3.1. Characterization of extracted SF
Extracted SF was characterized using XRD and FTIR analysis before further use. Accordingly, the XRD pattern (Fig. 2a) showed that the structure of SF was predominantly amorphous. The eminent single peak observed at 2θ= 20.1° was an indication of the silk II crystalline structure, similar to previous reports [33].
The FTIR spectrum resulted from analyzing the chemical structure of extracted SF is shown in Fig. 2b. Accordingly, the peaks observed at 1063, 1230, and 1445 cm-1 could be ascribed to the stretching vibrations of C-O-C bonds, stretching vibrations of C-N bonds, and bending vibrations of C-H of SF, respectively. Moreover, peaks located at 1525 and 1650 cm-1 were related to bending vibrations of N-H groups and bending vibrations of O-H groups, respectively. Peaks observed in the 3000-3600 cm-1 range were related to stretching vibrations of N-H and O-H bonds. The results were in line with previous work reported in literatures [34- 36].
3.2. Morphological analysis
3.2.1. Morphological study on nanofibrous layer
Similar to many natural biopolymers, SF cannot be easily electrospun alone and thus is commonly mixed with other natural or synthetic polymers for electrospinning [37]. SF has a negative charge on its backbone and when it is exposed to an electrical field, similarly-charged chains cause repulsion and hence prevent the success of electrospinning. PVA acts as a lubricant and neutralizes the repulsive forces of negative charges by slipping between SF chains. Thus, uniform electrospun SF-PVA fibers could be obtained [23].
In order to investigate the effect of the mass ratio of polymer contents on the morphology of fibers, SF/PVA nanofibers were electrospun at 1:1, 2:1 and 3:1 ratio at the constant electric voltage of 18 kV and nozzle to collector distance of 15 cm and resulting mats were investigated using SEM in fig. 3. As shown in Fig. 3., the SF/PVA nanofibers with 1:1 ratio were uniform, smooth and bead-less with random orientation. Increasing the PVA content (in other words, lower SF content in the solution) increased the total concentration from 8.5% for 3:1 solution to 8.7% and 9% for 2:1 and 1:1 solutions, which increased the viscosity. Hence, it was suggested that for the 1:1 solution, PVA polymer chains slipped between SF chains more uniformly and could lead to more uniform fiber diameters by neutralizing electrical charges and hence creating a stable jet that could be formed more readily under the force of the electric field.
While for the sample with 1:1 ratio an average fiber diameter of 199±28 nm was obtained, for samples with 2:1 and 3:1 SF/PVA ratios, the average fiber diameter values decreased, resulting in 130±36 and 125±41 nm diameters due to the lower concentration and viscosity of the solutions. This could be due to the fact that decrement of solution concentration followed by reduction of viscosity, diminished the chance of the formation of a stable jet on the nozzle and hence led to the formation of fibers that had structural defects. Hence, the scaffold resulted from the 1:1 SF/PVA solution was chosen as the optimum sample.
In order to determine the optimum electrospinning conditions, SF/PVA solution with 1:1 ratio was electrospun at 15 kV and 18 kV electrical voltages and at nozzle to collector distance of 10 and 15 cm and results is shown in Fig. 4. At the electrospinning condition of 15 kV applied voltage and 10 cm distance, beads were observed in the fibrous structure, and a high variation in fiber diameter was observed. It seems that increasing electrical voltage and nozzle to collector distance had a positive effect to obtain uniform and bead-free structure. Additionally, the average electrospun fibers diameters at voltages of 15 kV and 18 kV were found to be 206±60 nm and 202±44 nm for nozzle to collector distance of 10 cm and 211±51 and 199±28 nm for nozzle to collector distance of 15 cm, respectively. At a constant nozzle to collector distance, lower average fiber diameters were obtained in a stronger electrical field due to higher tension of the field. Additionally, increasing the nozzle to collector distance forced the solution to undergo higher stretching, which caused lower average fiber diameters at a constant voltage. The result is in line with the study reported by Fathi et al [37].
To conclude, the optimum electrospun conditions were obtained at a nozzle to collector distance of 15 cm and electrospinning voltage of 18 kV for the SF/PVA solution with 1:1 ratio, which resulted in the average fiber diameter of 199±28 nm with uniform and bead-free structure.
3.2.2. Morphological study on hybrid structures
SEM analysis was also used to investigate the morphology of hybrid hydrogel based on SA/GT incorporated with SF/PVA nanofibrous layer, as shown in Fig. 5. Accordingly, the SF/PVA nanofibrous layer was observed on the surface of the SA/GT blend hydrogel. Not only did the hydrogel encompass the nanofibrous layer, but also penetrated into the fibrous structure, which could enhance the properties of the hybrid wound dressing.
3.3. Crystalline and chemical structure of SF/PVA nanofibers
XRD analysis was used to evaluate the crystalline structure of SF, PVA, and their mixture, as shown in Fig. 6a. For pure PVA, two peaks at 2θ=19.1°, 40.9° were observed which could be related to the semi-crystalline nature of PVA. On the other hand, SF/PVA sample had a peak at 19.8° corresponding to the β-crystalline lattice in addition to the peak at 19.9°, which was attributed to the apparent superposition of both materials. In the SF/PVA mixture, these peaks were attributed to the β-sheet silk II structure. Silk II is the pleated form of silk comprising of β-form or anti-parallel crystals [38]. Results showed that the crystalline structure of SF was affected by the PVA in the mixture, and shorter SF chains could initiate the crystallization of PVA, resulting in more distinctive lattices. These results have also been observed in previous reports [39].
Chemical interactions between the components of nanofibers were analyzed using FTIR spectroscopy and results are shown in Figure 6b. For the PVA sample, the peak observed at 852 cm-1 was related to the rocking vibrations of C-H bond of PVA. Also, peaks observed at 1327, 1419, and 1635 cm-1 were related to bending and stretching vibrations of C-H of methyl and methylene bonds, and the bending vibrations of O-H of PVA, respectively [38]. Peaks observed at 1095 and 1720 cm-1 were both related to the C-O stretching bonds, respectively; and peaks observed at 2850 and 2929 cm-1 were related to the symmetric and asymmetric stretching vibrations of the C-H bond. In addition, peaks in the 3300-3600 cm-1 range were related to the stretching vibrations of O-H bonds due to the presence of hydroxyl groups [40].
In FTIR spectrum of SF/PVA sample, peaks observed at 1230, 1520, and 1650 cm-1, were related to the stretching vibrations of C-N bonds, bending vibrations of N-H, and the bending vibrations of O-H in SF, as previously reported by others [38]. In addition, peaks observed at 2850 cm-1 and 2920 cm-1 were related to the symmetric and asymmetric vibrations of C-H bonds of PVA. Finally, peaks observed in the 3600-3300 cm-1 range were related to the stretching vibrations of O-H and N-H bonds due to the presence of hydroxyl groups of PVA and amine groups of SF [34, 41].
3.4. Thermal properties of hybrid wound dressing
The thermal properties of prepared hybrid wound dressing were investigated using TGA and results are shown in Fig. 6c. The final char yield of the sample in the air conditions was 45.65%, showing the desired thermal stability of prepared hybrid structure. Initially, the sample demonstrated a small (6%) weight loss due to the evaporation of physically bound water and moisture, followed by a 4% weight loss up to 210 °C. The following endotherm region at about 220 °C on the TGA curves of the sample was attributed to a physical transition, such as the melting of crystalline phase, and not to a chemical decomposition. The weight loss in the 250 °C region was attributed to GT, and major decomposition occurred up to 307 °C. In general, the decomposition process included desorption of physically-bound water, removal of structural water (dehydration process), depolymerization as well as the rupture of C-O and C-C bonds in the ring units, which led to the formation of CO, CO2 and H2O, and finally, the formation of graphitic carbon substances (char). In addition, semi-plateau regions could be attributed to the hydrophilic nature of functional groups in polymers [42].
3.5. Swelling behaviour of hybrid wound dressing
Swelling capacity of hydrogels is one of their most important properties when used in biomedical applications such as drug delivery, tissue engineering, and wound healing due to its resemblance with the host tissue. When crosslinked polymer hydrogels are immersed in water or other solvents, they can swell, but they do not dissolve [43]. The swelling property of hydrogels depends on many parameters such as crosslinking density, solvent structure, and polymer-solvent interactions as well as the content of hydrophilic groups in the hydrogel structure. Swelling behaviour of hydrogels also controls biodegradation and sustainable drug release as well [44]. Accordingly, the swelling behaviour of hydrogel and hybrid wound dressing (hydrogel/nanofibers) were investigated and the results were shown in fig. 7 a, b and c.
As photographically shown in Fig. 7a. and Fig. 7b., and according to the obtained diagram (Fig. 7c), hydrogels swelled in distilled water without any changes in their apparent structure after 24 h. The average thickness of samples increased from 1±0.1 mm to 14±0.1 mm after swelling. Accordingly, 1251% and 1446% swelling values were obtained for the hybrid wound dressing (hydrogel/nanofibers) and hydrogel without nanofibers, respectively. The result concluded that the produced hydrogel can provide a moist environment to accelerate the wound healing process by increasing the rate of mass transfer of biologic substances to the wound site. In addition, hydrogels with favourable swelling properties can absorb the wound exudates, which in turn, prevent infections [45].
3.6. Drug release studies
In order to investigate the drug release behavior from hybrid wound dressing, UV-Vis spectroscopy was utilized and results are shown in Fig. 7d. Accordingly, calibration curve was first obtained by diluting cardamom oil extract in ethanol and the released content was studied at 0, 3, 6, 9, 18, 24, 48, 72, and 96 h at 240 nm. According to the results, the released content increased over time to 85% release of the drug in total. The release behaviour followed a two-step profile. In the first step (initial 12 h), the drug diffused slowly from the hybrid wound dressing and in the second step (after 12 h), initial burst was observed due to the rapid release from the scaffold, which could be due to the initiation of degradation process of biodegradable constituents. It has been reported that release of pharmaceutical substances from biodegradable polymers can be controlled by the porosity of hydrogel, swelling behaviour, drug loading content and the degradation process as well [46].
3.7. Antibacterial activity
Antibacterial performance of hybrid hydrogel/nanofiber was investigated against Gram-negative (E. coli) and Gram-positive (S. Aureus) using the disk diffusion method (Fig. 7e and Fig. 7f). Results revealed that the hybrid wound dressing (hydrogel/nanofiber) without extract had no antibacterial activity against both bacteria. While for the gentamycin sample as the control, inhibition zones of 17 mm and 16 mm against E. coli and S. Aureus were observed, this value for the hybrid wound dressing with cardamom oil extract was found to be 14 mm and 12 mm against E. coli and S. Aureus, respectively. The obtained results are in agreement with the results reported by Najafi et al. [47], who reported that cardamom extract improved the antibacterial properties of alginate/PEA scaffold.
The activity of sample containing cardamom oil extract was higher against the Gram-positive bacterium (85.35%) compared to the Gram-negative bacterium (75%). Similar results have been observed in other studies, where a higher activity against S. Aureus have been observed for cardamom essential oil compared to other bacteria [48]. The activity of cardamom oil extract against bacteria and microorganisms is primarily caused by disturbing the cytoplasmic membrane due to the difference in the surface charges of cell membrane and cyclic hydrocarbons present in the constituents of cardamom, which finally lead to coagulation of cell contents of the bacteria and hence, cell death [47]. It should be mentioned that cardamom contains different bioactive metabolites such as flavonoids, carotenoids, and terpenes which have potential pharmaceutical and clinical properties such as antioxidant, anti-inflammatory, antimicrobial, antivirus, and antibacterial properties [48, 49].
3.8. Cell viability and attachment
Cell viability, as an index of biocompatibility, was investigated using fibroblast cells for hybrid wound dressing with (E) and without cardamom oil extract (WE) and results are shown in Fig. 8a. In general, the samples had cell viability in the initial 24 h. However, the sample without extract caused higher cell viability than its counterpart with extract after 48 and 72 h of cell culture. Jamil et al. reported that for systems in which cardamom oil extract was encapsulated in chitosan nanocomposites, the extract reduced cell viability [50].
Cell attachment studies were also carried out after 1, 2, 3 days using SEM, and results are shown in Fig. 8b. In agreement with MTT studies, the number of alive cells on the surface of samples decreased when cardamom extract was added to the hybrid wound dressing. However, cell flattening and spreading was more evident in sample containing extract, particularly after 48 and 72 h. In addition, attachment studies showed that the surface of the sample was favourable host for cells to grow and proliferate that showed their potential for skin tissue regeneration.