Multifunctional hybrid poly(ester-urethane)urea/resveratrol electrospun nanofibers for a potential vascularizing matrix

Abstract

The challenges for clinical application of small-diameter vascular graft are mainly acute/chronic thrombosis, inadequate endothelialization, intimal hyperplasia caused by inflammation, oxidative stress, and the mismatch of mechanical compliance after transplantation. How to construct an effective regenerative microenvironment through a material with uniform dispersion of active components is the premise of maintaining patency of a vascular graft. In this study, we have compounded poly(ester-urethane)urea (PEUU) with various optimized concentrations of resveratrol (Res) by homogeneous emulsion blending, followed by electrospinning into the hybrid PEUU/Res nanofibers (P/R-0, P/R-0.5, P/R-1.0, and P/R-1.5). Then the microstructure, surface wettability, mechanical properties, degradation, Res sustained release properties, hemocompatibility, and cytocompatibility of P/R were evaluated comprehensively. The results indicate that Res can be gradually released from the P/R, and both the hydrophilicity and antioxidant ability of the nanofiber gradually increase with the increase of Res content. Moreover, with the increase of Res, the viability and proliferation behavior of HUVECs were significantly improved. Meanwhile, tube formation and migration experiments showed that Res promoted the formation of a neovascularization network. In brief, it is concluded that P/R-1.0 is the optimal candidate with a uniform microstructure, moderate wettability, optimized mechanical properties, reliable hemocompatibility and cytocompatibility, and strongest ability to promote endothelial growth for the vascularizing matrix.

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1. Introduction

Cardiovascular disease (CVD) remains a threat to human life and health and is one of the leading causes of death, accounting for 30% of the population.^1–3 For vessels that lose normal function, the most effective treatment currently focuses on bypass transplantation. Autologous vascular transplantation is regarded as the gold standard of bypass transplantation, but it cannot meet the needs of most surgeries due to factors such as lack of donors, trauma, anatomical abnormalities, and size mismatch.^4–6 Therefore, the use of artificial blood vessels as an alternative to autologous blood vessels for tissue repair has been widely studied.⁷

The existing large-diameter artificial blood vessels, including polytetrafluoroethylene (ePTFE), poly(ethylene-terephthalate) (PET), and other materials of artificial blood vessels, have achieved unprecedented success in clinical application.^8–10 However, due to both the slower blood flow rate and mismatch in the mechanical properties of small-diameter artificial blood vessels (<6 mm), early thrombosis, lumen stenosis, and intimal hyperplasia occurred frequently after vascular transplantation.^11–13 For this, there have been many studies in this aspect, such as pre-inoculation of cells and growth factors in the inner layer of small-diameter vascular grafts, or modification of the lumen surface with anticoagulant drugs. But these methods still need to be improved due to the complex production process, insufficient stability, and poor continuity.^14–16 Therefore, after decades of research, scientists have not found a suitable biomaterial to replace the matrix of small blood vessels.

Previous studies have shown that the rapid formation of complete endothelial cell layers after transplantation is still the primary consideration in the design of small-diameter artificial blood vessels.^17–20 The extracellular matrix plays a decisive role in cell induction and thus in vascular endothelial remodeling in vivo. Therefore, artificial blood vessel materials should have the same properties as a natural extracellular matrix, including mechanical properties, blood compatibility, topographical cues, biological signal transduction, etc., to guide vascular endothelial formation.^21–23

Resveratrol (Res) is a small molecule of natural polyphenols extracted from the traditional Chinese medicinal plant polygonum cuspidatum.²⁴ Its vascularization, anti-inflammatory, and antioxidant properties have been extensively studied.²⁵ Many studies have shown that Res plays a positive role in the growth of vascular endothelial cells and the generation of vascular networks at appropriate concentrations.²⁶ However, due to the low water solubility of Res (<1 mg mL⁻¹), and only 2% oral bioavailability of Res, the low bioaccumulation of Res in the cardiovascular system (such as in heart and aortic tissues) is another problem.²⁷ The concentration of Res achieved in the target organ is significantly lower than the effective dose applied in cell and animal studies.²⁸ Before reaching target cells, Res may degrade rapidly in the gastrointestinal tract, be over-metabolized by intestinal flora, or be over-consumed by other non-target cells, so a more efficient delivery system in vivo is needed.^29,30

Electrospinning is currently a relatively mature and simple method for preparing nanostructured materials. The nanofibers made by electrospinning have a three-dimensional porous structure similar to that of a natural tissue extracellular matrix, which is suitable for cell adhesion and growth, and can also encapsulate different types of drug molecules, as well as have been widely used in the preparation of tissue engineering scaffolds.³¹

Poly(ester-urethane)urea (PEUU) has biomechanical properties matching those of vascularized tissues, excellent processing properties, and stable biocompatibility, and has been increasingly used for tissue regeneration.^32,33 In this work, a double solvent method was introduced to prepare PEUU and Res co-blended electrospun solutions with different proportions for electrospinning nanofibers to overcome the limitation of uneven dispersion of Res in vascular material. With this, Res molecules could be efficiently combined into the nanofibers to achieve continuous drug delivery in targeted organs hence solving the low bioavailability of Res. The nanofibers were extensively characterized and the effect of nanofibers with different Res contents on the cell behavior of vascular endothelial cells was studied in detail.

2. Materials and methods

2.1 Materials

The materials used in the experiments are shown in the ESI† in Section S1.1.

2.2 Synthesis of the PEUU elastomer and preparation of hybrid P/R fibers

We prepared P/R nanofibers by using HFIP and DMF to dissolve PEUU and Res, respectively. The synthesis of PEUU elastomer and the preparation of P/R hybrid nanofibers were carried out according to the ESI† in Section S1.2.

2.3 Characterization and testing

The detailed tests and parameters for micromorphology, infrared spectroscopy, hydrophilicity, porosity and water absorption can be found in the ESI† in Section S1.3.

2.4 Degradation and Res release in vitro

The ethanol solution of Res was prepared by removing 5 mL of anhydrous ethanol in the centrifuge tube and adding a trace amount of Res. Anhydrous ethanol was used as the control group. The UV spectrum of Res was scanned by a UV-VIS spectrophotometer to determine the maximum UV absorption wavelength of Res.

The standard concentration curve of Res was drawn: a Res solution of 100 μg mL⁻¹ was prepared by accurately weighing 0.01 g Res powder and dissolving it in 10 mL ethanol, and then diluting the volume to 100 mL in a 100 mL volumetric bottle. After that, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, and 0.8 mL solutions were accurately removed using a pipette through the volume bottle to 5 mL Res solutions of 2, 4, 6, 8, 10, 12, 14, and 16 μg mL⁻¹ were prepared. The absorbance of Res at different concentrations was tested by a photometer, the standard concentration curve of Res was drawn and the standard concentration formula was calculated by Origin software.

The dry weight of four groups of nanofibers was recorded, and each group of nanofibers was immersed in 4 mL of protease K solution (2 U mL⁻¹ in PBS). It was then placed in a temperature vibrator at 37 °C (100 rpm). In order to maintain appropriate protease K activity throughout the process, the protease K solution was changed every 72 hours. The nanofibers were removed at the set time node, repeatedly cleaned with deionized (DI) water, frozen overnight in a −80 °C refrigerator, and lyophilized. The weight of the lyophilized nanofibers was recorded and three samples were taken in parallel from each set of nanofibers at each time point for testing. The weight of the nanofibers for four weeks was recorded and the residual mass ratio was calculated using the following eqn (1):

where W₀ and W₁ are the nanofibers’ weight before and after degradation, respectively.

The 10 mg nanofiber sample was weighed and placed into a 15 mL centrifuge tube containing 10 mL of PBS solution. The centrifuge tubes were then incubated in a vibrator at 37 °C. At the designated time point, 4 mL of slow-release solution was withdrawn from each centrifuge tube, and promptly replaced with 4 mL of fresh PBS solution to maintain a total volume of 10 mL. Three parallel samples were included in each P/R group. The absorbance of Res was measured at 315 nm, and the cumulative release (m_c) and cumulative release rate (R_c) of Res were calculated by eqn (2) and (3):

where C_n and C_x represent the concentration of resveratrol in the supernatant removed at n time and x time shifts.where m₀ is the total P/R nanofiber mass, m_c is the cumulative release at the corresponding time point, and p is the mass ratio of resveratrol to PEUU.

2.5 Antioxidant activity test in vitro

The antioxidant activity of P/R nanofibers was evaluated by the free radical scavenging efficiency of 1,1-diphenyl-2-picrohydrazine (DPPH). A purple DPPH solution was first prepared by dissolving 100 μg of DPPH in 50 mL of absolute ethanol. 4 mL DPPH solution was removed from a 10 mL centrifuge tube, and 5 mg nanofiber was added, respectively. DPPH solution without P/R nanofiber was used as a blank control group. It was incubated in a dark environment for 30 min. After incubation, absorbance (OD value) at 517 nm was measured by a UV-VIS spectrophotometer. Finally, the antioxidant efficiency of the P/R nanofibers was calculated using the following eqn (4):where OD_c and OD_h are the absorbance of the blank control group and experimental group respectively.

2.6 Hemocompatibility test

All animal experimental protocols are in accordance with the policy of the Institutional Animal Care and Use Committee (IACUC) of Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine and approved by the IACUC. The ethical principles were followed throughout all experimental procedures. All animal experiments were performed according to the Animal Management Regulations of China (1988 and revised in 2001, Ministry of Science and Technology) and all animals were purchased from Shanghai Slaccas Experimental Animal Co., Ltd (Shanghai, China).

The hemocompatibility of the fibers was analyzed through hemolysis and platelet adhesion tests. Rabbit blood taken from healthy New Zealand white rabbit ear veins was diluted in normal saline at a ratio of 8 : 10 (v/v) for use. P/R nanofibers were cut into 14 mm diameter fiber plates and put into a centrifuge tube containing 10 mL saline. For the positive control group, 10 mL of deionized water was used, and for the negative control group, 10 mL of PBS phosphate buffer was used. All centrifuge tubes were placed in a constant temperature shaker at 37 °C for 30 min, followed by the addition of 0.2 mL of diluted rabbit blood and incubation for 1 h. After incubation, the centrifugal tube in a low-speed centrifuge was centrifuged for 5 min at a speed of 1200 rpm. Finally, the supernatant was removed and the absorbance (OD value) at 545 nm was measured by a UV-visible spectrophotometer. The following eqn (5) was used to calculate the hemolysis rate:

where OD_s, OD_p, and OD_n are the absorbance of the sample, positive control group, and negative control group, respectively.

For the platelet adhesion experiment, platelet-rich plasma (PRP) and platelet-poor plasma (PPP) from fresh rabbit blood were separated by centrifugation at 1200 rpm. The P/R nanofibers were cut into 14 mm round sheets, placed on 24-well plates after alcohol fumigation and ultraviolet sterilization, added to 500 μL PRP, and incubated in a constant temperature shaking table at 37 °C for 2 h. Repeated rinsing with PBS phosphate buffer several times after incubation ensured that the platelets not adhering to the surface of the material were successfully washed away. 4% paraformaldehyde solution was added to fix for 4 h, and finally dehydrated (10%, 30%, 50%, 60%, 75%, 80%, 90%, and 100% after gradient alcohol dehydration treatment, respectively), soaked for 10 min each time, and the platelet adhesion on the surface of the three groups of materials was observed by scanning electron microscopy (SEM, Hitachi SU8010). Then, the area of platelets was measured in ImageJ software. Eqn (6) shows the calculation method of platelets coverage:

where A_p and A_m represent the platelet area and nanofiber area in the SEM image.

2.7 Cytocompatibility test

2.7.1 Cytotoxicity test by CCK-8. Cell culture, CCK-8 experiments, cell live and death staining, and laser confocal microscopy can be found in the ESI† in Section S1.4.

2.7.2 Transwell migration experiment. HUVECs were seeded in serum-free DMEM of transwell inserts with a density of 2.0 × 10⁴ cells per well, where the bottom chamber contained intact DMEM and different P/R nanofibrous membranes. The cell migration process was carried out at 37 °C and 5% CO₂ for 24 h. Cells that migrated to the bottom of transwell were immobilized with 4% paraformaldehyde solution for 30 min. Add 0.1% crystal violet and dye for 15 min.

2.7.3 HUVECs’ horizontal migration experiment. Experiments were accomplished according to previous experimental protocol.³⁴ For the ability test of HUVECs’ horizontal migration, each well of the 24-well plate was laminated with special stainless steel strips that divided the well into two sections, before the HUVECs were seeded into the 24-well plate with a density of 1.0 × 10⁵ cells per well. HUVECs, which have already been seeded on the surface of nanofibers for 12 h, may almost completely adhere to the surface of the sample. Afterwards, the laminated steel strips were carefully taken out and the cultivation was continued for another 48 h. After 48 h, 4% of paraformaldehyde was added to fix the cells in the well plate for more than 4 h. Then the samples in the well plate were washed with PBS over 3 times, followed by permeabilizing with 0.1% Triton X-100 for 20 min. With that, rhodamine-labeled phalloidin and DAPI staining solution were added to the well plate for 15 min in sequence. Finally, the creeping-in distances of HUVECs on the surface of nanofiber mats were observed and counted through inverted fluorescence microscope images using ImageJ software. We divided the cell migration distance into two length intervals: 0–250 μm and 250–500 μm. Within each designated time interval, three randomly chosen fields of view were examined, and the cellular count within each was meticulously quantified.

2.7.4 HUVECs’ tube formation experiment. To investigate the role of Res in the stimulation of angiogenesis, we conducted in vitro tube formation assays. In short, after thawing Matrigel at 4 °C overnight, each well of the 96-well plate was added with 50 μL and cultured at 37 °C for 60 min. P/R nanofibers sterilized 72 h in advance. According to the standard GBT16886.12-2017, the material was soaked at 37 °C for 72 h according to the ratio of surface area to medium of 1.25 cm² mL⁻¹ to obtain the extract. The cell suspension was adjusted to a concentration of 1.5 × 10⁴ cells per milliliter for each well, followed by incubation at 37 °C in an atmosphere containing 5% CO₂ for a period of 12 hours. Subsequently, the formation of tubular structures was examined under an optical microscope. The ImageJ software's angiogenesis analysis plug-in was used to detect the grid, calculate the total network length, the branching points, the number of meshes, the master segments, the master junctions, and the total length of the master segments.

2.8 Statistical analysis

Statistical analysis of the data was performed utilizing IBM SPSS statistics software. The results were articulated as the mean value accompanied by the standard deviation (SD) for each set of data. One-way ANOVA was performed on the results using Microsoft Office Excel. The obtained P-values were considered to be statistically significant. ns: not significant, *p < 0.05 statistical difference; **p < 0.01 significant statistical difference; ***p < 0.001 very significant statistical difference.

3. Results and discussion

The PEUU/HFIP solution was mixed with Res/DMF solution to obtain the solution and prepare the nanofibers by electrospinning. The electrospinning process in this method was stable and controllable, and the nanofibers P/R-0, P/R-0.5, P/R-1.0, and P/R-1.5 were prepared by this method. Electrospinning is a facile nanofiber fabricating method driven by electricity. Given a positive charge by an applied electric field, the polymer is stretched into ultra-fine fibers. With the volatilization of solvent, the tissue engineering scaffold obtained by electrospinning has the characteristics of large surface area, high porosity, and adjustable mechanical properties, which can simulate the three-dimensional mechanical environment of the extracellular matrix to a large extent. It is beneficial to cell adhesion, invasion, and growth, and promotes tissue regeneration.³⁵ Electrospinning has become one of the most common methods to manufacture artificial blood vessels.^36–38

Polyurethane elastomers are suitable as scaffolds for implantation in vivo because of their adjustable mechanical properties and excellent biocompatibility.³⁹ But certain limitations are also associated such as low cell adhesion, lack of anticoagulation and antiplatelet deposition ability, and easy thrombosis in the body.³³ Therefore, the rapid formation of endodermis after implantation has become the focus of current research.⁴⁰

3.1 Chemical properties, microstructure and surface wettability of nanofibers

PEUU was successfully synthesized by the two-step method (Fig. 1A). The synthesis of PEUU was accomplished through the utilization of PCL diol as the flexible chain segment, HDI as the rigid chain segment, and putrescine serving as the agent for chain extension. To observe the morphological characteristics of the fibrous scaffold surface, SEM observation showed that the fibers of the four groups of nanofibers were uniformly distributed and arranged in a random cross-network structure, and no similar drug recrystallization was found, which proved that the composite spinning of the two solutions was feasible (Fig. 1B). Electrospinning nanofiber diameter is affected by many processing parameters, including applied voltage, flow rate, concentration of spinning solution, conductivity, and distance from syringe to collector. The process parameters of electrospinning are also mentioned in the ESI.† They are all carried out under relatively the same conditions, and the obtained nanofiber membrane is directly used after waiting for the solvent to completely volatilize, without any post-treatment process. The clipping carried out in the course of various experiments will not affect the overall loading rate of Res, so the loading rate of Res is 100% in theory. Therefore, we believe that the load rate of resveratrol and the consistency of resveratrol load between different samples can be guaranteed. According to the SEM images, the average diameters of P/R-0, P/R-0.5, P/R-1.0, and P/R-1.5 nanofibers were 339.48 ± 93.01 nm, 395.25 ± 102.25 nm, 420.98 ± 110.83 nm, and 465.51 ± 99.90 nm, respectively (Fig. 1C). The nanofiber diameter increased slightly with the increase in Res molecule concentration. It has been noted that higher drug concentrations have led to an increase in viscosity resistance in the mixed solution resulting in an inability to maintain flow at the tip of the Taylor cone.⁴¹ In turn, large-diameter fibers were produced. The possible reason is that with the increase in the mass of Res, the viscosity of the solution system slightly rises due to the increase in the solid content. Under the same electrospinning process conditions, the fiber is difficult to be fully stretched under the condition of high viscosity, so the fiber diameter increases.

Fig. 1 Schematic illustration of the synthesis of the PEUU elastomer and the representative physicochemical characterization of the prepared nanofibers. (A) Synthesis method of PEUU; (B) SEM image of P/R-0, P/R-0.5, P/R-1.0, and P/R-1.5 nanofibers, respectively; (C) fiber diameter statistics; (D) FTIR spectra of P/R-0, P/R-0.5, P/R-1.0, P/R-1.5 nanofibers; (E) water contact angle; (F) porosity; (G) water absorption. (ns indicates not statistically significant; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; n = 3.)

The incorporation of Res into the nanofiber was determined by FTIR analysis (Fig. 1D). The FTIR spectra of P/R nanofibers showed that the wide absorption peaks at 3500–3200 cm⁻¹, 1680 cm⁻¹, and 1610 cm⁻¹ corresponded to O–H stretching of Res, –C=C– stretching, and –C=C– stretching (aromatic), respectively. Moreover, an obvious absorption peak of the carbamate bond was observed at 1800–1700 cm⁻¹, and a –N–H absorption peak appeared at 3300 cm⁻¹ in the spectrum of the P/R-0 nanofiber, which proved the successful synthesis of PEUU.

The general rule is that the hydrophilic scaffold material is more conducive to cell adhesion, and the hydrophilic surface will make the cell form more stretched, thus healthier and faster growing.^42,43 The measurement of dynamic water contact angle showed that the hydrophilicity of nanofibers changed to hydrophobicity with more Res added (Fig. 1E). Despite Res being classified as a hydrophobic substance, pertinent research has indicated that in scenarios where the hydrophilicity of the material remains relatively consistent, a decrease in the material's surface roughness correlates with a reduction in its contact angle.^44,45 As mentioned above, with the increase in Res load, the fiber diameter increases, the grooves between the fibers become shallow and the roughness decreases, so the contact angle of the nanofibers decreases.

Porosity is also an important consideration for the effect of nanofiber scaffolds on tissue regeneration, because appropriate space is necessary for the invasive growth and proliferation of cells. In general, porosity should not be too low for invasive cell growth.⁴⁶ Utilizing the solvent displacement method to ascertain porosity (Fig. 1F), it was observed that as the loading of Res incrementally increased, there was a corresponding decrease in the porosity of the nanofibers. The values of P/R-0, P/R-0.5, P/R-1.0, and P/R-1.5 are 85.30 ± 1.98%, 79.96 ± 3.60%, 74.55 ± 0.78%, and 67.96 ± 5.26%, respectively, which are also consistent with the results shown by SEM images and fiber diameter statistics above. The damaged part of the vascularized tissue is often infiltrated by a large amount of blood and exudate, which is not conducive to the long term repair of the tissue.⁴⁷ It can be seen from the water contact angle that although the four groups of nanofibers are not hydrophilic when the nanofibers are fully infiltrated by water, the water absorption rates of P/R-0, P/R-0.5, P/R-1.0, and P/R-1.5 could still reach 132.09 ± 10.38%, 110.52 ± 9.87%, 105.83 ± 10.86%, and 90.43 ± 10.03%, respectively (Fig. 1G). These results indicate that the P/R nanofiber can absorb the exudate of the damaged tissue and create a suitable environment for tissue regeneration.

3.2 Mechanical properties

The mechanical properties of tissue engineering scaffolds are one of the important guarantees to ensure that fiber scaffolds can have their expected effects in the body. Without an appropriate mechanical support, scaffolds may collapse, tear, and degrade in the body, thus losing their biological role, leading to repair failure, and even triggering the body's inflammatory response and rejection reaction.^48–51Fig. 2A shows the testing scheme of the mechanical properties of P/R nanofiber. After the fixtures equipped with mechanical terminals fix the two ends of the sample, they stretch in the opposite direction at a constant speed until they break. The optimum mechanical property of polyurethane elastomer is one of the reasons for choosing as scaffolds implanted materials. Studies report that rabbit carotid artery of the initial modulus range in 2.8–3.0 MPa.⁵²Fig. 2B shows the representative tensile stress–strain curve of P/R-0, P/R-0.5, P/R-1.0, and P/R-1.5. As the concentration of Res mixed with PEUU increased, Res small molecules produced a certain hydrogen bond crosslinking network after mixing with PEUU, and its structure contained two benzene rings with a certain rigidity. Therefore, the relative displacement of the elongation at break of the scaffold is somewhat constrained due to the enhanced intermolecular connectivity facilitated by hydrogen bonding (Fig. 2D), resulting in an observed increment in both initial modulus and tensile strength (Fig. 2E and F).

Fig. 2 Mechanical properties of the prepared P/R-0, P/R-0.5, P/R-1.0, P/R-1.5 nanofibers, respectively. (A) Schematic of the initial shape and fractured shape of the fiber strips; (B) and (C) are representative stress–strain curves of the dry state and wet state, respectively; (D) elongation at break; (E) tensile strength, (F) initial modulus and wet state, (G) elongation at break, (H) tensile strength, and (I) initial modulus (ns indicates not statistically significant; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; n = 5).

In addition, to simulate the environment in which the nanofibers fully infiltrated in the body, the nanofibers were soaked in PBS buffer and then taken out for mechanical testing in the wet state. As shown in Fig. 2C, the representative stress–strain curves of the four groups of nanofibers in a wet state are shown. Fig. 2G–I show the corresponding elongation at break, tensile strength, and initial modulus in the wet state, respectively. After water absorption, the expansion volume of the fibers increased, resulting in an increase in the contact area between single fibers and thus an increase in friction. Therefore, the tensile strength and initial modulus of the four groups of nanofibers after water absorption are higher than their dry state, indicating that the P/R nanofiber can still meet the needs of use in the in vivo environment.

3.3 Degradation and resveratrol sustained release properties in vitro

Fig. 3A shows the release diagram of Res from P/R nanofibers in PBS solution. According to UV-visible absorption spectrum analysis, the maximum absorption wavelength of Res is 315 nm (Fig. 3B). Then, the standard concentration curve of Res is drawn at 315 nm (Fig. 3C). The formula of the standard concentration curve obtained by the test is y = 0.12866x − 0.0046. R² = 0.99955, and the linear relationship is highly satisfactory. Fig. 3D and E show the drug release of the three groups of P/R nanofibers loaded with Res. The effect of different Res loading content on its release from the nanofibers is obvious. The cumulative release rates of P/R-0.5, P/R-1.0, and P/R-1.5 in the fourth week were 81.43 ± 3.10%, 55.44 ± 1.83%, and 55.19 ± 2.31%, respectively (Fig. 3F). It can be seen that Res was released in large quantities within the first day, followed by slow release. The cumulative release rates of P/R-0.5, P/R-1.0, and P/R-1.5 in 25 h were 49.76 ± 1.07%, 37.87 ± 1.15%, and 38.26 ± 1.12%, respectively (Fig. 3G). Due to their rapid short-term release, it is assumed that P/R nanofiber can effectively inhibit short-term acute coagulation after implantation. The higher cumulative release rate of Res in P/R-0.5 than in the other two groups may be attributed to the fact that Res is mainly distributed on the fiber surface or in shallow layers and the fiber structure has a larger porosity allowing easier penetration of PBS. The P.R-1.0 and P/R-1.5 nanofibers, on the other hand, tended to have similar release rates and lower overall release rates due to the deep embedding of Res molecules, reduced porosity, and possible molecular interactions. Resveratrol release is closely related to fiber degradation, which in turn is influenced by synergistic effects of hydrophilicity, porosity, matrix material properties and three-dimensional structure. As the resveratrol content increased, the fiber diameter also increased. This may be due to the fact that resveratrol plays a certain plasticizing or viscosity-enhancing role in the spinning process, which makes the fibers more difficult to stretch during the forming process, resulting in the formation of thicker fibers. The increase in fiber diameter usually leads to a decrease in the void space between the fibers, which may be the reason for the decrease in porosity. The tensile strength of P/R nanofibers decreased with the increase in Res content, and the elongation at break increased slowly, which also proved that the increase in Res content may play a plasticizing role for the intermolecular PEUU, and reduce the intermolecular force of PEUU, thus reducing the water resistance and stability of the P/R nanofibers. Secondly, with the increase in resveratrol content, the contact angle of the fiber membrane gradually decreases, indicating that the hydrophilicity of the material surface increases. This may be due to the fact that the hydrophobicity of resveratrol is relatively low compared with that of PEUU, and the doping will reduce the hydrophobicity of the whole fiber membrane, which makes it easier to adsorb water, so it is more likely to be released from the material, and with the release of resveratrol in this process, micropores or cracks may appear on the surface of the material, which will make the material unstable and accelerate the degradation, and further enhance the release of resveratrol. The above reasons may have contributed to the result that the mass residual rate of P/R nanofibers shown in Fig. 3H decreased with the increase of Res addition.

Fig. 3 Characterization of resveratrol release from P/R nanofibers. (A) Schematic representation of the principle of resveratrol release from fibers; P/R nanofiber (B) UV spectrum and (C) standard concentration curve of resveratrol; resveratrol of P/R-0, P/R-0.5, P/R-1.0, and P/R-1.5 nanofibers (D) 4 weeks release and (E) 25 hours release, (F) 4 weeks cumulative release rate, and (G) 25 hours cumulative release rate; (H) 4 weeks mass residual rate.

3.4 Antioxidation and hemocompatibility in vitro

Overproduction of reactive oxygen species (ROS) is the main cause of intimal hyperplasia and eventually vascular restenosis.^53,54 Therefore, it is necessary to evaluate the oxidation resistance of the materials. As a phenolic compound, Res has been widely studied and proved to have antioxidant effects such as scavenging or inhibiting the generation of free radicals, inhibiting lipid peroxidation,⁵⁵ and regulating the activity of oxidation-related enzymes.⁵⁶

DPPH is a stable free radical with unpaired valence electrons on one atom of the nitrogen bridge.⁵⁷ DPPH free radical scavenging is a common method for determining antioxidant activity. The antioxidant activity of four groups of nanofibers were evaluated by the DPPH clearance test. As shown in Fig. 4A, it was found that the antioxidant capacity of the nanofibers increased with the addition of Res, and the purple color of DPPH could hardly be observed at P/R-1.0 and P/R-1.5. The clearance rates of P/R-0 and P/R-0.5 were 43.98 ± 3.61% and 55.51 ± 2.87%, respectively, which showed obvious antioxidant activity increased from 5.58 ± 0.58% to 37.90 ± 1.95% (Fig. 4D).

Fig. 4 Antioxidant properties and blood compatibility of P/R nanofibrous scaffolds. (A) Digital imagery was obtained, depicting the interaction of DPPH with four distinct sample sets as well as with PBS; (B) digital imaging captured the supernatant and solution from centrifuged rabbit carotid blood, which had been treated with four distinct sample sets, deionized water, and PBS, with designated positive (+) and negative (−) controls; (C) SEM images of platelet adhesion of the four groups of samples (platelets were treated with red pseudo-color); (D) DPPH scavenging activity quantified by absorbance at 405 nm; (E) hemolysis rate; (F) quantitative analysis of platelet adhesion rate based on SEM images (ns indicates not statistically significant; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; n = 3).

In addition, as a blood contact material, it should not cause symptoms such as hemolysis, organ ischemia or hypoxia, nor should it activate platelets to make them gather in large numbers to form thrombosis, so the evaluation of blood compatibility is crucial.⁵⁸ We performed a series of blood compatibility assessments on four groups of P/R. Fig. 4B shows the hemolysis test digital photograph of the material and the SEM image of platelets attached to the P/R nanofibers as shown in Fig. 4C. The hemolysis rates of P/R in the four groups were 0.36 ± 0.24% for P/R-0, 0.68 ± 0.24% for P/R-0.5, 1.35 ± 0.18% for P/R-1.0 and 1.72 ± 0.16% for P/R-1.5, respectively. By comparing with the ISO10993-4 standard, observations indicate that the hemolytic percentage associated with the P/R in each of the four groups significantly falls below the 5% threshold as per the established criterion (Fig. 4E). The platelet coverage on the nanofiber surface of each group is quantitatively analyzed in Fig. 4F. Analysis reveals that the platelet adhesion rate of P/R-1.0 was 0.25 ± 0.09%, which was far less than the rates of 1.88 ± 0.43%, 2.08 ± 0.37%, and 2.32 ± 0.13% for P/R-0, P/R-0.5 and P/R-1.5. Compared with the other three groups of nanofibers, P/R-1.0 has a good antithrombin production effect, greatly reducing the adhesion and activation of platelets, and has obvious anticoagulant potential. A large number of studies have shown that the inhibition of platelet adhesion becomes stronger with the increase in resveratrol concentration.^59,60 However, the results of this study are ambiguous, and P/R-1.5 does not seem to show the inhibition of platelet adhesion. The possible reason is that during the platelet adhesion experiment, the co-culture time of platelets and P/R nanofibers was 2 h. Combined with the release results of Res in Fig. 3E, it can be seen that the release amounts of P/R-1.0 and P/R-1.5 Res in the first two hours were not very different and both were greater than P/R-0.5. Compared with P/R-1.5, the nanofiber diameter of P/R-1.0 nanofibers is smaller, and the P/R-1.0 nanofiber membrane has larger porosity, so it has a larger specific surface area. Under short-term conditions, more Res molecules will migrate from the pores to the surface of the nanofiber membrane and act on platelets, resulting in a local concentration of more than P/R-1.5 on the surface of the nanofiber membrane, so P/R-1.0 showed a better ability to inhibit platelet activation and adhesion.

3.5 Cytocompatibility

As a temporary substitute for damaged tissues, the nanofibers’ mechanical properties are pre-requisite. Apart from this, they are also very important for the adhesion and growth of cells. As a vascular graft, firstly, they do not have obvious cytotoxicity toward cells. Secondly, if the cells can be well attached to the surface of the graft for proliferation and growth, it plays a crucial role in the rapid in situ repair of damaged tissues.^61–63

Studies have shown that Res has a biphasic effect on angiogenesis, and plays an active role in promoting angiogenesis at lower concentrations, while excessive Res accumulation causes damage to vascular cells.²⁶ HUVECs were co-cultured with the nanofibers to evaluate its cytocompatibility. As shown in Fig. 5A, after co-culture with a P/R nanofiber membrane for 5 days, the cell viability of HUVECs was evaluated by live/dead staining. It is evident from the data that the proliferation effect of cells on the nanofiber membrane P/R-1.0 and P/R-1.5 is better than that of P/R-0 and P/R-0.5, and it can be seen that P/R-0, P/R-0.5 and P/R-1.0 have no obvious toxicity toward cells, while a few dead cells (red) are observed on the nanofiber membrane P/R-1.5. At the same time, combining with the three-dimensional reconstruction images of laser confocal microscopy (Fig. 5B), it can be seen that the HUVECs coverage area on the P/R-1.0 nanofiber membrane is the largest, and monolayer endothelial structures have been formed, and the endothelization degree is the best. Meanwhile, the results of CCK-8 were analyzed (Fig. 5C). There was no difference between P/R-0 and P/R-0.5 in promoting the proliferation of HUVECs. HUVECs co-cultured in P/R-1.0 and P/R-1.5 groups all prolificated gradually with time, and there was no significant difference on the 1st and 5th day, while the survival rate of P/R-1.0 cells was higher than that of P/R-1.5 cells at 3 d. The cell viability of the two P/R culture groups with high Res content was considerably higher than that of the two P/R culture groups with low Res content. Although the P/R-1.0 and P/R-1.5 groups did not exhibit a notable difference in terms of cell survival rates, the growth of HUVECs on different nanofibers obtained by laser confocal microscopy layer scanning on the 5th day of co-culture showed the most extended HUVEC morphology on the P/R-1.0 nanofibers. However, the cells on the P/R-1.5 nanofiber membrane tended to curl up and aggregate. These results suggest that, as in many previous studies, increasing the concentration of Res may not always be beneficial for the proliferation and growth of HUVECs and the degree of endothelialization.

Fig. 5 Evaluation of the cytocompatibility of P/R nanofibers: (A) images of live/dead cell staining after confocal 2D layer scanning; (B) images of live/dead cell staining after confocal 3D reconstruction; (C) CCK-8 assay of the proliferation viability of HUVECs cultured onto P/R-0, P/R-0.5, P/R-1.0, and P/R-1.5 after 1, 3, and 5 days of culture, respectively (ns indicates not statistically significant; * indicates p < 0.05; *** indicates p < 0.001; n = 3).

3.6 Cell migration assay

Cell migration, a fundamental cellular process, is integral to a spectrum of physiological functions, including tissue regeneration, embryonic development, repair of wounds, immune system reactions, and the metastatic spread of cancer, with particular significance in the context of vascular tissue regeneration.^64–67 To a large extent, the enduring patency rate of synthetic conduits with a small caliber, designed to emulate blood vessels, remains a significant concern. It depends on whether the endothelial cells on the side of the autologous blood vessel can quickly migrate to the suture site of the artificial blood vessel and form the inner cortex in the middle of the artificial blood vessel.^68,69

Fluorescent staining images of HUVECs cultured on P/R-0, P/R-0.5, P/R-1.0 and P/R-1.5 for 48 h are presented in Fig. 6A and Fig. S1 (ESI†). The experimental findings indicated that, under the conditions associated with the P/R-1.0 formulation, the migration of HUVECs towards the central migration zone from opposing sides was superior to that observed in the remaining trio of groups. Furthermore, within the migration zone, the morphology of HUVECs exhibited distinct pseudopodia and a spindle-like form. In contrast, a majority of HUVECs in the non-migration zone maintained their initial round configuration, characteristic of the early developmental phase of cellular growth. We also performed quantitative analysis on the migratory ability of HUVECs across the aforementioned four samples. In assessing cellular migration capacity, we focused our quantitative analysis on two parameters: the cell area within the migration zone, as illustrated in Fig. 6B, and the proportion of cells exhibiting varying migration distances relative to the entire population of migratory cells, as depicted in Fig. 6C. As delineated by the quantitative data presented in Fig. 6C, the P/R-1.0 group demonstrated the highest prevalence within the migration distance categories of 0–250 μm and 250–500 μm. This finding corroborates the superior migratory capacity of the HUVECs in this group, specifically highlighting the chemotactic allure of the P/R-1.0 in attracting cells. In conclusion, cellular growth and expansion are orchestrated processes that encompass cell adhesion, migration, and proliferation. These processes are directed towards establishing a rapid in situ cellular presence on the material surface, thereby facilitating effective tissue regeneration and structural formation.

Fig. 6 Migration assay of HUVECs seeded onto P/R-0, P/R-0.5, P/R-1.0, and P/R-1.5 after incubation of the cells for 48 h, respectively: (A) the nucleus (blue) are stained blue by DAPI and the cytoskeletons (red) are stained red by rhodamine-conjugated phalloidin; (B) quantitative spreading area measurements of HUVECs on different fiber after culturing for 48 h; (C) percentage of migrated HUVECs at 0–250 μm and 250–500 μm after 48 h co-culture (ns indicates not statistically significant; * indicates p < 0.05; ** indicates p < 0.01; n = 5).

The transwell cell migration experiment seeded HUVECs into the upper chamber of the transwell. The migration effect of the cells from the upper chamber to the lower chamber was observed by using fibrous membrane soaking medium for 30 min (Fig. 7A). The transwell cell migration experiment was conducted to evaluate the induced migration effect of P/R nanofibers on HUVECs, which was used to show whether P/R nanofibers can promote the rapid migration of HUVECs to form the inner cortex and the bridging of the vascular suture connecting artificial blood vessels and autologous blood vessels during vascular regeneration. We can observe representative images of cells migrating from the upper chamber of the transwell to the lower surface of the transwell through light microscopy (Fig. 7B). Three fields were randomly selected in each group, and the number of HUVECs in the P/R-1.0 and P/R-1.5 nanofibers were 43.7 ± 3.1 and 46.3 ± 6.1, respectively, which showed no significant difference but were much higher than the 29 ± 5.2 and 23.3 ± 1.2 cells in P/R-0 and P/R-0.5 (Fig. 7C). The cell coverage of HUVECs was also calculated as 8.36 ± 0.96% for P/R-0, 9.16 ± 0.28% for P/R-0.5, 14.45 ± 0.48% for P/R-1.0, and 14.11 ± 1.69% for P/R-1.5, respectively (Fig. 7D). The results showed that P/R nanofiber with Res loading of 1 wt% and 1.5 wt% had a positive effect on cell migration.

Fig. 7 (A) Schematic diagram of the transwell cell migration experiment; (B) microscope image of HUVEC migration in the transwell; (C) and (D) number of migrated cells and the migrated cell coverage, respectively (ns indicates not statistically significant; *** indicates p < 0.001; n = 3).

Combined with the results of Fig. 3E, it can be seen that when the P/R nanofibers release Res for 1 hour, there is little difference in the release amount of P/R-1.0 and P/R-1.5 Res, and both are greater than P/R-0.5. The possible reason is that compared with P/R-1.5, P/R-1.0 has a finer nanofiber diameter and larger porosity and specific surface area. Coupled with the hydrophobic property of the PEUU material itself, P/R-1.5 is not fully infiltrated by the medium in a short period of time, and Res molecules with the same mass as P/R-1.0 are released. There was no significant difference in the ability of P/R-1.0 and P/R-1.5 to induce vertical cell migration. When HUVECs are co-cultured with P/R nanofibers as shown in the horizontal migration results mentioned above, HUVECs on P/R-1.0 perform best. Different from the transwell migration experiment, HUVECs are in direct contact with P/R nanofibers in the horizontal migration experiment, and Res on the surface of P/R nanofibers can directly affect HUVECs. It can be inferred that P/R-1.0 nanofibers may be more favorable to the migration of HUVECs.

3.7 In vitro tube formation of HUVECs

The formation of the capillary-like structure of endothelial cells in vitro involves the steps of cell adhesion, migration, arrangement, protease secretion, and tube formation, which is of great reference value for evaluating the vascularization ability of active molecules.^70–72 The activity of Res on angiogenesis was measured by an in vitro tube formation experiment, and the total network length, branching points, number of meshes, master segments, and master junctions and the total master segment length were counted. The longer the total network length, the stronger the angiogenesis activity, and branching points refer to the nodes where new branches of the vascular network form. The number of branching points can reflect the complexity and maturity of the vascular network. In the process of angiogenesis, the formation of branching points is a key step in the expansion of the vascular network. The number of meshes refers to the number of individual grids, or loops, formed in the network of blood vessels. This index can reflect the connectivity and stability of the vascular network. Master segments refer to the vascular segments that form the backbone of the vascular network. This index can reflect the main structural and functional parts of the vascular network. Master junctions refer to branching points that play major roles in the blood vessel network; these points play a key role in the formation and expansion of the network. Total master segment length refers to the total length of the vascular segments that form the backbone of the vascular network. This index can reflect the development degree of the main blood vessels and the main blood vessel supply capacity of the network. As shown in Fig. 8A, the influence of four groups of nanofibers on the formation of tubes in HUVECs can be directly seen. It can be seen that under the condition of P/R-0 culture, HUVECs are almost all spherical, scattered and locally clustered, and can hardly form a continuous grid connected to each other. HUVECs are connected on P/R-0.5, forming a small number of dendritic structures. In contrast, P/R-1.0 and P/R-1.5 obviously observed that HUVECs were in a good growth state, and the cell morphology was extended in long strips with obvious pseudopods, and the cells formed tubes connected to each other, and finally formed an impressive grid-like structure. Then, the image was further quantitatively analyzed, as shown in Fig. 8B–G. There was no significant difference between P/R-1.0 and P/R-1.5 in the total network length, branching points, number of meshes, master segments, master junctions and the total master segment length, but both were much higher than the other two groups. The master segments, master junctions and the total master segment length of P/R-0.5 are higher than those of P/R-0. Therefore, it can be preliminarily concluded that a small amount of Res loading is beneficial for tube formation, and P/R-1.0 and P/R-1.5 may be the most beneficial for tube formation in HUVECs.

Fig. 8 HUVECs tube formation experiment: (A) representative images of HUVEC tube formation measurement; (B)–(G) statistics of the total network length, branching points, number of master junctions, number of master segments, total length of master segments, and number of meshes, respectively (ns indicates not statistically significant; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; n = 3).

4. Conclusions

In this study, a series of P/R nanofibers loaded with resveratrol (Res) were prepared by dual-solution electrospinning for enhancing the bioactivity of vascular endothelial cells and improving blood compatibility. The mechanical properties of P/R nanofiber in both dry and wet conditions were matched with the implantation requirements. The anticoagulation and anti-oxidation effects of P/R nanofibers increase with the increase of Res content. Moreover, P/R nanofibers with 1 wt% loading capacity had the most significant effect on promoting endothelial cell activity, and endothelial cells could form a continuous single layer of endodermal structure on the surface of the nanofibers. Combined with the results of the platelet adhesion experiment, it indicated that the P/R nanofiber presented a continuous and effective anticoagulation effect. In conclusion, these multifunctional hybrid poly(ester-urethane)urea/resveratrol electrospun nanofibers (P/R-1.0) may be a potential candidate for potential vascularizing matrices.

Author contributions

Cheng Liang: writing – original draft; data curation; validation; visualization; formal analysis. Yanan Wang: visualization; investigation; visualization. Renliang Zhao: visualization; investigation; visualization. Juan Du: resources; investigation. Jin Yao: visualization; formal analysis. Atta ur Rehman Khan: writing – review & editing. Tonghe Zhu: conceptualization; supervision; project administration; methodology; writing – review & editing; formal analysis. Huitang Xia: resources; investigation. Youwei Zhu: funding acquisition.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of interest

All authors declare no conflict of interest.

Acknowledgements

This work was financially supported by the Science and Technology Commission of Shanghai Municipality (22S31904700), the National Natural Science Foundation of China (82303265), Shanghai Sailing Program (22YF1426200), and Shanghai College Students Innovation Training Project (202410856019).

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4sm00937a

‡ Co-first authors.

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