Animals
All animal experiments concerned on this examine 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) had been bought from the Animal Heart, Anhui Medical College. Earlier than the experiment, 90 feminine rats had been acclimated at 20–25 ? for one week with a relative humidity of 35–65%. Feminine and male mice had 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 had been cultured in an incubator (HERAcell150i, Thermo, China) containing 5% carbon dioxide utilizing ?-Minimal Important Medium (MEM) (Hyclone, USA) and recognized through 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) had been used to measure the differentiation capacity of hUCMSCs. HUVECs had been recognized utilizing immunofluorescence staining. The antibodies concerned included anti-CD31 (342,553, Biolegend, China) and anti-vWF (371,979, Biolegend, China). HUVECs had been cultured in a endothelial cell medium (ScienCell, USA) supplemented with 5% fetal bovine serum.
Exosome extraction and identification
Exosomes had been obtained through speedy centrifugation. First, the cell tradition supernatants of hUCMSCs (cells passaged three – 5 instances) had been collected and centrifuged at 2,000 × g for 10 min and useless cells had been eliminated. For the remaining supernatant, the centrifugal pressure 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 had been obtained through centrifugation at 100,000 × g for 75 min. Exosomes had been precipitated with PBS once more at 100,000 × g and centrifuged for 75 min, and purified exosomes had been obtained. Lastly, the extracted exosomes had been resuspended utilizing 200 ?L of chilly PBS. The morphology of hUCMSC-Exos was noticed through TEM (JEM-ARM300F, JEOL, Japan). The scale of hUCMSC-Exos and expression of floor markers (CD63, CD9, and CD81) had been decided utilizing a NanoSight (Malvern, England) and thru western blotting, respectively.
Synthesis of GelMA-Exos hydrogel
Primarily based on earlier literature stories, we synthesized GelMA (12). To 50 mL of PBS, 10 g of gelatin and eight mL of methacrylate had been added individually and completely blended. 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 bathtub at 40 ? for 20 min. Subsequent, the exosome answer (300 ?g of exosome protein suspended in 200 ?L of PBS) was added and stirred for 30 s. GelMA-Exos answer was irradiated below ultraviolet gentle (wavelength 405 nm) for 40 s and saved at ??20 °C after gelation.
Hydrogel degradation assay
GelMA and GelMA-Exos had 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 had 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 had 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 had 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 had 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 had 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 had been euthanized, and their hearts, lungs, livers, spleens, and kidneys had 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 had been taken from totally different organs, and a complete of 45 tissue sections had been analyzed. Then, we used a microscope (X200) (Nikon TS2, Nikon, Japan) to look at the totally different tissue sections. The spleens of the mice had been eliminated individually and rinsed with PBS. A single-cell suspension was ready by putting mouse spleens on a 100-target cell display screen; they had been then subjected to urgent and grinding on the cell display screen utilizing a syringe needle core for 30 s, and the display screen was washed by including 10 mL of sterile PBS primarily based on three batches. Immune cells within the single-cell suspension had been collected through centrifugation (200 × g, 10 min). The cells in every group had been precipitated with 0.5 mL of PBS, 3 ?L of stream cytometry (CytoFLEX, Beckman, USA) antibodies had been added, and the pattern was incubated at 4 ? for 15 min in the dead of night. 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 had been obtained utilizing a Sigma 300 area emission scanning electron microscope (EVO10, Zeiss, German). Hydrogel samples had 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 via argon sputtering for a couple of seconds. Pictures had been then captured. GelMA and GelMA-Exos had been adequately dried utilizing a freeze-dryer, and the dried merchandise had been collected and milled right into a powder. Each GelMA and GelMA-Exos had been characterised utilizing Fourier-transform infrared spectrophotometry (Nicolet™ iS50, Thermo, USA). The rheological properties of the GelMA hydrogel, together with the G’ and G’’, had 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 blended 100 mg of hUCMSCs-Exos with 20% GelMA at 4 °C and positioned them within the higher compartment of a transwell, with 100 ?L 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 methodology was used to detect the protein focus in PBS answer and calculate the degrees of launched exosomes.
Uptake of hUCMSC-Exos
Exosomes had been stained and labeled utilizing the Paul Karl Horan (PKH)26 package (Sigma, USA). Exosomes had been fluorescently double-stained with an anti-CD63 (Sigma, USA) antibody. The 25% GelMA was blended with double-labeled exosomes in a water bathtub. HUVECs had been inoculated in 12-well plates at roughly 105 cells per properly. HUVECs had 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 commentary of HUVECs in 12-well plates had been carried out utilizing a confocal microscope (Energy HyD, Leica®, Germany).
Surgical process
The animal vein graft mannequin used on this examine was constructed in keeping with the literature (2). The surgical process used was as follows: SD rats had been anesthetized intraperitoneally and heparinization was induced. The necks of SD rats had been reduce (roughly 1 cm) alongside the path of the trachea, and unilateral veins had been remoted. Subsequent, the jugular vein was grafted onto the carotid artery. After venous transplantation, the rats had been fed warfarin water in keeping with their physique weight. To check the mixed results of hUCMSC exosomes and GelMA, SD rats had 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 had been retained, and the rats had been euthanized.
Shade 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 had been paraffin-embedded, dewaxed, hydrated, and baked in an oven at 62 ? for 60 min. They had been then soaked in xylene (10,023,418, China) 3 times, for about 10 min every time. Then, they had 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 coloration returned after soaking the pattern in faucet water for 15–30 min. The slices had been immersed in a 1% hydrochloric acid ethanol answer for about 5–30 s, rinsed once more in faucet water to revive the blue coloration, after which stained with 0.5% eosin ethanol answer (BASO, China) for one – three minutes. The slices had been immersed in absolute ethanol three – 5 instances and dried at 25 ?. The slices had been allowed to dry completely, positioned in xylene for 5 minutes, and noticed after sealing.
Masson staining
The vascular tissue samples had been fastened 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 had 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 organize 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 major antibodies had been used: anti-?-SMA, anti-PCNA, and anti-CD31 (1:1,000, Beyotime, China). The first antibodies on the remaining sections had been washed with PBS, after which the secondary antibody (1:500, Beyotime, China) was added and incubated. The secondary antibodies had been eliminated by washing as soon as with PBS. A fluorescence quencher was added, and the cells had been noticed.
Cell proliferation assay (CCK8)
HUVECs had 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 blended and added to the 96-well plate with 100 ?L per properly, and the sting holes had been full of sterile PBS. The inoculated 96-well plates had been positioned in an incubator and continued to develop till HUVECs coated your entire backside of the wells. PBS, GelMA, exosomes, and GelMA-Exos had been added to every properly. After continued tradition within the incubator for twenty-four h, 10 ?L of CCK-8 (Bioss, China) was added to every properly. HUVECs had been continued to be cultured for 4 hours, and optical densities (OD) values of every properly at 450 nm had 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 had been inoculated in every properly and cultured in a single day till cells utterly coated the underside of the plate. PBS, GelMA hydrogels, and GelMA-Exos had been added individually to every properly. With a ruler, cell scratches had been made utilizing a 20 ?L pipette gun tip perpendicular to the properly plate and line in order that the scratch intersected the marked line. The top of the gun was vertical, to attempt to make sure that the width of every scratch was constant. The cells had been washed and added serum-free medium. Pictures had been acquired after 24 h.
Stream cytometric evaluation of annexin V
HUVECs had 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 had been suspended with 500 ?l 1× binding buffer. 5 ?l FITC and 10 ?l propyl iodide had 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 had been extracted with radioimmunoprecipitation assay (RIPA) and quantified utilizing BCA. The proteins had 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 had 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 had been downloaded from the GEO database as follows: GSE159814 (40), GSE209966 (41), and GSE69909 (42). The degrees of miRNAs within the prime 10–30%, when it comes to expression, had been screened from the three datasets, and the intersection was chosen. This miRNA was thought of the dominant miRNA in exosomes. Two algorithms, MiRWalk and TargetScan, had been used to foretell the interactions between miRNAs and mRNAs. The outcomes of the 2 algorithms had been mixed to display screen for genes that may 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 could possibly be concerned of their regulation, with P?<?0.05 thought of statistically important. We used the ggplot2 bundle to visualise the outcomes.
Statistical evaluation
The information had been expressed because the imply?±?customary deviation and had been processed utilizing Statistical Product and Service Options (SPSS) model 21.0 software program (Chicago, IL, USA). Fisher’s take a look at of the minimal important distinction was used to match two teams. P?<?0.05 was thought of statistically important.