Bioengineered cardiovascular bypass grafts via in vivo self-assembly o…

Materials and reagents

The bio-based high-precision resin eResin-PLA Pro (PH100) was obtained from eSUN^®. Primary antibodies including rat monoclonal anti-CD11b (ab8878), rabbit polyclonal anti-fibronectin (FN, ab2413), mouse monoclonal anti-CD73 (ab257311), mouse monoclonal anti-CD31 (ab9498), rabbit polyclonal anti-VE cadherin (CDH5, ab232880), mouse monoclonal anti-N cadherin (CDH2, ab19348), and rabbit monoclonal anti-smooth muscle myosin heavy chain 11 (SM-MHC, ab133567) were purchased from Abcam. Additional primary antibodies included rabbit polyclonal anti-CD105 (PA5-46971), recombinant rabbit monoclonal anti-Ki67 (ab15580), rabbit polyclonal anti-arginase-1 (arg-1, PA5-85267), mouse monoclonal anti-iNOS (MA5-17139). Antibodies used for flow cytometry were as follows: CD45 monoclonal antibody (clone 30-F11, pacific blue, MCD4528), CD11b monoclonal antibody (OX-42, PE, MA5-17511), CD86 monoclonal antibody (BU63, APC, MA1-10294), and CD206 (mannose receptor, MMR) monoclonal antibody (19.2, PE, 12206942). Fluorescence-labeled secondary antibodies included goat anti-rat IgG (H + L) (Alexa fluor 488, ab150157), Goat anti-rabbit or mouse IgG (H + L) (Alexa fluor 594, ab150076, ab150116), goat anti-mouse or rabbit IgG (H + L) (Alexa fluor 647, ab150115, ab150079). Phosphate-buffered saline (PBS, pH 7.4, G4202), diamidino-2-phenylindole (DAPI, D21490) was also obtained from Gibco. Histochemical staining kits included hematoxylin and eosin (HE, G1005), masson’s trichrome (MTC, G1006), sirius red (SR, G1078), verhoeff–van gieson (VVG, GP1035), alcian blue (AB, GP1041), and periodic acid–schiff (PAS, G1008). Other reagents included electron microscopy fixative (G1102), 10% Triton X-100 (G3068), bovine serum albumin (BSA, GC305010), and sodium citrate antigen retrieval solution (G1201), all obtained from Servicebio^®. Iohexol contrast agent (2023B02025) was obtained from Hanson Pharmaceutical. The calcium-sensitive contrast agent Eu-DOTA-4AmC (CAS number: 481668-57-9, CB12563218) was purchased from Xi’an Kaixin Biotech Co., Ltd.

Scaffold design and 3D printing

A cylindrical vascular scaffold with an inner diameter of 2 mm and a wall thickness of 0.5 mm was designed using SolidWorks software (Dassault Systèmes, France). The scaffold featured a grid-like porous structure to mimic the volumetric characteristics of native blood vessels. The final model was exported in standard tessellation language format (Fig. S1) for subsequent 3D printing. The bio-based high-precision resin eResin-PLA Pro was selected as the printing material due to its good biocompatibility, favorable mechanical properties, and suitability for micro-scale 3D printing. According to the manufacturer’s documentation, the material has passed biocompatibility testing in accordance with ISO 10,993 standards. Printing was performed using an M-dental U60 DLP-based 3D printer (Ningbo Inteplast, China) equipped with a 405 nm light source. The standard tessellation language file was imported into the slicing software, and the printing parameters were set as follows: layer thickness = 25 μm, exposure time per layer = 8–10 s, bottom exposure time = 60 s. Support structures were automatically generated. The printing platform was calibrated before starting the print job.

After printing, the scaffolds were carefully removed from the build platform and immersed in 90% isopropyl alcohol for 10 min, followed by ultrasonic cleaning for 5 min to remove residual uncured resin. Post-curing was carried out under UV light at 60 °C for 30 min, following the manufacturer’s recommendations. Dimensional accuracy was verified using digital calipers and precision gauges, and the results (Fig. S2) were compared with the original CAD design (inner diameter: 2 mm; wall thickness: 0.5 mm).

Animal approval and anesthesia protocols

A total of 40 male Sprague-Dawley rats (8–10 weeks old, weighing 280–320 g) were used in this study. All experimental protocols were approved by the Animal Ethics Committee of Dongguan University of Technology and conducted in accordance with the guidelines of the National Institutes of Health (NIH) for the care and use of laboratory animals, ensuring humane treatment throughout the experimental procedures.

Prior to surgery, the rats were anesthetized via intraperitoneal injection of pentobarbital sodium (50 mg/kg). The depth of anesthesia was confirmed by the absence of a paw withdrawal reflex. Vital signs were continuously monitored during the procedure to ensure the absence of pain responses. Postoperative analgesia was provided, and the animals were observed daily for recovery and welfare assessment.

Preparation of BTC

A sterile PLA vascular scaffold with an inner diameter of 2 mm and a wall thickness of 0.5 mm was implanted subcutaneously into the abdominal region of SD rats to prepare the BTC. During surgery, a small incision was made on the abdominal skin, and the pre-measured scaffold was inserted into the subcutaneous pocket. The incision was then closed using intradermal sutures. On day 14 post-implantation, the original implantation site was reopened under anesthesia, and the scaffold together with the surrounding tissue capsule was carefully retrieved. A cylindrical segment approximately 0.2 mm in length was excised from the middle portion of the construct and longitudinally sectioned to obtain both inner and outer surface samples.

The samples were immediately fixed in electron microscopy fixative for 2 h, followed by graded ethanol dehydration, critical point drying, and sputter coating with gold using a HITACHI MC1000 ion sputtering device. Surface morphology of the inner and outer layers of the BTC was then examined using a HITACHI Regulus 8100 scanning electron microscope (SEM, Hitachi, Japan). During the experimental period, body weight and body temperature of each rat were recorded before surgery and at different time points post-implantation to evaluate systemic responses. Non-analyzed samples were stored at − 20 °C for further analysis.

Construction of AA defect model and vascular transplantation surgery

A total of 40 healthy adult male rats were used, with 20 rats allocated to each group: BTC graft group and autologous transplantation group. Five minutes before surgery, heparin (100 U/kg) was intraperitoneally injected for anticoagulation. After anesthesia, rats were fixed in a supine position, and the abdominal area was shaved and disinfected with iodophor. A midline abdominal incision was made through the skin and fascia to expose the AA, followed by careful dissection of the perivascular tissues. The aorta was clamped at both ends, and a 13-mm longitudinal incision was made on the anterior wall to create a localized defect. The lumen was flushed with heparinized saline to remove thrombi. Sterile pre-cut BTC grafts (13 mm in length, 2 mm in inner diameter, 500 μm in wall thickness) were implanted and sutured to the defect with 6 − 0/7 − 0 vascular sutures (continuous or interrupted suturing). For the autologous transplantation group, a 13-mm segment of the rat’s own aortic tissue was harvested and sutured in the same manner.

After checking for suture leakage, the distal vascular clamp was first released to expel air, followed by the proximal clamp. The abdominal cavity was irrigated with warm saline, and the peritoneum, muscle, and skin were sutured layer by layer. For AA samples without transplantation, the aorta was longitudinally dissected along the midline to obtain inner and outer surface tissues, which were immediately fixed in 2.5% glutaraldehyde solution for 2 h. Samples were sequentially dehydrated in gradient ethanol, freeze-dried, sputter-coated with gold, and observed for surface morphology using a SEM following the same protocol as BTC samples, to analyze the microstructural characteristics of the aortic wall. Unused AA samples were stored at −20 °C for future use.

BTC and AA samples were taken out from the refrigerator, rewarmed in a 37 °C water bath for 2 h, cut into 5-mm specimens, and fixed on the stage of an atomic force microscope (AFM, Bruker Dimension FastScan, USA). PBS was added to keep the samples moist. In tapping mode, a ScanAsyst-Air probe (tip radius a) and root-mean-square roughness (R_q). Nanoscale topographical differences in the inner/outer surfaces of BTC and AA (e.g., microvilli density, collagen fiber arrangement) were compared. Each group of samples was measured in triplicate, with 3 regions selected for analysis each time.

Water contact angle and surface morphology characterization

Samples stored at − 20 °C were thawed in a 37 °C water bath for 1 h, cut into 10 mm × 2 mm squares, and ultrasonically cleaned with ethanol and deionized water (15 min each), then dried for testing. Using a micro-syringe, 5–10 µL of distilled water droplets were dispensed onto the sample surface. The samples were placed on a SL250 contact angle goniometer (KINO Industry Co., Ltd., USA). After adjusting the position for a clear image, the droplet behavior was recorded at 5 frames/s until stable. The water contact angle (WCA) was calculated by the instrument’s tangent or height-width method.

The recorded images were processed with interpolation software to reconstruct the three-phase boundary, obtaining surface roughness (Ra, Rq), autocorrelation length, and spatial frequency. A model integrating surface height and contact angle data analyzed wetting properties. Droplet volume was calculated via multi-layer slice interpolation, with hierarchical algorithms used for multi-layer samples. Each sample was measured five times, and the average and standard deviation were reported to ensure data reliability.

2D small-angle X-ray scattering and X-ray diffraction analysis

The inner surfaces of BTC and AA were carefully separated using a micro-scalpel and fixed on the sample stage of a 2D small-angle X-ray scattering (SAXS) diffractometer (Rigaku, Japan), ensuring the surfaces were flat and free of obvious defects. The X-ray wavelength was set to 1.5406 Å (Cu Kα), and measurements were performed in the small-angle range (scattering angle θ 

For raw data processing, background subtraction and normalization were conducted using Rigaku software to generate two-dimensional scattering patterns. The scattering vector Q was calculated using the formula:

where λ is the X-ray wavelength and θ is the scattering angle.

Radial integration of the scattering patterns was performed to obtain the intensity vs. Q (I(Q)) curves. These curves were fitted using OriginPro to extract key parameters, such as average pore size and grain size.

Using a X-ray diffraction (XRD) diffractometer (6100, Shimadzu, Japan), test round, flat samples of BTC and AA with a diameter of 15 mm and a thickness of less than 1 mm. The test parameters were set as follows: tube voltage 40 kV, tube current 30 mA, scanning range 5°−80° (2θ), scanning speed 10°/min, and step size 0.02° [20, 21]. After installing the samples, the instrument was initialized, the parameters were input for measurement, and the data were saved after completion.

2.8 Zeta potential determination

The inner surface membranes of BTC and AA samples were cut into strips (width ≤ 1.5 mm, length 20–30 mm), curled into spirals, and tightly packed into a quartz capillary (inner diameter: 2 mm, length: 50 mm) without visible gaps.

A 10⁻³ M KCl solution (pH 7.0 ± 0.1), filtered through a 0.22 μm membrane, was used as the electrolyte. Prior to sample measurement, blank calibration was performed using an empty capillary to eliminate background effects. Instrument accuracy was verified using a standard glass capillary with a known zeta potential (− 30 ± 2 mV).

The streaming potential measurements were conducted using an Anton Paar SurPASS instrument. The membrane-filled capillary was fixed in the streaming potential measurement module, with both ends connected to electrodes and a peristaltic pump. The KCl solution was pumped at a constant flow rate of 0.1 mL/min. Steady-state streaming potential (ΔV) and pressure difference (ΔP) were recorded for each sample, and each sample was tested five times.

After subtracting the blank value, the zeta potential (ζ) was calculated using the following formula:

where: η: solution viscosity, r: capillary radius, ε: relative permittivity, ε₀ ​: vacuum permittivity.

Cyclic elasticity and suture retention strength testing

BTC and AA samples rewarmed in a 37 °C water bath were trimmed to dimensions of 12 mm in length, 2 mm in width, and 1 mm in thickness. Using a DMA dynamic thermomechanical analyzer (Q800, TA Instruments, USA), the samples were fixed in a single-cantilever fixture. The temperature was programmed to rise from room temperature to 37 °C and held constant. In strain-controlled mode, a strain of 25% was applied for 10 cycles, with stress-strain curves and elastic recovery data recorded.

Cut BTC (inner diameter 2 mm, wall thickness 0.5 mm) and AA (inner diameter 2 mm, wall thickness 0.34 mm) into 13 mm-long tubes. Perform interrupted sutures using 8 − 0 absorbable sutures with a stitch spacing of 3 mm. Each sample should have ≥ 5 suture points. Prepare 4 samples per group. Use a Zwick/Roell Z005 universal testing machine equipped with a 1 N load cell and custom arc-shaped fixtures. Set the tensile speed to 1 mm/min, ensuring alignment between the sample axis and tensile direction. Clamp the sample and initiate tensile testing until failure. Record the force-displacement curve, maximum failure load, and failure mode (suture pullout/material rupture).

Histochemical staining

For BTC samples (excised subcutaneously), AA, and BTC/autograft samples collected at corresponding time points post-transplantation, the tissues underwent sequential ethanol gradient dehydration, xylene clearing, and paraffin embedding after SEM detection [22]. Using a PM-24 paraffin microtome (Saiweier, China), 4-µm-thick tissue sections were prepared. The paraffin sections were first immersed in xylene I and II for 10 min each, followed by sequential incubation in absolute ethanol, 95%, 85%, and 75% ethanol for 5 min each, and finally washed three times with PBS buffer for 5 min each. Various histochemical staining kits were used according to the manufacturer’s instructions [23]. Stained images were acquired using a whole-slide scanner (3DHISTECH, Hungary). Quantification of components in stained sections was performed using FiJi ImageJ-win 64 software (based on ImageJ 2.x, developed by the National Institutes of Health, USA), with three individual biological samples per group.

Immunocytochemistry

Deparaffinized sections were first immersed in antigen retrieval buffer, boiled at medium heat for 15 min, and then allowed to cool naturally. Subsequently, the sections were washed three times with PBS buffer for 5 min each. To block non – specific binding, the sections were incubated with 5% goat serum in PBS for 10 min at 37 °C.

Primary antibodies were then applied to the sections, which were incubated overnight at 4 °C. After removing the primary antibodies, the sections were equilibrated to room temperature for 30 min and gently washed with PBS to remove any unbound antibodies. Next, secondary antibodies were applied to the sections and incubated for 1 h at room temperature in the dark to prevent photobleaching. Finally, the sections were stained with DAPI for 5 min to visualize the cell nuclei.

All primary and secondary antibodies were used in strict accordance with the manufacturer’s instructions. Immunofluorescence images of the stained samples were acquired using a 3DHISTECH whole – slide scanner. For subsequent image analysis, Fiji ImageJ – win64 software was employed to process and quantify the data.

Transmission electron microscopy observation

BTC and autologous transplantation samples retrieved at 7 days post-surgery were immediately fixed in 2.5% glutaraldehyde/1% paraformaldehyde electron microscopy fixative at 4 °C for 2 h. Samples were rinsed 3 times with 0.1 M PBS (10 min each), post-fixed with 1% osmium tetroxide for 2 h, and dehydrated in gradient ethanol (50%→70%→90%→100%) for 15 min each. After two 15-min transitions with 100% acetone, samples were infiltrated with epoxy resin (acetone: resin = 1:1 for 1 h; 1:2 for 2 h; pure resin overnight) and polymerized at 60 °C for 48 h. Ultra-thin Sects. (60–80 nm) were cut using an ultramicrotome and double-stained with uranyl acetate and lead citrate for 15 min each. Vascular ultrastructure images were acquired using a Transmission electron microscopy (TEM, HT7700,HITACHI, Japan) at an acceleration voltage of 100 kV.

Flow cytometry

Tissues from BTC and autologous grafts at 7 days post-surgery were minced and digested in a solution containing 0.25% trypsin-EDTA and 1 mg/mL collagenase IV at 37 °C for 30 min with gentle agitation. The cell suspension was filtered through a 70-µm strainer, centrifuged (300 g, 5 min), and the supernatant discarded. Cells were lysed using 1 mL of RBC Lysis Buffer (eBioscience) for 5 min at room temperature, followed by two washes with PBS. The cell concentration was adjusted to 1 × 10⁶ cells/mL.

For immunostaining, 100 µL of cell suspension was incubated with Fc receptor blocker (Mouse BD Fc Block™) for 15 min at 4 °C. Fluorescently conjugated antibodies against CD45, CD11b, CD68, CD206, and CD86 were added according to the manufacturer’s instructions and incubated for 30 min in the dark. Cells were washed twice with 2 mL of PBS. Samples were fixed with 4% paraformaldehyde for 20 min, washed again, and resuspended in 300 µL of PBS.

Flow cytometric analysis was performed using a Coulter CytoFLEX flow cytometer (Beckman, USA), acquiring 10,000 events per sample. Data were analyzed using FlowJo software (v10.8.1), with isotype controls used to gate out non-specific binding.

Doppler ultrasound and micro-computed tomography detection

At postoperative weeks 1, 4, and 12, after anesthesia, the animals were placed in a supine position and fixed. The abdominal area was shaved and coated with coupling gel. A MyLab SigmaPVET color doppler ultrasound system (CDU, Esaote, Italy) was used to measure the internal diameter, external diameter, and wall thickness (WT) of the graft. Color Doppler imaging was employed to assess blood flow velocity, including peak systolic velocity (PSV) and end-diastolic velocity (EDV), as well as the resistance index (RI). Spectral Doppler was used to record the Doppler spectra across three cardiac cycles.

At postoperative week 24, basal spontaneous pulse frequency of the artery was continuously recorded for 10 min using a biological signal acquisition system (BL420, YiLian Medicine, Shanghai, China), with electrodes closely attached to the arterial surface. The average value was calculated and used as a reference for subsequent electrical field stimulation parameter settings.

Following baseline data collection, the experimental animals were subjected to general anesthesia. Iohexol contrast agent was then administered via tail vein injection at a dose of 1.5 mL/kg. Subsequently, whole-artery computed tomography(CT) scanning was performed using a Quantum GX2 Micro-CT system (PerkinElmer). Scanning parameters were set as follows: voltage at 50–70 kVp to ensure image resolution while minimizing radiation exposure; current at 400–500 µA to maintain stable X-ray output; and slice thickness controlled between 50 and 100 μm to clearly capture structural details of the artery.

Next, the calcium-sensitive contrast agent Eu-DOTA-4AmC was injected via the tail vein at a dose of 1.3 mL/kg. After an appropriate waiting period of 15–30 min to allow full distribution and binding of the contrast agent to calcium ions, the same Micro-CT scanning protocol was repeated.

After completing both scans, the electrodes of the electrical stimulation device were precisely fixed at both ends of the artery. The stimulation intensity and frequency were gradually adjusted to stabilize the arterial pulsation at a pacing frequency of 1 Hz. The imaging view was switched to a top-down perspective, and secondary dynamic imaging of the abdominal aorta was conducted using the built-in professional image analysis software of the Quantum GX2 Micro-CT system. This software, based on 3D registration and optical flow algorithms, performed frame-by-frame analysis of the continuous image sequences, enabling high-precision quantification of the motion trajectories of both the intima and adventitia.

Finally, three-dimensional reconstruction was carried out based on data from both scans. Using image processing algorithms, key functional indicators—including vascular patency, calcium wave conduction velocity, and anisotropy ratio—were systematically analyzed.

Blood pressure and compliance assessment

At 24 weeks post-surgery, rats were anesthetized via intraperitoneal injection of sodium pentobarbital (50 mg/kg), placed in a supine position on the operating table, and the abdomen was routinely disinfected. A PE-50 catheter pre-filled with heparinized saline (10 U/mL) was inserted into the proximal segment of the AA replacement (approximately 5 mm from the anastomosis) and secured with silk sutures. A TMY-203B pressure sensor (TaiMeng, China) was connected to the BL420 Biological Signal Acquisition System, configured with: Sampling frequency: 100 Hz, Time constant: 0.001 s, Filtering frequency: 100 Hz. After air was purged from the sensor-catheter system, blood pressure waveforms were recorded using the Blood Pressure Recording module of the BL420 software. Following signal stabilization, continuous recordings were acquired 2 s, with synchronous marking of systolic blood pressure (SBP) and diastolic blood pressure (DBP) peaks during the cardiac cycle.

Vascular diameter measurements were performed using a high-frequency Doppler ultrasound system (VisualSonics Vevo 3100, 30 MHz probe). Anesthetized rats were positioned supine, and ultrasonic coupling agent was applied to the abdomen. Longitudinal sections of the replaced aortic segment were obtained 10 mm from the anastomosis. Using M-mode imaging, diameter changes were recorded over at least three consecutive cardiac cycles to measure the maximum systolic diameter (D_max) and minimum diastolic diameter (D_min). Each measurement was repeated three times, and the mean values were used for analysis. Vascular compliance (C) was calculated using the following formula:

where D_max,D_min​: Systolic and diastolic vessel diameters (mm), P_sys​,P_dia​: Systolic and diastolic blood pressures (mmHg), L: Length of the measured vessel segment (fixed at 13 mm). All measurements were performed independently by two experienced investigators, and the results were averaged.

Statistical analysis

All data are presented as mean ± standard deviation (SD). For comparisons among multiple groups (≥ 3 groups), a one-way analysis of variance (ANOVA) was first performed to assess overall differences, followed by Tukey’s post hoc test for pairwise comparisons if the ANOVA was significant. For interactions between two variables, a two-way ANOVA was used with Tukey’s or Bonferroni’s correction for multiple comparisons. Two-group comparisons were analyzed using Student’s t-test (parametric data) or the Mann-Whitney U test (nonparametric data). All statistical analyses were conducted using GraphPad Prism 10.0 (GraphPad Software, San Diego, CA, USA), with statistical significance set at P