All starting materials and solvents were obtained from commercial suppliers and used without further purification. L/D-phenylalanine methyl ester hydrochloride, 1,4-benzenedicarbonyl dichloride and diglycol were purchased from Aladdin Chemicals. LPHEG and its enantiomer DPHEG were synthesized according to a previous report (Dou et al., 2019). ¹H NMR (400 M Hz, DMSO-d₆, δ) results are as follows: δ = 3.1 (m, 4H, CH₂), 3.4 (m, 16H, CH₂), 4.2 (q, 2H, OH), 4.7 (q, 2H, CH), 7.3 (m, 10H, Ar H), 7.8 (s, 4H, Ar H), 8.9 (d, 2H, NH) ppm. EI-MS for L(D)PFEG calcd. 636.71; found 637.28 (M + H)⁺.

The D(L)PHEG powder was suspended in deionized water (2 mg/ml). The solution was then heated until a transparent solution was obtained. The hydrogel was formed when the solution was cooled down to 25°C.

PCL (MW 80 000, Sigma–Aldrich, United States ) and D(L)PHEG were dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) to prepare the D(L)PHEG and PCL mixture. PCL was dissolved at a concentration of 12% w/v at different LPHEG or DPHEG concentrations at 1, 2, 3, and 4 mg/ml. Moreover, 12% w/v PCL/HFIP was prepared as a control. An electrospinning platform was used to generate chiral hybrid scaffolds, which consisted of a grounded collector covered by an aluminum foil, a high-voltage power supply and a syringe driven by a syringe pump. Electrospinning was conducted at an applied voltage of 10.0 kV, solution flow rate of 1.0 ml/h, needle tip-to-collector gap distance of 20 cm, and a collection speed of 200 revolutions per minute. Each of the scaffolds was collected on the aluminum foil after 6 h of electrospinning followed by drying in vacuum.

First, the DPHEG and LPHEG powders were suspended in deionized water (2 mg/ml) to form hydrogels. The DPHEG and LPHEG powders were also dissolved in HFIP (2 mg/ml). CD and UV-Vis signals of DPHEG hydrogel, LPHEG hydrogel, DPHEG HFIP solution, and LPHEG HFIP solution were collected using the JACSO J-815 CD spectrometer. The CD spectra of all samples were recorded in the UV region (200–400 nm) with a bandwidth of 0.5 nm.

FTIR spectra of the DPHEG and LPHEG powders and ATR-FTIR spectra of all chiral hybrid scaffolds were obtained using a Bruck EQUINOX55 instrument. The PCL scaffold was used as a control. The KBr disk technique was used for solid-state measurements to collect FTIR spectra from D(L)PHEG powder samples. Samples were scanned in the wavelength range between 4,000 and 600 cm⁻¹ at intervals of 0.4821 cm⁻¹. To obtain ATR-FTIR spectra, chiral hybrid scaffolds were scanned in the wavelength range of 4,000–600 cm⁻¹ at an interval of 1.9285 cm⁻¹.

The hydrophilicity of different chiral hybrid scaffolds and PCL was assessed based on water contact angle measurements using a JC 2000D optical contact angle meter (Zhongchen, Shanghai). Results are indicative of the surface wettability of the scaffold. The PCL scaffold was used as a control. The water droplet (volume: 2.0 μl) was placed on the surfaces of different scaffolds for 1 and 10 s, and the shapes of the water droplet were recorded by a camera. The static water contact angles of the different chiral hybrid scaffolds were also recorded. For each scaffold, the measurements were conducted three times at room temperature (25°C) and the results were averaged (n = 3).

SEM measurements were performed using an FEI QUANTA 250 microscope. LPHEG and DPHEG samples were prepared by depositing dilute solutions of hydrogel on silicon wafers and by drying them in an oven at 40°C overnight. The chiral hybrid scaffolds and PCL scaffolds were measured directly. The operating voltage was 10 kV.

The mechanical properties of the chiral hybrid scaffolds and PCL scaffolds were characterized by tensile tests performed on a universal tester (H5 K-S, Hounsfield, United Kingdom). The samples used for tensile testing were cut into 1 cm wide and 5 cm long stripes, and the thickness of each sample was measured using an electronic digital micrometer. The samples were stretched at a constant tensile rate of 90 mm/min. The stress–strain curves of all scaffolds were plotted. Young’s modulus, fracture strain, and ultimate tensile stress were analyzed statistically (n = 5).

Primary HUVECs were obtained from Shanghai Jiao Tong University in Shanghai. They were cultured in DEME/F12 (HyClone, United States ) and supplemented with 5% (v/v) fetal bovine serum and 1% (v/v) antibiotics at 37°C in an incubator with 5% CO₂ in a humidified atmosphere. HUVECs were trypsinized for cell seeding. The 24-well plates were coated with DPHEG and LPHEG (100 μl). The plates and scaffolds were pre-sterilized by exposure to ultraviolet (UV) light, with the plates exposed for 40 min.

Cell proliferation was evaluated based on the cell counting kit-8 assay (CCK-8, Beyotime, Shanghai, China). HUVEC suspensions (1 × 10⁵ cells per well) were seeded on 96-well plates, which contained chiral hybrid scaffolds and PCL scaffolds. CCK-8 reagent was added according to the recommended measurements after 1–5 days of incubation. After incubation for 2 h, a microplate reader (Thermo Fisher, United States ) was used to measure the optical density at 450 nm (630 nm was used as a reference). The results were analyzed statistically (n = 3).

HUVECs (1 × 10⁵ cells per well) were seeded on 24-well plates coated with DPHEG hydrogel and LPHEG hydrogel cultured in an incubator for 2 days. In addition, 24-well plates containing chiral hybrid scaffolds and PCL scaffolds at the same cell number were cultured for 5 days. Subsequently, according to the recommended measurements, each well was stained using a Calcein/PI Live/Dead Viability/Cytotoxicity Assay Kit (Beyotime, Shanghai, China) for 30 min. A fluorescence microscope (LEICA, Germany) was used to acquire images of the DPHEG hydrogel and LPHEG hydrogel for 2 days. Chiral hybrid scaffolds were observed using a confocal laser scanning microscope (TCS SP5 II, Leica, Germany) on days 1 and 5.

HUVECs (5 × 10⁴ cells per well) were seeded in 48-well plates with chiral hybrid and PCL scaffolds cultured for 1 day. The cytoskeletal organization and cell nuclei of HUVECs on the different scaffolds after 1 day of culture were analyzed by fluorescent staining. Samples for fluorescent staining were washed three times with phosphate buffer solution (PBS). HUVECs were then fixed with 4% (v/v) paraformaldehyde solution for 30 min. Subsequently, according to the recommended measurements, each well was stained with FITC–phalloidin (Servicebio, Wuhan, China) (that stains the actin cytoskeleton) for 120 min at room temperature, and then incubated with 4,6-diamidino-2-phenylindole (DAPI) (Servicebio, Wuhan, China) (that stains live nuclei) at 25°C for 10 min. The stained samples were washed and visualized using a confocal laser-scanning microscope. Image J was used to calculate the number of cells.

The RNeasy Mini Kit (Qiagen GmbH, Germany) was used to extract ribonucleic acid from HUVECs cultured on the D/PCL-2 and PCL scaffolds according to the manufacturer’s specifications. A blank control (tissue culture plastic) was used. Reverse transcription was achieved using the PrimeScript™ RT reagent synthesis kit with gDNA Eraser (Perfect Real Time) (Takara, Japan) by MiniOptiCon real time PCR detection system (BioRad, United States). All operations were performed according to the manufacturer’s instructions. The 2× SYBR Green qPCR Master Mix (Low Rox) (Quanta Biosciences, United States) and ABI 7300 real-time PCR system (Thermo Fisher, United States) were used for RT-qPCR. The primer sequences used are listed in Supplementary Table S1. Glyceraldehyde-3-phosphate dehydrogenase served as an internal control. All fold changes in gene expression were normalized to those in the control group.

All quantitative data are expressed as mean ± standard deviation. Statistical analysis was conducted using one-way analysis of variance (ANOVA) and Student’s t-test with p < 0.05 accepted as statistically significant.

DPHEG and LPHEG molecules can self-assemble in helical nanofibers in water. The chirality of the hydrogels was characterized by CD and SEM. The SEM images demonstrated that DPHEG and LPHEG self-assembled to form right-handed and left-handed helical structures (Figures 2A,B). The CD/UV-Visible (Vis) absorption spectra of the chiral molecules were quantified (Figures 2C–F). The CD signals of the LPHEG and DPHEG hydrogels in H₂O showed Cotton effects at 226 and 265 nm (Figures 2C,D), which further confirmed the chirality of DPHEG and LPHEG. In the HFIP solution, DPHEG, and LPHEG molecules also can self-assemble into helical nanofibers. The CD spectra of the DPHEG and LPHEG enantiomers exhibited significant Cotton effects at 217 nm (Figures 2E,F). Furthermore, the shape of the CD spectra changed drastically from one solvent to another, but they all showed chirality.

To explore the influence of the chiral hydrogel on cell spreading and proliferation, HUVECs were cultured on presterilized 24-well plates coated with DPHEG and LPHEG hydrogels. After incubation for 2 days, we found that the number of HUVECs increased (Figures 2G,H; Supplementary Figure S2). The cell densities on the DPHEG and LPHEG hydrogels were (3.29 ± 0.29) × 10⁴/cm² and (1.24 ± 0.23) × 10⁴/cm², respectively, (Figure 2I). The difference in cell density between the two different chiral hydrogels showed that the DPHEG hydrogel could significantly improve cell proliferation, compared with the LPHEG hydrogel.

By blend electrospinning, the resulting chiral hybrid scaffolds at the concentrations of D(L)PHEG (1, 2, 3, and 4 mg/ml) were named as D/PCL-1, D/PCL-2, D/PCL-3, and D/PCL-4 scaffolds, and L/PCL-1, L/PCL-2, L/PCL-3, and L/PCL-4 scaffolds, respectively (Supplementary Figure S3). The PCL scaffold was used as a control. The structure of D(L)PHEG was examined by FTIR spectroscopy (Figure 3A). The D(L)PHEG characteristic absorptions appeared at 1,640 and 1,541 cm⁻¹, which can be attributed to the amide I and II bands (Figure 3A). The structures of the resulting scaffolds were characterized by ATR-FTIR. The PCL scaffold was used as the control. The ATR-FTIR characteristic absorption of the PCL scaffold appeared at 1,723 cm⁻¹ (Figure 3B) owing to the stretching of the C = O bond in the ester groups that were abundant in the structure of PCL, which also appeared in the spectra of the D/PCL and L/PCL scaffolds. Meanwhile, the ATR-FTIR spectra of the D/PCL and L/PCL scaffolds showed characteristic absorptions at 1,635, 1,540, and 1723 cm⁻¹ (Figure 3B), thus revealing the successful integration of D(L)PHEG and PCL. The morphologies of the D/PCL and L/PCL scaffolds were characterized by SEM. We observed similar nanofibrous architectures in all chiral hybrid scaffolds. All scaffolds yielded disordered nanofibers (Figures 3C–E; Supplementary Figure S4).

Hydrophilicity tests were conducted to determine changes in the wettability of the scaffolds (Figures 4A,B). The PCL scaffold was hydrophobic with a water contact angle of 123.7 ± 3.6 (Figure 4A). In comparison, the hydrophilicities of the D(L)/PCL scaffolds improved because of the addition of LPHEG and DPHEG. After 1 s, the water contact angles of the D/PCL-1, D/PCL-2, D/PCL-3, D/PCL-4, L/PCL-1, L/PCL-2, L/PCL-3, and L/PCL-4 scaffolds decreased slightly to 120.6 ± 2.3, 71.0 ± 3.8, 70.4 ± 3.5, 77.8 ± 2.0, 95.4 ± 8.4, 86.1 ± 10.2, 63.9 ± 3.0, and 78.9 ± 6.2°, respectively (Figure 4A). After 10 s, the contact angles of all the scaffolds (except D/PCL-1 with water contact angle of 85.9 ± 7.6°)decreased to 0°. Finally, the water contact angles of the D/PCL and L/PCL scaffolds were 0° (Supplementary Figure S5). The water contact angles of the D(L)/PCL scaffolds were decreased, which also suggests the successful integration of D(L)PHEG and PCL.

The mechanical properties of tissue-engineered vessels are important for maintaining their structural and functional integrity. The mechanical properties, including Young’s modulus, ultimate tensile stress, and fracture strain of different chiral hybrid scaffolds were evaluated by tensile tests. All scaffolds displayed similar mechanical characteristics (Figure 5). PCL scaffolds yielded a Young’s modulus of 37.17 ± 3.74 MPa (Figure 5B), fracture stain of 599.45 ± 38.32% (Figure 5C) and ultimate tensile stress of 6.53 ± 0.53 MPa (Figure 5D). Compared with the PCL scaffold, the chiral hybrid scaffolds L/PCL-1, L/PCL-2, L/PCL-3, L/PCL-4, D/PCL-1, D/PCL-2, D/PCL-3, and D/PCL-4 scaffolds exhibited similar Young’s modulus, fracture strain, and ultimate tensile stress outcomes (Figures 5B–D).

We found that the cell density of the DPHEG hydrogel was higher than that of the LPHEG hydrogel. Chirality is an important influencing factor for the endothelial remodeling of TEVGs (Figure 2I). To further explore the influence of chiral hybrid scaffolds with chiral molecules on endothelium remodeling, HUVECs were incubated on PCL, D/PCL-1, D/PCL-2, D/PCL-3, D/PCL-4, L/PCL-1, L/PCL-2, L/PCL-3, and L/PCL-4 scaffolds. The adhesion and proliferation of HUVECs were tested using the CCK-8 assay and fluorescence staining. As depicted in Figure 6, HUVECs could adhere and proliferate on PCL, D/PCL-1, D/PCL-2, D/PCL-3, D/PCL-4, and L/PCL-1 scaffolds. The number of HUVECs increased slightly after successive 24-h periods (Figure 6). Therefore, on D/PCL scaffolds, the number of cells was relatively higher than those on L/PCL and PCL scaffolds. The number of cells on D/PCL-2 was greater than those on the other scaffolds. When the concentration of DPHEG was >2 mg/ml, compared to that of D/PCL scaffolds, the number of cells decreased as the DPHEG level increased. However, for L/PCL scaffolds, cells adhered to and proliferated only on L/PCL-1 scaffolds.

Subsequently, a similar finding was observed; the cell density on D/PCL scaffolds was higher than that on L/PCL scaffolds based on fluorescence staining (Figures 7A,B). After 1 day of culture, the average number of cells on the PCL, D/PCL-1, D/PCL-2, D/PCL-3, D/PCL-4, and L/PCL-1 scaffolds were (3.56 ± 1.28) × 10³/cm², (1.27 ± 0.03) × 10⁴/cm², (1.95 ± 0.16) × 10⁴/cm², (1.71 ± 0.19) × 10⁴/cm², (1.07 ± 0.10) × 10⁴/cm², and (1.64 ± 0.08) × 10⁴/cm², respectively, (Figure 7C). The D/PCL-2 scaffold possessed the highest cell density. The cell density of the PCL scaffold was the lowest. Compared with the findings on day 1, the number of cells increased slightly after day 5. The cell densities of the PCL, D/PCL-1, D/PCL-2, D/PCL-3, D/PCL-4, and L/PCL-1 scaffolds were (7.75 ± 0.90) × 10³/cm², (3.58 ± 0.45) × 10⁴/cm², (5.74 ± 0.24) × 10⁴/cm², (5.20 ± 0.38) × 10⁴/cm², (2.59 ± 0.21) × 10⁴/cm² and (2.97 ± 0.23) × 10⁴/cm², respectively, (Figure 7D). Similarly, the D/PCL-2 scaffold possessed the highest cell density. The fluorescence staining images demonstrated a higher HUVEC density when the cells were grown on D/PCL scaffolds compared with those on the L/PCL or PCL scaffolds. Among them, the D/PCL-2 scaffold yielded the highest cell density.

After 1 day of culture, the cells on PCL, D/PCL-1, D/PCL-2, D/PCL-3, D/PCL-4, and L/PCL-1 scaffolds appeared polygonal in shape and attached well, as documented by confocal laser microscopy (Figures 8A–F). The average cell spreading areas per cell were 524.76 ± 155.48, 526.84 ± 129.24, 855.67 ± 99.23, 737.94 ± 104.92, 665.43 ± 155.62, and 599.89 ± 148.23 μm² (Figure 8G). The cells on the D/PCL-2 scaffolds had the largest average spreading area.

The aforementioned results indicate that chirality was an important influencing factor for endothelial remodeling, and HUVECs had the highest cell viability in the D/PCL-2 scaffold. Therefore, the D/PCL-2 scaffold was selected for the TEVGs. To detect how the D/PCL-2 scaffold influences the process of endothelial remodeling, HUVECs were seeded on a D/PCL-2 scaffold to detect relative protein expression levels using RT-qPCR. Fibronectin-1 (FN-1) and vinculin are two well-known proteins that promote cell adhesion (Bays and DeMali, 2017; Sun et al., 2020; Yang et al., 2020). FN-1 and vinculin were upregulated in the D/PCL-2 scaffold (Figure 8H), which suggests that the D/PCL-2 scaffold promoted cell adhesion and proliferation by enhancing the two proteins. In addition, we measured Bcl-2 and caspase-3 and found that the two proteins did not increase (Supplementary Figure S6).