 s, rinsed once more in faucet water to revive the blue colour, after which stained with 0.5% eosin ethanol answer (BASO, China) for one – three minutes. The slices have been immersed in absolute ethanol three – 5 occasions and dried at 25 ℃. The slices have been allowed to dry completely, positioned in xylene for 5 minutes, and noticed after sealing.
Masson staining
The vascular tissue samples have been mounted with 4% paraformaldehyde, sliced after paraffin embedding, stained with Weigert iron hematoxylin for 5 minutes, rinsed with operating water, differentiated in 1% alcohol hydrochloride for 10 s, and rinsed with faucet water for 5 minutes to return to blue. The pattern was stained with ponceau fuchsin stain answer for 5 minutes and washed with weak acid working answer for one minute. Phosphomolybdic acid aqueous answer was handled for 3 minutes. Aniline blue answer was re-dyed for 5 minutes. The pattern was dehydrated and sealed for microscopic examination.
Immunofluorescence staining
The samples have been soaked in 4% paraformaldehyde answer after which soaked in 0.3% TritonX-100 (Sigma, USA) at room temperature for 15–20 min. The TritonX-100 was then eliminated and washed with PBS. Then, 0.25 g of bovine serum albumin (BSA) was added to five mL of PBS to arrange the sealing answer, and the pattern was incubated within the sealing answer at 37 ℃ for 30 min. The first antibody was added to the sealing answer and incubated at 4 ℃ in a single day. The next main antibodies have been used: anti-α-SMA, anti-PCNA, and anti-CD31 (1:1,000, Beyotime, China). The first antibodies on the remaining sections have been washed with PBS, after which the secondary antibody (1:500, Beyotime, China) was added and incubated. The secondary antibodies have been eliminated by washing as soon as with PBS. A fluorescence quencher was added, and the cells have been noticed.
Cell proliferation assay (CCK8)
HUVECs have been collected by centrifugation, made right into a single cell suspension, and the cell focus was diluted to five–10 × 104 cells/mL. The cell suspension was gently combined and added to the 96-well plate with 100 μL per properly, and the sting holes have been crammed with sterile PBS. The inoculated 96-well plates have been positioned in an incubator and continued to develop till HUVECs lined your entire backside of the wells. PBS, GelMA, exosomes, and GelMA-Exos have been added to every properly. After continued tradition within the incubator for twenty-four h, 10 μL of CCK-8 (Bioss, China) was added to every properly. HUVECs have been continued to be cultured for 4 hours, and optical densities (OD) values of every properly at 450 nm have been measured utilizing enzyme-labeler (Multiskan FC, Thermo, USA).
Cell migration assay
GelMA-Exos was soaked in PBS and saved in a cell incubator. On day 0, day 3, day 7, and day 14 after soaking, the GelMA-Exos was eliminated and experimented. A marker was used to attract even horizontal traces roughly 0.5–1 cm aside on the again of the six-well plate. Roughly 5 × 105 HUVECs have been inoculated in every properly and cultured in a single day till cells fully lined the underside of the plate. PBS, GelMA hydrogels, and GelMA-Exos have been added individually to every properly. With a ruler, cell scratches have been made utilizing a 20 μL pipette gun tip perpendicular to the properly plate and line in order that the scratch intersected the marked line. The pinnacle of the gun was vertical, to attempt to make sure that the width of every scratch was constant. The cells have been washed and added serum-free medium. Pictures have been acquired after 24 h.
Stream cytometric evaluation of annexin V
HUVECs have been collected and washed with chilly PBS. Apoptosis was detected utilizing Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide package (AP101, Multi Sciences, China). Dilution of 5× binding buffer to 1× binding buffer was carried out with double steaming water. The cells have been suspended with 500 μl 1× binding buffer. 5 μl FITC and 10 μl propyl iodide have been added to every tube of cells. After gently mixing, the combination was incubated for 5 minutes at room temperature. Evaluation was carried out utilizing stream cytometry (CytoFLEX, Beckman, USA).
Western blotting
The proteins have been extracted with radioimmunoprecipitation assay (RIPA) and quantified utilizing BCA. The proteins have been separated and electrophoresed utilizing 12% protein prefabricated glue (Beyotime, China), after which transferred to a Hybond-P polyvinylidene difluoride membrane. The membrane was eliminated after completion, put into 5% milk sealing answer, and sealed in a low-speed shaker at room temperature for one hour. After eluting the blocking answer, the first antibody was added and incubated at 4 °C in a single day. The antibodies used have been anti-CD81 (1:1,000, Abcam, USA), anti-CD9 (1:1,000, Abcam, USA), anti-CD63 (1:1,000, Abcam, USA), anti-AKT (1:8,000, Abcam, USA), anti-p-AKT (1:8,000, Abcam, USA), anti-mTOR (1:5,000, Abcam, USA), and anti-p-mTOR (1:5,000, Abcam, USA). After eluting the first antibody, the secondary antibody was added and incubated at 4 °C for 45 min. The strip place was analyzed after publicity.
Bioinformatics evaluation
Three datasets have been downloaded from the GEO database as follows: GSE159814 (40), GSE209966 (41), and GSE69909 (42). The degrees of miRNAs within the high 10–30%, when it comes to expression, have been screened from the three datasets, and the intersection was chosen. This miRNA was thought-about the dominant miRNA in exosomes. Two algorithms, MiRWalk and TargetScan, have been used to foretell the interactions between miRNAs and mRNAs. The outcomes of the 2 algorithms have been mixed to display for genes that is likely to be regulated by miRNAs. STRING (https://cn.string-db.org/) was used to investigate gene interactions, and a miRNA–mRNA–mRNA interplay community was established. Utilizing the clusterProfiler bundle of R language (3.6), GO/KEGG evaluation was used to investigate the purposeful enrichment of genes and discover pathways that might be concerned of their regulation, with P < 0.05 thought-about statistically vital. We used the ggplot2 bundle to visualise the outcomes.
Statistical evaluation
The info have been expressed because the imply ± commonplace deviation and have been processed utilizing Statistical Product and Service Options (SPSS) model 21.0 software program (Chicago, IL, USA). Fisher’s check of the minimal vital distinction was used to check two teams. P < 0.05 was thought-about statistically vital.
