Extraction and cultivation of endothelial progenitor cells
Endothelial progenitor cells (EPCs) were extracted and cultivated using the whole bone marrow adherent method. Bone marrow was isolated from the femurs of 3-week-old female rabbits and transferred to a sterile 15 mL centrifuge tube. The tube was centrifuged at 800 rpm for 5 min, and the supernatant was discarded. The pellet was then resuspended in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DME/F12, Hyclone, USA) culture medium supplemented with 10% fetal bovine serum (Gibco, USA). The cell culture bottle was placed in a CO2 incubator at 37 °C with a 5% CO2 atmosphere for 20 h. Subsequently, non-adherent cells were collected by centrifugation at 800 rpm for 5 min and resuspended in EBM-2MV culture medium (Lonza, Basel, Switzerland). The cells were transferred to a new cell culture bottle and continued to be cultured in the CO2 incubator to obtain EPCs. The culture medium was replaced every 48 h to obtain second-generation cells for subsequent characterization.
Extraction of endothelial progenitor cell-derived exosomes
The cells were cultured until reaching a density of 70–80%, and then the medium was replaced with exosome-depleted serum culture medium for an additional 2 days of cultivation. Afterward, the culture supernatant was collected into a 50 mL centrifuge tube. The collected supernatant was subjected to centrifugation to remove cell debris and other large particles. Centrifugation was performed using a Beckman Coulter centrifuge at 2000 × g for 20 min to remove cell debris, followed by centrifugation at 10,000 × g for 30 min, and finally at 100,000 × g for 120 min. After centrifugation, the pellet was resuspended in 15 mL of PBS (Servicebio, Chian). The suspension was filtered through a 0.22 μm pore-sized membrane filter to remove residual cell debris and other impurities, obtaining a purer supernatant. Finally, the filtrate was centrifuged at 4000 × g to obtain a 200 µL pellet for subsequent experiments.
Measurement of exosome particle size
The exosomes were transferred to centrifuge tubes and diluted with PBS buffer, ensuring thorough mixing of the sample to achieve a homogeneous suspension. A Malvern particle size analyzer was set up according to the manufacturer’s instructions. The sample was transferred to the sample chamber of the Malvern particle size analyzer, ensuring the cleanliness of the sample chamber and avoiding any air bubbles that could affect the measurement results. Appropriate parameters were set based on the instrument’s requirements and the nature of the sample for particle size measurement. The particle size distribution of the exosomes was analyzed by measuring the intensity of light scattered by the sample. For nanoparticle tracking analysis (NTA) of exosome size, exosome samples were prepared following the same procedure as with the Malvern particle size analyzer. Exosome samples were collected from the culture supernatant and transferred to centrifuge tubes. The pellet was resuspended using a buffer solution (PBS) and ensured thorough mixing of the sample. According to the manufacturer’s instructions, the sample was injected into the sample chamber of the NTA instrument. Laser power and camera exposure time were adjusted based on the sample’s properties and instrument requirements. The instrument automatically captured and tracked the motion trajectories of the exosomes and analyzed their size and concentration based on their Brownian motion. After the exosomes were fixed with glutaraldehyde, their morphology and particle size were analyzed using transmission electron microscopy (TEM, Hitachi, Japan).
Preparation of decellularized extracellular matrix (dECM)
New Zealand rabbits were euthanized, and their tracheas were immediately harvested. The connective tissue on the outer wall of the trachea was meticulously removed, and the fresh trachea was then cut into small fragments. The fragments underwent a thorough washing with PBS solution, followed by immersion in sterile deionized water at 4 °C for 24 h. Subsequently, the fragments were incubated in a solution containing 0.25% Triton X-100 and 0.25% sodium deoxycholate (SD) at 37 °C for 24 h. After the incubation period, the fragments were rinsed with sterile distilled water for 30 min and then incubated in a 1 M NaCl solution containing 1 kU/mL DNase I (Sigma, USA) and 2 U/mL RNase (Sigma, USA) for 24 h. The entire process was performed in a constant-temperature shaking incubator at 37 °C, set to a speed of 60 revolutions per minute. Following the incubation, the fragments underwent a 72-hour washing process with sterile deionized water, with water being changed every 12 h. The washed tracheal fragments were subsequently frozen at -80 °C for 24 h and then subjected to freeze-drying in a vacuum freeze dryer for 24 h. After the freeze-drying process, the resulting dried dECM particles were ground using liquid nitrogen. The ground particles were then filtered through a stainless steel mesh until they could pass through completely. Finally, the ground and filtered dECM powder was transferred into tubes and stored at -20 °C for future use.
Preparation of GMN-dECM-Exos hydrogel scaffold
To prepare the GMN-dECM-Exos hydrogel, 0.15 g/mL GelMA and 0.04 g/mL Nanoclay were added to PBS buffer and stirred at room temperature until fully dissolved. A magnetic stirrer was employed to facilitate the dissolution process. The mixture was then heated to 50℃ and stirring was continued until complete dissolution. Subsequently, the dECM freeze-dried powder was added to the solution to achieve a 5% concentration. Next, 50 µg of exosomes per milliliter of solution was added and stirred at 37℃until fully dissolved. The pH of the solution was measured using a pH meter and adjusted to pH 7. The dissolved solution was then filtered through a 0.22 μm filter membrane to obtain a clear hydrogel solution. The filtered solution was transferred into a mold, and a photoinitiator, 0.25% (w/v) lithium acylphosphinate photo-initiator (LAP, EFL, China), was added for crosslinking. The scaffold underwent rapid visible light-induced crosslinking with an irradiation intensity of 11mW/cm2 for a duration of 1 min.
Detection of porosity
The scaffold is vacuum-dried to constant weight, and its dry mass Wd is measured. Subsequently, the scaffold is placed in anhydrous ethanol with a known density ρe to ensure complete coverage. It is soaked for 24 h to allow the ethanol to fully permeate the scaffold’s pores, after which the wet weight Ww is measured. The scaffold is then removed, and any residual ethanol on the surface is gently blotted away, followed by measuring its wet mass W w. The volume of the scaffold is V, and the porosity P(%) is calculated as:
$$\:P\left(\%\right)=(Ww-Wd)/\rho e\:V\times\:100\%$$
Biomechanical evaluation of scaffolds
The biomechanical properties of various scaffolds were assessed using a universal testing machine (AGS-X, Shimadzu Corporation, Japan). Each group consisted of three samples (n = 3). The selected scaffold samples underwent measurements using a vernier caliper to determine their length, thickness, outer diameter, and inner diameter. These dimensional parameters were meticulously recorded. Subsequently, the scaffold samples were subjected to compression testing on the universal testing machine. Incremental loads were gradually applied until the point of maximum deformation or failure of the scaffold was reached. The corresponding maximum load value and elastic modulus were recorded at this critical stage.
Live/dead staining assay
For the Live/Dead Staining Assay, cells were directly cultured on the surface of the biomaterial. After a 3-day incubation period, the cultured cells were treated with Calcein AM (Beyotime, China) and incubated at 37 °C for 30 min. Subsequently, propidium iodide dye (Sigma, USA) was added, followed by a 10-minute incubation at 4 °C in the dark. Fluorescence microscopy was then used to capture images for observation.
Western blot
Exosomes were isolated from the culture medium of endothelial progenitor cells through ultracentrifugation. Subsequently, the exosome samples diluted in PBS were collected and dissolved in RIPA protein extraction buffer to release proteins. Protease and phosphatase inhibitors were added to preserve protein integrity. The protein concentration of the extracted vesicles was determined using a BCA protein assay kit (Thermo, USA). A suitable gel concentration, such as 10–15% SDS-PAGE gel, was chosen based on protein size and target protein molecular weight. The vesicle protein sample was mixed with protein loading buffer, heated, and loaded into gel wells. Following electrophoresis, proteins were transferred onto a PVDF or nitrocellulose membrane using a wet transfer system. The membrane was then incubated in blocking buffer, followed by incubation with primary and secondary antibodies. After washing, the membrane was developed using chemiluminescent reagents (Thermo, USA).
DNA quantification
Genomic DNA was extracted from fresh and decellularized tracheal samples using the Genomic DNA Purification Kit (Shenergy Biocolor, Shanghai, China). Briefly, 50 mg of dry tissue was ground in liquid nitrogen and digested in a buffer containing proteinase K at 55℃ for 3 h. The mixture was centrifuged at 12,000 rpm for 5 min at room temperature, and the supernatant was collected. After adding 300µL of solution B, the sample was mixed and centrifuged again under the same conditions. Following three washing steps to remove contaminants, DNA was eluted in 100µL of Tris-EDTA solution. The DNA concentration was quantified by measuring absorbance at 260 nm and 280 nm using a microplate reader (Epoch, BioTek, USA).
Flow cytometry
EPCs were harvested at the second passage (P2), washed with phosphate-buffered saline (PBS), and resuspended in a staining buffer. The cell suspension was incubated with fluorescence-conjugated antibodies: CD31/APC (Bioss, Beijing, China), CD34/PE (Genetex, SC, USA), CD45/PE (Bioss, Beijing, China), and CD105/FITC (Genetex, SC, USA). Following incubation, the cells were washed to remove unbound antibodies and then resuspended in PBS for analysis. Data were acquired using a flow cytometer and analyzed to identify the cell populations.
In vivo angiogenesis assay
The chicken chorioallantoic membrane (CAM) assay was employed as an in vivo model to evaluate the angiogenic properties of the sample groups [51]. Fertilized chicken eggs were incubated under controlled conditions at a constant temperature of 37.8 °C and humidity of 60%. On the 8th day of incubation, a window was created approximately 1 cm away from the embryo’s head on the CAM surface, marked by a pencil. After iodine disinfection, a central area of approximately 5 mm×5 mm was designated, and the eggshell and shell membrane were meticulously removed using ophthalmic forceps and scissors. Tissue samples measuring 2 mm×2 mm were placed in the avascular region between the two anterior vitelline veins, around 1 cm away from the embryo’s head. All procedures were conducted under sterile conditions, and CAMs were photographed daily for the subsequent 4 days. The number of blood vessels covering the grafts and the sponge was evaluated using an image analyzer to assess the angiogenic response.
Immunofluorescence staining
Paraffin-embedded sections were deparaffinized and hydrated. Antigen retrieval was performed by incubating the sections at 37℃ for 30 min in a 0.1% trypsin solution, followed by rinsing with PBS. Excess surface water was gently removed from the sections, and circles were drawn around the tissue. Within these circles, 5% bovine serum albumin (BSA) was applied. The sections were then incubated at 37℃in a humidified chamber for 30 min. Subsequently, the blocking solution was carefully removed, and the pre-diluted primary antibody was applied to the sections. The sections were placed in a humid box with a small amount of water and incubated overnight at 4℃. The sections were washed with PBS, after which the corresponding fluorescent secondary antibody was applied within the circles, covering the specimen tissue. The sections were incubated at room temperature in the dark for 60 min and then washed with PBS. Next, the DAPI staining solution (Solarbio, China) was added within the circles, followed by incubation at room temperature in the dark for 2 min. The sections were washed with PBS and an appropriate amount of anti-fade mounting agent was added. Finally, coverslips were applied using neutral resin. The sections were observed and images were captured using a fluorescence microscope.
Immunohistochemical staining observation
The paraffin-embedded sections were subjected to deparaffinization and hydration. Antigen retrieval was performed by incubating the sections at 37℃for 30 min in a 0.1% trypsin solution, followed by rinsing with PBS. A 3% hydrogen peroxide solution, serving as an endogenous peroxidase inhibitor, was added to the slides and allowed to incubate at room temperature for 10 min. Each slide was then treated with normal non-immune animal serum, a non-specific staining inhibitor, at room temperature for 10 min. Then the primary antibody was applied to the sections. The slides were incubated overnight at 4℃ followed by washing with PBS. Subsequently, a biotinylated secondary antibody, specifically a biotin-labeled goat anti-rabbit IgG polymer, was added and incubated at 37℃ for 30 min. After another round of PBS washing, the slides were exposed to a streptavidin-biotin-peroxidase complex solution. The slides were then processed for DAB staining (Solarbio, China) according to the provided instructions, with a staining time ranging from 30 s to 2 min. Following an 8-minute incubation with hematoxylin solution at room temperature, the slides were rinsed with tap water and counterstained with a bluing reagent. Subsequently, the sections were dehydrated, cleared, mounted, and observed under a microscope.
Tracheal scaffold implantation
New Zealand rabbits were anesthetized via intravenous injection of 2.5 mL/kg of 20% (w/v) ethyl carbamate (Aladdin, China), followed by maintenance with isoflurane throughout the surgical procedure. Following shaving and disinfection, an incision was made at the ventral midline of the neck caudal to the larynx. The neck muscles were carefully dissected layer by layer, with meticulous separation of fascia and blood vessels to fully expose the trachea. A window defect measuring 5 mm in length and 2 mm in width, involving two tracheal rings, was created approximately 2 cm below the cricoid cartilage. A tracheal scaffold measuring 6 mm in length and 3 mm in width was implanted at the site, and the defect and scaffold were secured using 4 − 0 absorbable surgical sutures (Jinhuan, China) with interrupted suturing. After suctioning, the wound was closed in layers using 3 − 0 silk sutures ((Jinhuan, China), and a local anesthetic (2% (w/v) lidocaine; Aladdin, China) was applied around the wound site. Postoperatively, the animals received an intramuscular injection of 8 million U/day penicillin G for three consecutive days to prevent infection. Finally, tracheal samples were collected following euthanasia of the rabbits via intravenous injection of an overdose of pentobarbital sodium (200 mg/kg). All animal experiments conducted in this study were approved by the Ethics Committee of the Yangzhou University School of Medicine.
Scanning Electron microscope (SEM) observation
Fresh tissue samples were collected from each group and prepared into 3 mm × 3 mm tissue blocks. The samples were fixed in 2.5% glutaraldehyde at 4 °C for 24 h. After fixation, the samples underwent three washes with PBS, each lasting 15 min. Subsequently, the tissue samples were dehydrated in a graded alcohol series for 10 min at each level. The dehydrated samples were then dried using a critical point dryer (CPD-300, Leica, Germany) with CO2. The dried samples were mounted with the observation surface facing upward and affixed to a specimen holder. A thin layer of gold was deposited on the sample surfaces using an ion sputtering instrument (SCD500, Bal-Tel, USA). Finally, the samples were examined and photographed using a field emission scanning electron microscope (Gemini 300, Hitachi, Japan).
Statistical analysis
Statistical analysis was conducted using one-way analysis of variance (ANOVA) or a two-tailed Student’s t-test, employing GraphPad Prism 7 software (San Diego, USA). Each experiment was replicated three times, and the results are reported as mean ± standard deviation.
