1. Introduction
Skin is the body’s largest organ, with multiple critical functions. It serves as a protective barrier against harmful microorganisms and mechanical damage, helps maintain the body’s temperature balance, and aids in the excretion of waste products [
1,
2,
3,
4,
5]. The integrity of the skin is affected by burns, trauma, and diabetes which can lead to skin inflammation and bacterial infection.
Wounds, caused by a lack of skin integrity, are a major issue in the world, and have caused economic and social problems in all societies [
6,
7,
8]. The human skin has the ability to regenerate itself against small and superficial wounds. However, in the case of severe and deep wounds, the natural healing process is not enough, and protection of the injured area is needed until the wound is completely healed. Therefore, the fabrication of proper wound dressing to accelerate the wound healing process is necessary [
9]. The ideal wound dressing should perform tasks such as providing a moist environment around the wound, exchanging gas and nutrients, collecting exudate from the wound site, not adhering to the wound, and to prevent bacterial infection and allergic reactions. Accordingly, a wound dressing should be biocompatible, biodegradable, swellable, elastic, and antibacterial. In addition, having a porous structure is necessary for a dressing to improve cell growth, proliferation, migration, and angiogenesis. As a result, the fabrication of an ideal wound dressing to have all these items require great effort and precision [
10,
11].
There are various methods, such as solvent casting [
12], salt leaching [
13], freeze-drying [
14], and electrospinning [
15] in the fabrication of wound dressings. Electrospinning is an effective technique that can use a wide variety of biomaterials to produce nanofibrous structures with uniform and continuous nanofibers and high specific surface area. This method fabricates highly porous structures, which simplify gas and nutrient exchange and provide moisture around the wound, preventing excessive drying of the wound. Also, electrospun nanofibers have similar flexibility and tensile strength as human skin and can simulate the extracellular matrix (ECM) structure of the skin tissue, providing a microenvironment which promotes cell attachment, growth, and proliferation [
16,
17,
18]. Hadizadeh et al. [
19] fabricated a poly (ɛ-caprolactone) (PCL)/gelatin (Gel)-based electrospun scaffold, incorporated with surfactin (Sur) and curcumin (Cur). The PCL/0.2Sur-Gel/3%Cur scaffold showed suitable wettability, mechanical properties, degradation rate, antibacterial activity, and biological properties (
in vitro and
in vivo). Also, Bao et al. [
20] fabricated an electrospun scaffold composed of hyaluronic acid (HA), graphene (Gr), and polyphenolic tannic acid (TA). The in vivo results indicated that after 14 days, the wound area of the scaffold loaded with 0.3 w/v% TA was 1.12 ± 0.54 mm
2, was significantly (
p < 0.05) better than that of the HA and control groups. However, in addition to the fabrication method of the scaffolds, the biomaterials used for fabricating the scaffolds also have a significant effect on their final properties.
Poly (glycerol sebacate) (PGS) is a tough polyester which is synthesized by polycondensation of glycerol and sebacic acid. It is a biocompatible, elastic, and biodegradable polyester, and its degradation products can be removed by the body’s metabolism. However, this polymer does not have the ideal hydrophilicity and degradation rate and cannot form a nanofibrous structure due to its low spinnability [
21,
22,
23]. Thus, it should be blended with hydrophilic polymers.
Gelatin (Gel) is a naturally occurring hydrophilic polymer that can be extracted from collagen, the most abundant protein of the extracellular matrix (ECM). Gel offers several advantages, including biocompatibility, non-immunogenicity, and biodegradability, making it a suitable material for supporting cell attachment, growth, and proliferation. Moreover, Gel is readily available at a relatively low cost. It can be electrospun using aqueous solutions of acetic acid, formic acid, or ethanol. [
24,
25]. In a study, Farahani et al. [
26] fabricated a cellulose acetate/gelatin/
Zataria multiflora nanoemulsion (CA/Gel/
ZM-nano) wound dressing using the electrospinning method. The results showed that adding Gel decreased the rate of drug release and increased the cell viability of the dressing. Also, in another study, Sanhueza et al. [
25] prepared an electrospun scaffold composed of Gel/poly-3-hydroxybutyrate (PHB) nano/microfibers for the healing of diabetic wounds. In vivo results showed that for 14 days, the wound area decreased significantly (
p < 0.1) after adding Gel to the scaffolds. Moreover, the histological analysis indicated that hypodermis was formed after treatment of the wounds with the Gel-PHB scaffold.
In this paper, we fabricated and evaluated a PGS/Gel nanofibrous structure using electrospinning to assess its morphological, physical, and biological properties. To the best of our knowledge, this study is the first to examine the fabrication and characterization of a PGS/Gel electrospun scaffold for wound healing applications. Our findings can provide valuable insights for biomedical engineers seeking to develop superior wound dressings with optimal properties.
2. Materials and Methods
2.1. Materials
The applied materials in this study have been shown in
Table 1.
2.2. Synthesis of poly (glycerol sebacate) (PGS) prepolymer
Based on the method presented by Kharaziha et al. [
27] for synthesizing PGS prepolymer, firstly, a glycerin bath was prepared at 130 °C in a glass container, which had been placed on a magnetic stirrer equipped with a heating system (IKI/Germany). Then, glycerol, which had been degassed under a nitrogen atmosphere and at 130 °C for 2 h, was added to a three-necked flask placed in this bath. After the temperature of glycerol reached the temperature of the bath, sebacic acid powder, which had been dried at 55 °C in a vacuum oven (Memmert/Germany), was introduced to glycerol with a molar ratio of 1:1. This system was stirred on a magnetic stirrer under a slow flow of nitrogen gas and at 130 °C for 24 h. After the mentioned period and the cooling of the system, a paste-like material was obtained, which was the PGS prepolymer. The schematic of the synthesis process has been shown in
Figure 1.
2.3. Fabrication of the electrospun scaffolds
Firstly, the PGS/Gel solution with a ratio of 3:1 was prepared using acetic acid 75 v/v% at 38-42 °C, and the final polymer concentration was set in the range of 10-30 wt. %. As a crosslinking agent, EDC/NHS was dissolved in ethanol and then introduced to the polymeric solution, followed by stirring for 30 min to complete the cross-linking process. Finally, all the samples were loaded in a 1-ml plastic syringe with a 21-gauge needle and electrospun using an electrospinning device (GMS300/Iran), and the concentration of the solution was optimized according to the solution spinnability and the quality of the nanofibers electrospun on laboratory slides. The electrospinning process was carried out at 25 °C. The prepared optimum scaffold was placed in a vacuum oven (Memmert/Germany) at room temperature for 6 h.
2.4. Morphological assessment
To assess the structural features of the scaffolds, such as morphology, porosity, and integrity, scanning electron microscopy (SEM, Seron AIS 2300C/Korea) was used. After cutting the scaffolds into the 1×1 cm
2 squares, they were fixed on a grid, followed by coating their surface with gold. Using SEM micrographs of the scaffolds, the diameter of 100 fibers was measured with Image J software (Wayne Rasband, National Institute of Health/USA), and the range of fiber diameter was determined [
28]. Also, MATLAB (R2017b) software was applied to calculate the porosity of the scaffolds from their SEM micrographs [
29].
2.5. Fourier transform infrared spectrometry
To evaluate the possible interactions between the two polymers and the chemical structure of the scaffolds, the Fourier transform infrared spectrometry (FTIR, JASCO FT/IR-6300/Japan) test was applied. The test was performed over a wavenumber range between 400 to 4000 cm-1 at room temperature.
2.6. Measurement of contact angle
Using a contact angle meter (XCA-50, PMC/Iran), the measurement of water contact angle (WCA) was applied to assess the hydrophilicity of all the scaffolds. The analysis was carried out at room temperature, and the sessile drop technique was used to record the contact angle. The droplet volume was set at 4 µl, and the measurements were performed after 15 s of dropping distilled water on the surface of the scaffolds.
2.7. In vitro degradation
Based on the ASTM F1635 standard, an in vitro degradation test was conducted to study the degradation rate of the scaffolds. In this regard, the samples were cut into 1×1 cm
2, and after weighing (
W0) them with a laboratory scale of 5 decimal places (PLS510-3A/Germany), they were immersed in 5 ml of phosphate buffer saline (PBS) solution, followed by incubation (Memmert/Germany) at 37 °C. On days 1, 4, 7, 14, and 21 of the incubation period, the samples were removed from the solution, rinsed with distilled water to remove the formed salts, and dried in a vacuum oven (Memmert/Germany) at 37 °C for 2 h before weighing (
Wt), and the following formula was used to calculate the weight loss percentage:
To avoid the effects of the degradation products on the quality of the solution that may affect the weight loss profile of the samples, the buffer solution was completely replaced every 24 h.
2.8. In vitro cellular studies
After cutting the scaffolds into circles with a diameter of 1.5 cm and sterilizing them with UV light, ethanol, and PBS, respectively, they were fixed in the wells of 24-well plates and prepared for cell seeding. The prepared human dermal fibroblast (HDF) cells were transferred to the flasks containing DMEM culture medium, which was supplemented with 10% FBS and 1% penicillin-streptomycin, and the flasks were kept in an incubator (Memmert/Germany) with 5% CO2 at 37 °C for 3 weeks. Until the cell confluency reached 80%, the culture medium was renewed every 2 days. Then, the cells were trypsinized, and after counting the cells, 5 × 103 cells were seeded on the prepared scaffolds, followed by incubating the plates. The culture medium was renewed every other day until the end of the cell culture period.
According to the ISO-10993-5 standard, the MTT assay was applied to evaluate the viability of the cultured cells on the scaffolds, and the results were compared to the scaffold-free control sample. On the 3rd and 5th days of cell culture, the culture medium was removed, and the cells were rinsed twice with the PBS solution to remove the dead cells. Next, 200 µl of serum-free culture medium containing 20 µl of the MTT solution (5 mg/ml) was added to each well. After 4 h of incubation at 37 ℃ and 5% CO2, the insoluble blue crystals of formazan were formed due to the presence of mitochondrial enzymes in living cells, and the color of the medium turned from yellow to dark blue. To dissolve the formazan crystals, 200 µl of DMSO solution was replaced with the MTT solution, and the pipetting process was performed for the complete dissolution of the crystals. Finally, the formazan solution was transferred to a 96-well plate, and the absorbance at the wavelength of 570 nm was measured with a microplate reader (BioTek-FLx800/USA).
The cells were cultured on the prepared scaffolds for up to 5 days to observe cell attachment and proliferation. After removing the culture medium on the 5th day of incubation, the samples were rinsed 3 times with the PBS solution, and 4% glutaraldehyde was applied to fix the cells, followed by keeping the samples at 4 °C for 2h. After that, the scaffolds were dehydrated using ethanol solution with concentrations of 50, 70, 80, 90, and 100 v/v%, respectively. After drying the scaffolds at room temperature, cell attachment and proliferation were assessed using SEM (Seron AIS 2300C/Korea) analysis.
2.9. Statistical analysis
Using SPSS software (n = 3; version 22) for statistical analysis, the data was evaluated via one-way ANOVA. The results (n = 3) were reported as mean ± standard deviation (SD). The significance level was defined where the p-value is less than 0.05 (p < 0.05).
4. Discussion
Fiber uniformity and integrity are among the main morphological factors of electrospun scaffold to provide an appropriate surface, promoting cell attachment and proliferation [
33,
34]. Adding gelatin has resulted in the production of a more uniform nanofibrous structure, possibly due to the hydrogen bonding between PGS and Gel [
35], which has been further discussed below. Yang et al. confirmed the formation of hydrogen bonds between Gel and poly (3-hydroxybutyrate-co-4-hydroxybutyrate) (P3,4HB) polyester [
36]. Another factor, porosity percentage, is important in scaffold designing. A porous 3D scaffold with a porosity percentage of above 70% has an interconnected structure, allowing the cells to penetrate in its depth as well as facilitating nutrient and waste exchange [
37]. All the fabricated scaffolds have a porosity percentage above 75%, showing a suitable 3D structure which could be applied in biomedical applications. Based on FTIR results, the shift of the carbonyl functional group peak to lower wavenumbers and a decrease in its intensity could be attributed to the formation of hydrogen bonds between the carbonyl groups of PGS and amide groups of Gel. Other study showed that hydrogen bonds were formed between Gel and Polycaprolacton (PCl) electrospun multilayer nanofibrous scaffold [
38].
Surface properties, such as hydrophilicity, should be optimized to promote cell attachment, spreading, and proliferation. Water contact angle measurement is a general technique to assess the hydrophilicity of the scaffolds’ surface [
33]. According to other studies [
34,
37], the surfaces with water contact angles in the range of 40°-80° have appropriate hydrophilicity and can support cell adhesion and proliferation. Based on the results, adding gelatin has significantly (
p < 0.05) decreased the water contact angle of the PGS/Gel scaffold, probably due to its hydrophilic nature due to the presence of hydroxyl groups, which has been confirmed by other studies [
39,
40]. Also, the formation of hydrogen bonds in the PGS/Gel scaffold (based on FTIR results) could be another reason for the increasing trend of the hydrophilicity of the scaffold, proved by another study [
33].
The biodegradation rate of an ideal scaffold should be matched with the rate of the wound healing process [
37]. Due to their high porosity and nano-sized fibers, electrospun structures possess relatively high surface area, making them suitable to be used as a biodegradable scaffold [
34]. The results of in vitro biodegradation analysis show that the pure PGS scaffold has a very slow degradation rate over 21 days due to its hydrophobicity, which is not desirable for wound healing applications [
41]. On the other hand, adding Gel increased the biodegradation rate of the PGS/Gel scaffold because of the Gel hydrophilic nature [
38,
42,
43] and the formation of hydrogen bonding between Gel and PGS [
34]. Also, the MTT results indicate that adding Gel has significantly (
p < 0.05) increased viability of the cells cultured on the PGS/Gel scaffold, which could be attributed to the hydrophilic nature of Gel, the presence of sufficient hydroxyl groups on the surface of the scaffold [
37], and the formation of hydrogen bonding interactions between PGS and Gel [
34]. Wang et al. showed that the presence of Gel in the PCL/Gel hierarchical scaffolds caused an increment in the viability of the cultured cells on the blend scaffolds [
44]. Moreover, confirming the MTT results, the SEM micrographs of the 5th day of cell culture show the appropriate cell attachment and proliferation on the surface of the PGS/Gel scaffolds. Other studies indicated a superior cellular behavior of the PGS/Gel scaffold, making it suitable for cardiac tissue engineering applications [
27,
45].