Morphology of Electrospun Membranes

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As ideal cardiovascular grafts, it is necessary to have abilities of rapid endothelialization and antibacterial activity to cope with the risks of restenosis, thrombosis and bacterial infections. In this work, dual functional electrospun membranes of poly(ε-caprolactone) (PCL)/gelatin were developed. A short cell active peptide, REDV, was covalently conjugated with low molecular weight PCL and introduced to improve the adhesion of vascular endothelial cells (VECs) on the membranes, meanwhile a plant-extracted antibacterial agent, eugenol, was loaded to provide antibacterial activity as well. A ~25% burst release of eugenol was detected and the release reached ~85% at final. The electrospun membranes displayed significant antibacterial activity with about 75% and 85% of growth inhibition rate for Escherichia coil and Staphylococcus aureus, respectively, and low cytotoxicity with more than 80% of relative viability for L929 fibroblast cells. The cell culture assays indicated that REDV decorated on the electrospun membranes could conspicuously promote VEC adhesion and proliferation. Therefore, the dual function of rapid endothelialization and antibacterial activity endowed by the combination of REDV and eugenol impelled the electrospun membranes promising as cardiovascular grafts.

1. Introduction

Recent years, cardiovascular diseases have been the number one cause of death around the world as described by the World Health Organization. With the rapid development of tissue engineering and modern medicine, a number of cardiovascular-associated implants such as small-diameter vascular grafts and heart stents have been designed and applied to treating with the related diseases [1-3]. However, the challenges from restenosis and thrombosis often result in a short-term patency for cardiovascular grafts, and then limit their ultimate success and ubiquitous clinical use [4, 5].

Rapid endothelialization has been extensively investigated as an effective way to restrain restenosis and thrombosis, and prolong the long-term patency [6-8]. Therefore, that vascular endothelial cells (VECs) attach to and consequentially proliferate on the surface of cardiovascular grafts to form endothelial layer is of importance. Many strategies have been developed to facilitate grafts specially and selectively adhering VECs and most of them involved immobilizing various cell active peptides such as QK, RGD, REDV and YIGSR, which can optimize the graft surfaces to promote VEC adhesion, migration and proliferation [7-9]. Among the peptides, REDV (Arg-Glu-Asp-Val), found in the alternatively-spliced IIICS-5 domain of human plasma fibronectin, appeals to a lot of attention as an endothelial cell specific adhesive ligand which can mediate the endothelial cell adhesion and migration via the α4β1 integrin [10, 11]. In the reported literatures, REDV was introduced onto different substrate surfaces and all the results showed the competitive growth of endothelial cells over smooth muscle cells [12-14]. In our previous study, REDV was grafted onto the electrospun poly(ethylene glycol)-b-poly(L-lactide-co-ε-caprolactone) membrane surfaces and the modified membranes also showed promoting effect on VECs adhesion and proliferation [15].

Furthermore, bacterial infection has been another threat in implantation operations, which can result in multiple clinical complications and even death, aggravating the patients’ healthcare burden, so that it is essential to fabricate the artificial scaffolds with antimicrobial activity [16-18]. Antibacterial agent loaded biomaterials with desired dose can ensure sustained release over a period of time and can minimize the toxicity, which are potential alternative for cardiovascular grafts to deal with the implant-associated bacterial infection. Numerous antibiotics and chemical antibacterial agents such as tetracycline hydrochloride, ciprofloxacin, chlorhexidine and triclosan have been loaded in electrospun nanofibers for wound dressing and other tissue engineering applications [19-22]. Whereas, considering the bacterial resistance to antibiotics and the toxic and side effects from chemical antibacterial agents, natural antimicrobial essential oils extracted from plants have become preferred. Its phenolic constituents have exhibited significant antibacterial activity and low toxicity and have been widely applied in textile, wound healing, tissue regeneration and food packaging [23-25]. Of these, eugenol (4-allyl-2-methoxyphenol), an amphipathic hydroxyphenyl propene, is one of the best candidates with excellent antiseptic and antibacterial properties [26]. Eugenol has been introduced onto the surface of polycarbonate urethane as cardiovascular implant by grafting polymerization of eugenyl methacrylate and showed high inhibitory effect on bacterial growth [14].

In the research for cardiovascular materials, poly(ε-caprolactone) (PCL) has been widely employed because of its good biocompatibility and suitable mechanical properties [27]. But, as a synthetic polymer, PCL often leads to low cell adhesion and proliferation for lack of bioactive sites [28]. Gelatin, however, a natural polymer derived from partial hydrolysis of native collagen, contains many integrin binding sites for cell adhesion, migration and differentiation, which can make up for the shortcomings of PCL [29], and meanwhile the quick degradation of gelatin can modulate the drug release in the hybrid PCL/gelatin material [30]. In fact, the hybrid PCL/gelatin electrospun scaffolds have shown more favorable interactions with mesenchymal stem cells in comparison with PCL scaffolds in the literature [31].

Based on the above understanding, bio-scaffolds with multiple functions could be more competitive in the applications. In this work, we developed a hybrid PCL/gelatin electrospun membrane containing REDV and eugenol. Different from common surface modification, REDV was covalently coupled with low molecular weight PCL and then electrospun with gelatin and high molecular weight PCL. The intact surface of the prepared membrane decorated by REDV via REDV-conjugated PCL migrating in the electrospinning process.(此处不宜说的太明白,因为结果中并没有体现REDV往表面迁移的证据) Eugenol was encapsulated taking the prepared membrane as a carrier to realize controlled release. The prepared membranes were expected to possess dual function of rapid endothelialization and antibacterial activity.

2. Experimental Methods

2.1. Materials

PCL (= 80 kDa) and gelatin (type B) were purchased from Sigma. The cysteine-terminated REDV peptide (REDVC, 98%) was purchased from Shanghai Science Peptide Biological Technology Co., Ltd., China. REDV-conjugated PCL (REDV-PCL-REDV, PR) was synthesized as described in Supplementary Information. Eugenol (Eg, 99%) was provided by Tianjin Heowns Biochemical Technology Co., Ltd., China. N, N’-Carbonyldiimidazole (CDI, 98%), 4-dimethylaminopyridine (DMAP, 99%), 2, 2-dimethoxy-2-phenylacetophenone (DMPA, 98%) and 2, 2, 2-trifluoroethanol (TFE, 99%) were purchased from Tokyo Chemical Industry Co., Ltd., Japan. Mueller-Hinton broth (MHB, BR) and nutrient agar (BR) were obtained from Beijing Aoboxing Biological Technology Co., Ltd., China. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) and endothelial cell medium (ECM) were supplied by ScienCell, USA. Cell counting kit-8 assay (CCK-8) and DiI dye were supplied by Jiangsu KeyGEN Biological Technology Co., Ltd., China. Other chemical reagents such as 2-hydroxyethyl methacrylate (HEMA, 98%), acetic acid (HAc, 99.5%)were purchased from Tianjin Kemiou Chemical Reagent Co., Ltd, China.

2.2. Preparation of electrospun membranes

A polymer blend solution was prepared by mixing PCL/PR/TFE and GT/TFE according to the settings in Table 1. A tiny amount (1 v/v % of TFE) of HAc was added to make the above solution homogeneous. Then a pre-set eugenol (5, 10, 20, and 30 wt% of polymer) was dropped into the solution under stirring. Then, the solutions were electrospun at a flow rate of 0.4 mL/h, a high voltage of 10 kV and a distance of 15 cm between the collection and capillary tip. Each solution was electrospun for 10 h, and the thickness of the electrospun membranes were about 50 μm. The electrospun samples were designated corresponding to their components in Table 1.

2.3. Characterization of electrospun membranes

2.3.1. Morphology

For morphology characterization, the electrospun fibers were observed by a scanning electron microscope (SEM, Hitachi SU1510, Japan) with an acceleration voltage of 10 kV after sprayed with gold for 40 s and the average fiber diameters were measured from SEM images using Image J software.

2.3.2 Chemical analyses

Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectra and X-ray photoelectron spectra (XPS) were obtained with an infrared spectrometer equipped with a ZeSe crystal and an liquid nitrogen cooling MCT detector (Perkin-Elmer Spectrum 100, USA) and an X-ray photoelectron spectroscope (Perkin-Elmer PHI 5000C ECSA, USA), respectively. The scan range of ATR-FTIR was 4000 cm-1 to 650 cm-1 with a resolution of 4 cm-1. The XPS were conducted under an ultra-high vacuum less than 5 × 10-8 Torr at 45° with Al K radiation (1486.6 eV) operating at 24.2 W. The tested area was a circle of 100 μm diameter.

2.3.3 Mechanical properties

The tensile tests of the membranes in both the dry state and the wet state were performed on a tensile testing machine (Testometric M350–20KN, UK) equipped with a 100 KN load cell at a crosshead speed of 5 mm/min, a gauge length of 40 mm. All samples were cut into 10-mm width and 60-mm length. The dry state specimens (n=5) were tested immediately at room temperature. The wet state specimens (n=5) were prepared by immersing into PBS for 1 h at room temperature before the test.

2.4. In vitro release behaviors of eugenol

2.4.1. Eugenol encapsulation efficiency

The membranes were cut into similar weight of pieces (n=3) and dissolved in 1 mL of TFE, respectively. Then, the solution was dropped in 20 mL of methanol. After the polymer was precipitated, the supernatant was detected on a UV-vis spectrometer (Shimadzu UV-3600 Plus, Japan) at the wavelength of 282 nm [32]. The amount of eugenol was obtained from the calibration curve of eugenol. The encapsulation efficiency was calculated using the following equation:

Encapsulation efficiency (%)=m/m0 × 100%

where m and m0 represent the actual and theoretical amount of eugenol in the electrospun membranes, respectively.

2.4.2. Eugenol release profile

The release behaviors of eugenol were determined based on a previous protocol [33]. Briefly, the eugenol-containing membranes were cut into similar weight of pieces (about 20 mg, n=3) and incubated in 3 mL of PBS at 37oC with shaking. At predetermined time intervals, 1 mL of extracting solution was replaced by 1 mL of fresh PBS. The removed solution was used to measure the release amount of eugenol on a UV-vis spectrometer (Shimadzu UV-3600 Plus, Japan) at the wavelength of 282 nm with a reference of PG release solution. The cumulative eugenol release amount was calculated by the following equations:

mt = m1 + m2 + m3 +… + 3mx (x=1,2,3…)

Cumulative release (%) = mt/m × 100%

where mt represents the cumulative amount of eugenol released from the membranes at predetermined time, mx represents the release amount of eugenol in 1 mL of PBS at the predetermined time, and m represents the total amount of eugenol loaded in the membranes.

2.5. Antibacterial activity of electrospun membranes

The antibacterial activity of the membranes against typical gram-negative bacteria Escherichia coil (E. coil ATCC 8739) and gram-positive bacteria Staphylococcus aureus (S. aureus ATCC 6538) was estimated by the modified “immersion” method according to the references [17, 34].Briefly, the bacterial strains were cultured in MHB overnight to reach the mid-log growth phase and then were diluted to an optical density (OD) of 0.07 at the wavelength of 600nm on a microplate reader (Tecan M200 Pro, Switzerland) corresponding to colony forming units (CFU) 3 × 108 mL-1.

The membranes were cut into circular discs (10 mm in diameter), sterilized under UV radiation for 1 h (30 min each side) and then placed in the 48-well microplate. The above bacterial solution was diluted to 3 × 105 CFU/mL and 50 μL of this bacterial solution was added to each well. Additionally, 150 μL MHB was added to each well to fill 200 μL total volume, and the 48-well microplate was incubated at 37 oC for 24 h. Inoculated wells without membranes and wells just containing pure MHB were used as positive control and negative control, respectively. After the incubation, 10 μL of the inoculum was taken out and diluted with an appropriate dilution factor. Then the bacterial solutions were spread on nutrient ager plates. The colonies formed after an incubation of 18 h at 37 oC were imaged and counted by Adobe Photoshop software. The bacterial growth inhibition rate was calculated by the colony counting using the following equation:

Growth inhibition rate (%) = × 100%

A SEM observation was carried out to examine the morphology of bacterial cell membranes after treated with the electrospun samples [35]. A certain mass of PG/Eg30 sample containing a lethal dose of eugenol was immersed into 1ml of bacterial solution (3 × 106 CFU/mL) at 37 oC for 4 h. PG sample was used as a contrast. After that, the bacteria were harvested by centrifugation at 3000 rpm for 10 min and rinsed thrice with PBS. Subsequently, the cells were fixed with 2.5% glutaraldehyde solution at 4 oC for 6h and dehydrated with ethanol solution at gradient concentrations (30%, 50%, 70%, 80%, 90% and 100%) for 10 min. Then the cells were pelleted down and mounted on silicon wafers to use for SEM observation.

2.6. Biocompatibility assays

2.6.1. Cytotoxicity assay

The cytotoxicity of membranes was evaluated using the extracting solutions of membranes [30]. The samples were sterilized under UV radiation for 1 h (30 min for each side), immersed in DMEM at a proportion of 5 mg/mL and then incubated at 37 oC for 48 h to produce the extracting solutions.

L929 fibroblast cells resuspended in DMEM containing 10 v/v % FBS were seeded in a 96-well microplate at a density of 1.0 × 104 cells/well. Subsequently, the FBS-containing DMEM was added to fill 200 μL total volume of each well. After incubated at 37 oC, 5% CO2 for 24 h, the medium was replaced by the above extracting solutions. The wells of which medium was replaced by pure DMEM were used as positive control. The plate was incubated for another 24 h, and then 200 μL CCK-8/DMEM (10 v/v %) solution was added to each well to replace the extracting solution. After further incubated for 4 h, the supernatant was withdrawn to measure OD at the wavelength of 460nm. The relative cell viability was calculated using the following equation:

Relative cell viability (%) = × 100%

2.6.2. Cell adhesion and proliferation assays

For investigating the effects of REDV incorporated in membranes on VECs, Human umbilical vein endothelial cells were cultured on membranes for different time to assess the adhesion and proliferation properties. The samples were cut into circular discs (10 mm in diameter), sterilized under UV radiation for 1 h (30 min each side) and then placed in 48-well microplates. 1.0 × 104 cells resuspended in ECM were seeded in each well. Then additional ECM was added to fill 300 μL total volume and refreshed every 2 days. The plates were incubated at 37 oC and 5% CO2.

After cells attaching to the samples for 4 h and 8 h, the non-adhered cells were removed and the adhered cells were fixed by methanol, stained by 0.1 v/v % crystal violet and lysed by 2 v/v% SDS. Then, the OD of supernatant was measured at the wavelength of 570 nm.

Furthermore, after the plates were incubated for 3, 6, 9 days respectively, 200 μL CCK-8/ECM solution was added to each well to replace existing ECM. After further incubated for 4 h, the OD of supernatant was measured at the wavelength of 460 nm. Cell morphology was observed by SEM and Laser scanning confocal microscope (LCSM, Leica TCS SP8) on day 6. Before SEM observation, cells on membranes were fixed with 4% polyformaldehyde and dehydrated with ethanol solution at gradient concentrations (30%, 50%, 70%, 80%, 90% and 100%). For LCSM, the cells were stained with DiI dye before seeded on the membranes. After cultured 6 days, the cells on the membranes were fixed with 4% polyformaldehyde and then mounted.

2.7. Statistical analysis

All quantitative data were expressed as mean ± standard deviation (SD). Each sample was conducted with at least three parallel tests for antibacterial and cytological experiments. Statistical analyses were performed using either one-way analysis of variance (ANOVA) or Student’s t-test. A p-value less than 0.05 was indicated statistically significant.


3. Results and Discussion

3.1. Morphology of electrospun membranes

Although either PCL or gelatin can be dissolved in TFE to form a transparent solution, phase separation occurred and the polymer solution became turbid when mixing together. This is due to the pH of solution is close to the isoelectric point of gelatin, and a tiny amount of HAc was dropped to make the solution homogeneous [36, 37]. The SEM images of the electrospun membranes were shown in Fig. 1. All the electrospun fibers were smooth and interconnected randomly with no breads detected. The diameters of the fibers were all in the range of 700-900 nm as shown in Table 1. Different eugenol content of PG/PR/Eg5 – PG/PR/Eg30 samples did not lead to an apparent variation trend for their fiber diameters. Whereas some ultrafine fibers (about 200 nm) were seen in all the membranes possibly resulting from the electrical conductivity of gelatin in the electrospining solution. As a polyelectrolyte polymer, gelatin have many ionizable groups such as amino and carboxylic groups, which can be ionized in protic solvent. Therefore, ultrafine fibers could be formed as a result of the charge mobility under a high direct current voltage [38, 39].

3.2. Mechanical properties of electrospun membranes

Mechanical properties of the prepared electrospun membranes were measured by the uniaxial tensile testing, to insure that the membranes could withstand the forces during surgical operation or from tissue growth and physiological activities. The typical stress-strain curves and the values of the tensile strength, the Young’s modulus and the elongation at break in both the dry and wet state were shown in Fig. 2(A-D), which revealed the behaviors of electrospun membranes under dynamic stress [27, 40]. Overall, the prepared electrospun membranes had the higher tensile strength (1.56-2.64 MPa), the higher Young’s modulus (43.6-72.9 MPa) and the lower elongation at break (7-31 %) in the dry state compared with those in the wet state. Nevertheless, we paid more attention to their behaviors in the wet state, which were similar with the physiological condition. In the wet state, the tensile strength and the Young’s modulus showed distinct increasing trends (Fig. 2B and 2C), while the elongation at break had a dramatic decreasing trend (Fig. 2D) with increased eugenol content in the PG/PR/Eg5 – PG/PR/Eg30 membranes. The results indicated that the incorporation of eugenol could make the electrospun membranes inelastic, probably resulting from the interactions between polymer and eugenol, which was similar with the results of chitosan-based films incorporated with cinnamon essential oil described by Hosseini [41]. In the wet state, the tensile strength, the Young’s modulus and the elongation at break were in the range of 1.03-1.69 MPa, 9.3-17.3 MPa and 50-142 %, respectively, which were comparable to the mechanical properties of human coronary arteries in the reported references [42, 43]. Therefore, these results suggested that the prepared electrospun membranes would be suitable for cardiovascular grafts in terms of mechanical properties.

3.3. Eugenol encapsulation efficiency and release behaviors

As show in Table 1, the eugenol encapsulation efficiencies of eugenol-containing electrospun membranes were 74.3%, 72.4%, 68.4%, 66.7% and 71.0%, respectively. There was no obvious correlation between encapsulation efficiency variation and eugenol content for the PG/PR/Eg5 – PG/PR/Eg30 samples. The loss of encapsulation efficiencies were probably due to the volatilization of eugenol in the electrospinning process, which wasn’t encapsulated into the membranes.

The eugenol release profiles of the eugenol-containing electrospun membranes PG/Eg30 and PG/PR/Eg5 – PG/PR/Eg30 were presented in Fig. 3A, which showed almost similar release patterns for different electrospun membranes. An initial burst release within 1 day about 25% of eugenol released from the membranes was observed, followed by a sustained release rate up to 21 days. Then the eugenol released at a slower rate until release equilibrium, when about 85% of eugenol was released. The eugenol release behavior was associated with eugenol diffusion and gelatin collapse. The eugenol on the surface of the membranes first burst released, and then, the eugenol sustainedly released with gradual collapse of gelatin. This can be demonstrated by the SEM morphologies of electrospun membranes after released 21 days (Fig. 3B), in which the fibers became rough and even banded after the collapse of gelatin. Later, the eugenol release was limited by the low degradation rate of PCL. The inner eugenol diffused to PBS through the PCL at a slower rate and reached the release equilibrium in the end [39].

3.4. Antibacterial activity of electrospun membranes

Implant-associated bacterial infection is severe in surgical operations, and the antimicrobial activity is preferred for implantable scaffolds. Eugenol has the accepted broad-spectrum antimicrobial activity and has been considered as a powerful potential alternative for its natural origin. The antibacterial activities of the prepared electrospun membranes against E. coil and S. aureus were evaluated using the “immersion” method, and the results calculated by the colony counting were shown in Fig. 4. There was an expected trend that the bacterial growth inhibition rate of electrospun membranes was enhanced with increased content of eugenol for both E. coli and S. aureus. The PG/Eg30 sample showed the highest bacterial growth inhibition rate, which was ~85% for S. aureus and ~75% for E. coli. For different test strains, the eugenol-containing membranes PG/Eg30 and PG/PR/Eg5 – PG/PR/Eg30 showed superior antibacterial activity against S. aureus to E. coil. This is possibly related to the lipopolysaccharide layer in the outer membrane of gram-negative bacteria, which surrounds the cell wall and restricts the penetration of hydrophobic eugenol [44]. The antibacterial tests indicated that the electrospun membranes containing over 10% eugenol display promise in coping with implant-associated infections.

SEM micrographs for the cell membrane morphology of E. coil and S. aureus after treated with PG and PG/Eg30 samples were shown in Fig. 5. Intact and smooth membranes were seen for test strains treated with PG sample. In contrast, malformed cells were found in the images of test strains treated with PG/Eg30. Significant morphological evolvements were witnessed in E. coil, in which the cells collapsed and cell membranes became wrinkled and corrugated. For S. aureus, many blebs enclosed on the cell surfaces from leakage of intracellular content and then the cells shrank. The phenomena can be ascribed to the hydrophobicity of eugenol, which makes eugenol accumulate in the cell surface, disrupting the cell membrane and increasing the non-specific permeability of cell membrane [26].

3.5. Cytotoxicity of electrospun membranes

In this work, to evaluate the effect of eugenol incorporated into electrospun membranes on cell viability, L929 cells were cultured in the prepared extracting solutions of electrospun membranes. The relative cell viability of L929 cells after exposure to the extracting solutions for 24 h was ascertained by the CCK-8 assay, as shown in Fig. 6. Obviously, the eugenol-free samples PG and PG/PR exhibited higher relative cell viability close to 100%, indicating that almost harmless substances to cells were released from these two samples [45]. It is well known that the cytotoxicity of eugenol depends on the eugenol dose, which are emerged at high concentrations of about 2 mM [46]. Also, the cytotoxicity results in Fig. 6 presented a dose-dependent effect for extracting solutions of electrospun membranes. That is, the relative cell viability of eugenol-loaded electrospun samples PG/PR/Eg5 – PG/PR/Eg30 gradually decreased as the increased content of eugenol. However, with relative cell viability more than 80%, it can be considered as cytocompatible for these eugenol-loaded samples, which partially can be attributed to that the fibers serve as a carrier vehicle for eugenol and prevent cells from damaging even at a higher eugenol content of 30% [47].

3.6. Adhesion and proliferation of VECs on electrospun membranes

Adhesion and proliferation first occur among a number of physiological responses to a substrate, which are the basis of biomedical materials application. In order to understand the effects of REDV on VECs, the adhesion and proliferation assays were conducted. Fig. 7 presented the adhesion of VECs on the electrospun membranes. A significant increase was observed for VECs adhesion on the REDV-containing samples PG/PR and PG/PR/Eg5 – PG/PR/Eg30 in comparison with the REDV-free samples PG and PG/Eg30 cultured for 4h and 8h. The electrospun membrane PG/PR without eugenol showed the highest adhesion in all samples, exhibiting the promoting effect of REDV on VEC adhesion. The adhesion of PG/PR/Eg5 – PG/PR/Eg30 samples gradually decreased, possibly resulting from the negative effect of eugenol in the surfaces of the samples. Fig. 8 and Fig. 9 showed the proliferation profile of VECs on electrospun membranes. As shown in Fig. 8, the cell viability evaluated by the CCK-8 assay increased with the culture time and revealed the proliferation. At day 3, the viability values of REDV-containing samples PG/PR and PG/PR/Eg5 – PG/PR/Eg30 were significantly higher than those of REDV-free samples PG and PG/Eg30. The negative effect of eugenol released from the membranes made a decreased viability values for the samples PG/PR/Eg5 – PG/PR/Eg30. Similar trends were also seen at day 6 and 9. Besides, the proliferation rates of REDV-containing samples were remarkably faster than those of REDV-free samples. These results proved that REDV could evidently facilitated VECs proliferation, which were coincident with our previous research [15]. Furthermore, above results can be supported by the visualized morphology images from SEM (Fig. 9A) and LCSM (Fig. 9B). From Fig. 9A, we can see that VECs adhered to and spread on the REDV-containing membranes well, and these phenomena were superior to the spread of VECs on the REDV-free samples. It also can be seen that some VECs spread over multiple fibers on the membranes, indicating the good cytocompatibility of electrospun membranes. In Fig. 9B, the plasma membranes were stained by DiI dye so that the cell contours could be easily observed. The VECs on the REDV-containing membranes showed obvious spreading morphology compared with the cells on REDV-free samples. Moreover, the cell numbers of REDV-containing samples were more than those of REDV-free samples. Once again, these results demonstrated the promoting effect of REDV on VEC adhesion and proliferation. Whereas, the eugenol in PG/PR/Eg5 – PG/PR/Eg30 samples resulted in imperceptible negative effect on VEC adhesion compared with the sample PG/PR from both SEM (Fig. 9A) and LCSM (Fig. 9B).

4. Conclusions

Eugenol-loaded hybrid PCL/gelatin electrospun membranes decorated with REDV were successfully prepared with fibrous morphology. The mechanical properties of electrospun membranes were evaluated and contented the requirement of tissue engineering scaffolds. The antibacterial agent eugenol were incorporated in the membranes with encapsulation efficiencies in the range of 65%-75%, and showed a regular release profile with ~25% of eugenol burst release and ~85% release equilibrium. The electrospun membranes displayed significant antibacterial activity with about 75% and 85% of growth inhibition rate for E. coil and S. aureus, respectively, via the “immersion” method, as well as low cytotoxicity with more than 80% of relative cell viability. And on the premise of not affecting the fiber morphology and not enhancing the cytotoxicity, we can expect that more eugenol would be incorporated in electrospun membranes to obtain more pronounced antibacterial activity. Cell adhesion and proliferation assays indicated that the electrospun membranes had good cytocompatibility, and REDV could evidently facilitate VECs adhesion and proliferation. The prepared elelctrospun membranes with dual function of rapid endothelialization and antibacterial activity have a potential in tissue engineering.

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