Human adipose-derived stem cell isolation and culture

Fresh human lipoaspirates were isolated from healthy patients following approval of the Research Ethics Committee of Shanghai Ninth People’s Hospital, as previously described21,35. All the experiments were performed in accordance with relevant guidelines and regulations and informed consent was obtained from all the patients. The hASCs were cultured in DMEM supplemented with 10% FBS (HyClone), 100 U/mL penicillin (Sigma Aldrich) and 100 mg/mL streptomycin (Sigma Aldrich), and cultured in 5% CO 2 at 37 °C. When the cells reached 80% confluence, the cells were further passaged.

Differentiation of hASCs (SMCs)

We previously investigated the status of isolated hASCs during differentiation under 5 ng/mL TGF- β1 (R&D Systems) and 2.5 ng/mL BMP4 (R&D Systems)21. The identity of SMCs was characterized by undulating morphology. They were evaluated by immunofluorescence for SMC markers. Fluorescent phallacidin (ThermoFisher, A12379) was used to label F-actin. FITC-conjugated anti-α-smooth muscle actin (αSMA), SM22α, calponin and smooth muscle-myosin heavy chain (SM-MHC) were used to identify differentiated hASCs (SMCs). When the cells reached confluence, they were subcultured and referred to as Passage 1 cells. Human umbilical artery smooth muscle cells (hUASMCs) served as the positive control.

Whole-cell patch clamp recording

Whole-cell patch clamp recordings were performed in the current clamp mode on an inverted microscope. Currents were recorded and amplified using a HEKA patch clamp amplifier (HEKA, Germany), and pClamp 10 software (MolecularDevices, Sunnyvale, CA). A coverslip containing the attached cells was placed in the recording chamber (1 mL). Cells were continuously perfused with extracellular solution (in mmol/L: NaCl 140, KCl 5, MgCl 2 2, CaCl 2 2, HEPES 10, Glucose 10, pH 7.3–7.4). Whole cell recordings were conducted using electrodes with a resistance of about 3–5 MΩ when filled with internal solution (in mmol/L: KCl 140, HEPES 10, EGTA 5, MgCl 2 2.5, Mg-ATP 2, pH 7.3–7.4). Pharmacological stimuli were added to the bathing medium in the vicinity of the cell using a fast gravity perfusion system containing 80 mM KCl; 100 μM acetylcholine (Ach); 10 nM endothelin-2(ET-2); 10 nM endothelin-1(ET-1); and 100 μM prostaglandin F2α (PGF2α).

Differentiation of hASCs (ECs) and characterization

Stimulation with 50 ng/mL VEGF and 100 ng/mL BMP4 under hypoxic conditions (37 °C, 5% CO 2 , and 2% O 2 ), induced hASCs differentiation into ECs. The ECs manifested cobblestone appearance after immunofluorescence staining for CD31, CD144 and von Willebrand factor (vWF), and endothelial tube formation assay, as described previously26,36. Human umbilical vein endothelial cells (HUVECs) served as the positive control.

Determination of LDL uptake and nitric oxide production by cultured ECs

LDL uptake was assessed by incubating cells with 2.5 μg/mL acetylated LDL labeled with Dil-Ac-LDL(Thermo Fisher, L3484), for 4 h at 37 °C. Cells were analyzed by fluorescence microscopy. Nitric oxide levels in the supernatants of differentiated hASCs (ECs) in the 14-day culture were measured using nitric acid reductase (Colorimetric Assay for Nitric Oxide, Neogen Corporation). The assays were performed by mixing 5–85 μL of supernatant with buffer to a total volume of 85 μL. Each solution was supplemented with 10 μL of the reconstituted nitrate reductase and 10 μL of 2 mM NADH, followed by incubation for 20 min at room temperature. After addition of 50 μL of Color Reagent #1 and 50 μL of Color Reagent #2, the absorbance of the sample was read at 540 nm, by spectrometry.

Preparation of PCL-gelatin biodegradable scaffolds

The PCL-gelatin mesh (Shanghai Ju Rui Biomaterials) was used as a template for seeding of hASCs. Electrospinning solutions (5% w/v) were prepared by mixing PCL and gelatin in a weight ratio of 50:50. The electrospinning apparatus was equipped with a Statitron® IFP™ power supply, which generated up to 50 KV (Tianjing High Voltage Power Supply Co., Tianjing, China). The steady flow rate of the solution was controlled at 0.7 mL/h. The electrospinning voltage was set at 16 kV. The electrospun scaffolds were collected on an aluminum foil, and vacuum-dried at room temperature for 24 h. The fiber diameter was measured based on 25 different fibers in the SEM images, and the total porosity was calculated according to previous methods37.

PCL-gelatin unwoven fibers were formatted into a 50 × 48 × 2 mm mesh. The scaffold was pre-sterilized with 75% ethanol, followed by overnight incubation with low glucose-DMEM (containing 10% FBS, penicillin–streptomycin). The medium was removed and the scaffold was allowed to air dry.

Assessment of scaffold cytotoxicity

To assess the cytotoxicity induced by scaffolds, the activity of lactate dehydrogenase (LDH) in the medium was determined using a commercially available kit (Sigma Diagnostics). Cytotoxicity was calculated based on a formula provided by the manufacturer38. Cells were cultured with the scaffolds for 10 days, and cell counts were obtained using a Cellometer Auto T4 Cell Counter (Nexcelom).

Seeding SMCs on PCL-gelatin scaffolds

Differentiated ASCs (SMCs) were collected and re-suspended in growth medium at a density of 5 × 107 cells/mL. Each PCL-gelatin mesh in the tissue culture dishes was seeded with 1 mL of cell suspension. The cell/PCL-gelatin complexes were incubated for 4 h to allow for adhesion of cells to PCL-gelatin fibers, and the culture medium was added. The complexes were stored in an incubator for an additional 7 days, and transferred to a vessel bioreactor for further incubation.

Pulsatile vascular bioreactor culture

The cell/PCL-gelatin complexes surrounded the silicone tubes with an outer diameter of 4 mm after 7 days in culture. In the dynamic system, the culture in the pulsatile bioreactor was performed as previously described21. The control samples were cultured in normal growth medium using the static culture system. The culture was maintained in the incubator at 37 °C, and the medium was refreshed every 3 days.

Seeding differentiated hASCs (ECs) on smooth muscle cell polymer

After another 6–8 weeks of incubation, the PCL-gelatin degraded and the luminal supporting silicone tube was removed, followed by injection of 5 × 106 differentiated ASCs (ECs), suspended in DMEM containing 25 mmol/L HEPES (Invitrogen), 10% FBS, 1% antibiotic-antimycotic, and 25 g/mL L-ascorbic acid, into the lumen. The outer area of the chamber was filled with the culture medium and cultured for another 1–2 weeks.

Histology, scanning electron microscopy and immunochemistry

Tissue samples were fixed in 10% formalin, embedded in paraffin, and 5-μm-thick sections were obtained. Samples were stained with Hematoxylin and Eosin (H&E), or Masson trichrome. Other tissue samples were fixed for 30 min with 2.5% glutaraldehyde in phosphate buffer (pH 7.2). After fixation, the samples were dehydrated in a graded ethanol series. The air-dried samples were sputter coated with platinum palladium, and examined with a scanning electron microscope (SEM, Quanta 200; FEI). Immunochemical staining was performed by staining frozen cross-sections of samples fixed in cold acetone with mouse monoclonal anti-αSMA (1:100, Sigma Aldrich), and rabbit monoclonal anti-CD31 primary antibodies (1:100; Abcam), followed by incubation with the appropriate HRP-linked secondary antibody, and visualization with DAB.

NO production, PGI 2 and hydroxyproline content of the tubular scaffold

PGI 2 content was measured by determining the concentration of its stable hydrolytic product. The assay was carried out according to the manufacturer’s instructions (PGI 2 EIA kit, Cayman Chemical, Ann Arbor, MI, USA). The production of PGI 2 was expressed as ng PGI 2 /mg protein.

Nitric oxide (NO) production was quantified using nitric acid reductase as described above, according to the manufacturer’s instructions (Colorimetric Assay for Nitric Oxide, Neogen Corporation). The absorbance of each sample was detected at 540 nm by spectrometry. The hydroxyproline content was determined using a hydroxyproline assay kit (Sigma, MAK008) to oxidize hydroxyproline with DMAB, and measure the product colorimetrically at 560 nm.

Biomechanical assessment

The mechanical properties of the bioengineered vessel were examined as previously described13. Briefly, the bioengineered vessels and normal human HSV with an inner diameter of 4 mm were sectioned circumferentially. Circumferential tissue strips measuring 3 × 8 mm were mounted on a customized holder in their relaxed state. A constant elongation rate of 4 mm/min was used along the longitudinal axis of each sample. The ultimate strength and elastic modulus were analyzed according to the specimen strain and stress. Suture retention strength was measured by placing 5–0 polypropylene sutures in the four quadrants of the vessel wall, 1 mm from the vessel edge. A constant elongation force was loaded at a rate of 3 mm/min until the sutures pulled through the vessel edge. Burst pressure was tested using a pressure tester (CST1006, Const, China). The vessel was cannulated proximally, filled with PBS, and occluded distantly with a silk tie. The intraluminal pressure was increased (0.02 MPa/s) until the vessel ruptured. The burst pressure was recorded.

Statistical analysis

Data were presented as means ± standard deviations. SPSS10.0 software was used for statistical analysis. A paired Student’s t-test was used to test the differences between the two groups. Significant differences (p-value < 0.05) are marked with asterisks in the figures.