1 Introduction

Regeneration of injured tissues by tissue engineering approaches via developing novel biomaterials has gotten great attention [1]. Tissue engineering provides the structural and functional matrix by the combination of scaffolds, bioactive molecules, and cells to develop responsive constructs for damaged tissues [2]. Scaffold design and components are important factors that can influence tissue engineering outcomes [3]. Various methods, such as electrospinning, gas foaming, 3D printing, and freeze-drying, have been reported to fabricate scaffolds [1, 2, 4]. Nowadays, polymeric electrospun scaffolds have attracted a great deal of attention for tissue engineering applications especially, skin regeneration. Electrospun nanofibers find good advantages such as large surface-to-volume ratio, highly porous extracellular matrix (ECM) resembling membrane, mimicking the fibrous structure of collagen, laminin, and elastin, supporting cell attachment, infiltration, and proliferation for developing tissue engineering scaffolds [5,6,7,8,9]. Recently, new approaches such as multicores-sheath nanostructures have also been developed by coaxial electrospinning technique to produce smart thermo-regulated textiles [10]. PUs, as synthetic bioactive materials, have been considered for fabricating scaffolds [11]. They are mostly used in biomedical and pharmaceutical applications because of their favorable characteristics, such as high oxygen permeability, biocompatibility, biodegradability, and mechanical properties comparable to natural tissues [12]. PU-based nanofibers are suitable for skin tissue engineering and wound dressing fields as PU offers advantages, including excellent mechanical properties, promoting epithelization, and porous structure for nutrient and gas exchanges [13,14,15]. In addition, the barrier properties of PU-based nanofibers do not allow bacteria to reach the internal surface of the wound [16]. Functional PUs with desired properties such as biodegradability, blood compatibility, and self-assembly has also been investigated for biomedical applications [17]. In a recent study [18], it was reported that the incorporation of PCL to PU electrospun solution in the ratio of 1:2 could present good pore size, nanofiber diameter size, and mechanical properties. Therefore, the development of a composite nanofiber with PU and PCL using electrospinning technology can address the multi-aspects of scaffolds for tissue engineering. However, the low hydrophilicity of PU-based scaffolds is one of their drawbacks that limit their applications [19]. The surface modification of PU nanofibers with nanoparticles can overcome the hydrophobicity of scaffolds thanks to the presence of different polar groups in PU [20]. Among candidates to be used for skin tissue engineering, GNPs can be considered as attractive nanostructures because of their biocompatibility, low toxicity, and electrical conductivity properties for affecting the cell behaviors [21,22,23]. It has been reported that electrical conductivity is another factor that can influence cell attachment and proliferation properties [24]. In addition, the positive effects of GNPs on neovascularization and accelerating the wound healing process have been mentioned [25]. Yang et al. reported the fabrication of GNPs doped PCL/gelatin fibers for wound healing application. Their results showed effective antimicrobial activity, biocompatibility, and adequate wound healing property [26]. However, there are no sufficient studies on using GNPs for skin tissue engineering.

In the past years, studies have been conducted on the fabricating scaffolds containing natural oils. Soybean is a natural material composed of proteins, carbohydrates, oil fractions, and isoflavones [27, 28]. The oil fraction of soybean include polyunsaturated oils (linoleic acid (51%), oleic acid (25%), palmitic acid (11%), linolenic acid (9%), and stearic acid (4%) residues) [29, 30]. SO possesses various properties that attract pharmaceutical, cosmetics, and food industries for the delivery of bioactive molecules [31]. SO with essential fatty acids can present antioxidant, anti-inflammatory, and epithelizing properties. Soybean-modified polyamide-6 mats were found to support a higher percentage of fibroblast attachment by visible contact points with the surrounding fibers and ECM release of cells, well-spread and elongated morphology, and migration [32]. Moreover, the promotion of cell adhesion and proliferation by SO-based PU networks has been reported [33]. The epoxidized form of SO has been applied for the cross-linking of α-cellulose-based scaffolds with high porosity and biocompatibility [34].

The present study aimed to develop biocompatible and cell responsive electrospun PU/PCL nanofiber scaffolds containing SO and GNPs for tissue engineering applications. For this purpose, the fabricated scaffolds were subjected to physicochemical characterization such as chemical composition, morphology, surface roughness, mechanical properties, wettability, and electroconductivity. The biocompatibility evaluations of the fabricated nanofibers were conducted by proliferation and attachment of the NIH-3T3 fibroblast cell line.

2 Materials and Methods

2.1 Materials

PU, PCL, HAuCl4, tri-sodium citrate, and (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) were supplied from Sigma-Aldrich. Tetrahydrofuran (THF), N, N Dimethylformamide (DMF), and Dimethyl Sulfoxide (DMSO) were purchased from Merck. Roswell Park Memorial Institute medium (RPMI) 1640 medium, Fetal Bovine Serum (FBS), Trypsin–EDTA, and Penicillin–Streptomycin (Pen-Strep) were obtained from GIBCO.

2.2 Methods

2.2.1 Synthesis of GNPs

GNPs were synthesized based on the reduction of Au ions by tri-sodium citrate. To synthesize the GNPs, the aqueous solution of HAuCl4 (1 mM, 20 ml) was stirred under 100–150 ºC to reach the boiling point. Then, tri-sodium citrate solution (1% w/v, 2 ml) was quickly added to the boiling HAuCl4 solution. After the color changed from light yellow to dark red, the reaction was stopped, and the obtained GNPs were cooled at room temperature.

2.2.2 Preparation of Different Nanofibrous Mats by Electrospinning

PU/PCL nanofibers were prepared with regards to the previous research [18] with some modifications. For preparation of the electrospinning solution, 0.189 g PU and 0.094 g PCL were dissolved in DMF and THF (4:1). The mixture was stirred for 18 h to get a homogeneous solution. To fabricate the SO-loaded nanofibers (PU/PCL/SO), initially, the PU/PCL solution was prepared similarly to the above section, and then SO with a concentration of 30% v/v was added to it. For preparation of the electrospun solution of PU/PCL/SO/GNP composite nanofibers, after obtaining PU/PCL solution, the centrifuged GNPs (2 ml) and SO (30% v/v) were added to it, respectively. The same electrospinning conditions were applied to all the samples. For electrospinning, 3 ml of the solution was placed in a syringe and fitted into the device. The solution was injected at the rate of 2 ml/h, and the collector distance from the needle tip was adjusted to 15 cm. To charge the solution, a 20 kV voltage was applied. The formed electrospun nanofibers were collected over the aluminum foil placed on the collector and dried in a vacuum for 24 h.

2.2.3 Physicochemical Characterization of Nanofibrous Mats

ATR-FTIR (Bruker) was used to indicate the surface chemical structure of the electrospun nanofibrous mats. In addition, this analysis was used to determine whether SO was successfully integrated into PU/PCL scaffolds. The spectrum was recorded within the range of 600–4000 cm−1.

The morphology and diameter size distribution of prepared nanofibers was investigated using SEM (MIRA3, Tescan). For this purpose, the gold sputter-coated samples were imaged by SEM. The nanofiber diameter size distribution was determined by ImageJ software.

To evaluate the hydrophilicity of the fabricated nanofibers, the water contact angle measurements were determined by a contact angle instrument (Dataphysics, OCA 15 plus). To this, the nanofibers were cut into square shapes (1 × 1 cm), and water droplets (4 µl) were gently placed on the nanofiber’s surfaces. The images were recorded by CCD camera and the contact angles were calculated by the image analysis software.

To assessment the surface roughness of the fabricated nanofibers, AFM (Nanosurf, Nanosurf Mobile- S) was used. The topographic and phase images were obtained.

For the electrical conductivity assessment of the fabricated nanofibers, the standard four-probe technique was used [35] by measuring the voltage in different currents. Finally, the resistance was calculated. The electrical conductivity of the nanofibers can be calculated by inverting the resistance value of them.

2.2.4 Mechanical Characterization

To evaluate the mechanical properties of the fabricated nanofibers, the tensile strength was measured using a mechanical instrument (Instron Z010, Zwick/Roell). For this purpose, the rectangular-shaped (50 mm × 10 mm) samples were placed into the testing machine, and tests were conducted at a strain rate of 5 mm/min.

2.2.5 Biocompatibility of the Nanofibers

We used fibroblast cell line to investigate the biocompatibility studies due to the importance of these cells in the injured tissues [13]. NIH-3T3 fibroblast cell lines were cultured in RPMI 1640 medium complemented with 10% v/v FBS and 1% v/v Pen-Strep and incubated at 37 °C under 5% CO2. All the nanofibers were punched into the small disk shapes and sterilized with UV light for 60 min. After reaching 70–80% confluency, the cells were detached with trypsin–EDTA and seeded on the surface of the scaffolds (5 × 103 cells/each well). The cell-seeded samples were incubated under standard culture conditions for several days. The biocompatibility of the fabricated nanofibers was evaluated by the MTT assay. At the predetermined times (3 and 5 days after seeding), the culture medium was replaced with MTT solution (3 mg/ml) and a new medium containing FBS. After incubation for 4 h at 37 °C, the supernatant was discarded and replaced with 150 µl DMSO to dissolve the formazan crystals. Finally, the absorbance of the samples was measured by a Microplate Reader (Awareness Technology).

The cell attachment on the surface of nanofibers was studied to evaluate the compatibility of the fabricated mats for fibroblast attachment, connection, and migration. For this investigation, the NIH-3T3 fibroblast cells were placed on the surface of each nanofiber according to the previous section and incubated for three days. Then, the medium was removed, the cell-seeded scaffolds were rinsed with fresh phosphate-buffered saline (PBS), and cells were fixed with glutaraldehyde (4% v/ v in PBS) for 20 min. In the next step, ethanol solutions (50, 60, 70, 80, 90, and 100% v/v) were applied to dehydrate the samples. Finally, the attached cells on the nanofiber’s surfaces were imaged by SEM.

2.3 Statistical Analysis

Statistical analysis was performed by one-way analysis of variance (ANOVA) via Graph PAD Prism software (version 8.0.2). All the experimental results were presented as means ± standard deviations, and p value < 0.05 was determined as the statistical significance.

3 Results and Discussion

3.1 Physicochemical Characterization of Nanofibrous Mats

3.1.1 ATR-FTIR Spectroscopy

In this work, ATR-FTIR spectroscopy was used to indicate the chemical composition of the nanofiber’s surfaces. Figure 1 displays the peaks of pristine SO, PU/PCL, PU/PCL/SO, and PU/PCL/SO/GNPs composite nanofibers. As shown in Fig. 1, the characteristic band of PCL was observed at 1725 cm−1 which was assigned to the stretching vibration of carbonyl groups (C=O). The peaks near 2868–2952 cm−1 were attributed to the C−H stretching vibration of PCL units. About PU, the characteristic absorption peak at 1531 cm−1 was corresponded to the amide groups of N−H and C−N. About SO, the bands around 2854–2924 cm−1 and 1462 cm−1 were attributed to the −CH3 and −CH2 stretching vibrations. In addition, peaks at 3010 (CH=CH), 1709 (−COOC−), and 1083 cm−1 (−C−O−C−) were observed, that could show the existence of unsaturated double bonds of SO [36]. From the spectra, it can be seen that the peak of hydrocarbon bonds (methyl stretching), which is seen around 2900 cm−1, has increased in SO and samples mixed with it. Since soy is a fatty acid, its integration into the PU/PCL mesh increases the elongation of methyl, which can be seen in the spectra of SO, PU/PCL/SO, and PU/PCL/SO/GNP. Therefore, SO forms chain entanglements instead of covalently binding to PU/PCL mass materials. This increase in methyl elongation and methyl groups indicates that SO has been successfully integrated into the samples.

Fig. 1
figure 1

FTIR spectra of pristine SO, PU/PCL, PU/PCL/SO, and PU/PCL/SO/GNPs composite nanofibers

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3.1.2 Morphological Characterization of Nanofibrous Mats

In tissue engineering and fabrication of appropriate fibers for biomedical applications, the study of their morphology is very important in terms of fiber diameter [37, 38]. Fiber diameter is an important parameter for cellular signaling that can ultimately significantly affect cell differentiation and function. Optimizing fiber diameters is critical to achieving a well function and biocompatible scaffold [39, 40]. In this work, the fabricated PU/PCL, PU/PCL/SO, and PU/PCL/SO/GNP composite nanofibers were morphologically investigated through SEM images. Figure 2 shows the different groups of nanofibers prepared in this study. The fibers showed suitable morphology and free of beads characteristics. The presence of a porous network was also observed despite the distinct fibers. In addition, based on the SEM images, it was observed that the presence of SO in the nanofiber structure has increased the diameter of the nanofibers and their fusion. It also appears that the fibrous structure has lost its homogeneous shape. But on the other hand, it was observed that the presence of GNPs in the PU/PCL/SO/GNP fibers has created a homogeneous and uniform fiber structure. Generally, the combination of SEM micrographs and ImageJ analysis of the nanofiber samples shows that the presence of SO and GNPs increases the diameter of the fibers. This phenomenon is thought to be due to the being ejected of more solutes at the tip of the Taylor cone, which increases the fiber diameter. This has been observed in previous studies [41, 42]. These results demonstrated the successful incorporation of SO into PU/PCL mesh materials as well as the successful incorporation of GNPs.

Fig. 2
figure 2

SEM images and size distribution of a PU/PCL nanofibers, b PU/PCL/SO nanofibers, c PU/PCL/SO/GNP nanocomposite fibers, and d GNPs

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3.1.3 Surface Water Absorption of Nanofibrous Mats

Surface hydrophilicity is one of the important parameters that affect the biological properties of fibers and provide improved properties in terms of cell growth and proliferation [43, 44]. For this purpose, in the present study, to evaluate the mentioned feature, measuring the contact angle of the water droplet was used. The contact angle assessment is based on the theta angle, and the lower theta angle lead to the more hydrophilicity of the surface. In addition, according to the previous studies, a contact angle close to at least 90 indicates a hydrophobic surface [41]. In the present study, the contact angle of the PU/PCL fibers is more than 113 degrees, which means incomplete wetting. On the other hand, contact angle measurements for the PU/PCL/SO and PU/PCL/SO/GNP fibers show 91 and 76 degrees, respectively, indicating good wetting of these scaffolds. In this part of the study, several measurements were performed on the samples at different spatial locations. As shown in Fig. 3, the fibers containing SO have more hydrophilic surface properties than oil-free fiber (PU/PCL fiber). In addition, as it was reported in previous studies that the use of GNPs decreases the contact angle [23, 45], in this study, it was also observed that the presence of GNP leads to a further decrease in the water contact angle and thus more wettability. This is most likely because the contact angle of GNPs is about 82.3° ± 8.0°, as reported by Lucio et al. [46]. Accordingly, the decrease in water contact angle can be related to the more hydrophilic nature of GNPs than PU/PCL. Finally, it was demonstrated that the presence of GNPs led to more wettability of the PU/PCL/SO/GNP nanofibers compared to the other samples. It was demonstrated that the presence of GNPs along with SO in the structure of PU/PCL/SO/GNP fibers caused a significant increase (p < 0.01) compared to the PU/PCL nanofiber in terms of surface hydrophilicity.

Fig. 3
figure 3

Contact angle images and measurements of a PU/PCL nanofibers, b PU/PCL/SO nanofibers, and c PU/PCL/SO/GNP nanocomposite fibers ***p < 0.001

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3.1.4 The Surface Roughness Study

To increase the biocompatibility of tissue engineering scaffolds, achieving higher hydrophilicity with surface roughness is critical, because it can create a favorable environment for the appropriate function of cells and their proliferation [47]. As shown in Fig. 4a, PU/PCL nanofibers show a surface with noticeable roughness. The rough surface of PU/PCL nanofibers was altered by the incorporation of SO (Fig. 4b). It has also been observed that the surface roughness of the sample encoded with PU/PCL/SO/GNP (Fig. 4c) is more balanced compared to the pure PU/PCL nanofibers or PU/PCL nanofibers loaded with SO. Therefore, it was proved that the great change in the surface is attributed to the presence of GNPs, which causes a significant surface smoothness. Meanwhile, SO does not have a considerable effect on the surface nature of PU/PCL nanofibers, and only their presence, along with GNPs, causes a more balanced surface roughness, which is essential for better interaction with cells.

Fig. 4
figure 4

AFM results of a PU/PCL nanofibers, b PU/PCL/SO nanofibers, and c PU/PCL/SO/GNP nanocomposite fibers

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3.1.5 Electrical Conductivity Measurement of the Nanofibers

The distribution of GNPs throughout the scaffold is an effective strategy to increase electrical conductivity [48]. GNPs, known as biocompatible nanostructures, have great potential for enhancing intercellular electrical communication. Various studies have shown that the presence of GNPs in various structures made of materials such as alginate, collagen, and poly (2-hydroxyethyl methacrylate) increases the electrical conductivity of the scaffold and better function of cardiomyocytes and effective differentiation of mesenchymal cells [49,50,51]. In this study, the electrical conductivity of PU/PCL, PU/PCL/SO, and PU/PCL/SO/GNP nanofibers was reported according to Table 1. It was observed that both oil and GNPs caused a significant increase in electrical conductivity and acted well as electrical stimuli.

Table 1 Electrical conductivity measurement of the nanofibers
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3.2 Mechanical Characterization of the Nanofibers

Mechanical properties strongly depend on the composition of the structure. To estimate the effect of GNP and SO on tensile properties, the stress–strain curves for various samples were plotted (Fig. 5). In addition, Table 2 reports the mechanical properties of the samples. It has been observed that the mechanical properties showed a lower value for PU/PCL/SO mesh compared to pure PU/PCL nanofibers. The results showed that the combination of SO leads to statistically significant data. This reduction is due to the greater flexibility that is more appropriate for some tissue engineering applications [52]. Various surface factors, such as porosity and fiber distribution, can be effective in the mechanical properties. A compound that increases porosity reduces the tensile strength of the nanofibers, although the excellent alignment of the nanofibers increases the strength and thus creates more elasticity.

Fig. 5
figure 5

Stress–strain curve of electrospun a PU/PCL nanofibers, b PU/PCL/SO nanofibers, and c PU/PCL/SO/GNP nanocomposite fibers

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Table 2 Mechanical properties of the prepared nanofibers
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3.3 Biocompatibility of the Nanofibers

3.3.1 MTT Assay

It is expected that antimicrobial substances such as GNPs should always be highly biocompatible when used in clinical applications and not be toxic to normal cells [53, 54]. Thus, to investigate the biocompatibility of the fabricated nanofibers, MTT assay was performed on days 3 and 5. On the third day, all the samples showed a significant enhancement in optical density (OD) compared to the control sample (Fig. 6). As can be seen, because of the excellent biocompatibility, the fabricated PU/PCL showed high cell viability. On the other hand, the PU/PCL mixture with SO did not change cell viability. This can be due to the SO’s biocompatibility with human cells. In addition, it was observed, when PU/PCL mixed with GNP (PU/PCL/SO/GNP), the cell viability slightly decreased, but it remained within acceptable limits. In addition, there is no significant difference between the study groups on the third and fifth days. These confirm that the presence of GNP and SO in the structure of nanofibers has not led to a negative effect on their biocompatibility. Our research, as well as that of others [41, 55, 56], has shown that cell proliferation increase in GNP-bound substances.

Fig. 6
figure 6

The MTT results of PU/PCL, PU/PCL/SO, and PU/PCL/SO/GNP nanocomposite fibers at 3 and 5 days after seeding

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3.3.2 Cell Attachment Study

Various surface properties, including different chemical groups, hydrophilicity, and roughness, can affect cell adhesion and alter cell viability [57, 58]. As various studies have shown an increase in cell binding following an increase in hydrophilicity [59, 60], our results have also demonstrated that the addition of SO and GNP increases hydrophilicity and ultimately increases cell adhesion and viability. As mentioned earlier, incorporating SO and GNP has resulted in a rougher surface, followed by increased cell attachment. This can be seen in Fig. 7.

Fig. 7
figure 7

SEM images shows the NIH-3T3 fibroblast cells attachment on the a PU/PCL, b PU/PCL/SO, and c PU/PCL/SO/GNP nanocomposite fibers at 3rd day after seeding

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4 Conclusion

In the present study, the electrospun PU/PCL, PU/PCL/SO, and PU/PCL/SO/GNP nanofibers were fabricated and characterized. The presence of GNPs in the PU/PCL/SO/GNP fibers has created a homogeneous and uniform fiber structure. In addition, the AFM results indicated a significant surface smoothness for PU/PCL/SO/GNP nanocomposite mats. The wettability behavior of the nanofibers was measured, and PU/PCL/SO/GNP nanocomposite fibers showed more hydrophilicity compared to the other groups. The reinforcement of PU/PCL nanofibers with SO and GNP leads to an increase in electroconductivity. The cytocompatibility studies demonstrated the fabricated scaffolds, especially PU/PCL/SO/GNP nanocomposite fibers, have great potential to be applied as biocompatible biomaterials to support cellular adhesion and tissue engineering.